# Patent

## Title
Composition comprising Xanthoceras sorbifolia extracts, compounds isolated from same, methods for preparing same and uses thereof

## Abstract

This invention provides compositions, methods and process of producing extracts from Xanthoceras sorbifolia. The extract comprises alkaloids, coumarins, saccharides, proteins, polysaccharides, glycosides, saponins, tannins, acid, flavonoids and others. The composition can be used for treating cancer, arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder, impotence and premature ejaculation; for preventing cerebral aging; for improving memory, cerebral functions; or for curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease or other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder. This invention provides compounds comprising at least one sugar, a triterpene, such as Sapogenin, and at least one side chains at Carbon 21 and 22, such as Angeloyl groups. The compounds of the present have various pharmaceutical and therapeutic applications.

## Background
This application (1) is a continuation of U.S. Ser. No. 10/906,303, Filed Feb. 14, 2005 now U.S. Pat. No. 7,524,824 which is a Continuation-In-Part of International Application No. PCT/US04/43465, Filed on Dec. 23, 2004, which is a Continuation-In-Part application of PCT/US04/33359, Filed Oct. 8, 2004, which claims benefit of U.S. Ser. No. 60/509,851, Filed Oct. 9, 2003 and U.S. Ser. No. 60/532,101, Filed Dec. 23, 2003; and (2) claims benefit of U.S. Ser. Nos. 60/617,379, Filed Oct. 8, 2004, 60/613,811, Filed Sep. 27, 2004; and 60/607,858, Filed Sep. 7, 2004; and (3) claims benefit of U.S. Ser. No. 10/471,384, Filed Sep. 4, 2003, corresponding to Int'l App'l No. PCT/IB02/04750, Filed Aug. 28, 2002, which claims priority of U.S. Ser. No. 09/944,805, Filed Aug. 31, 2001, now U.S. Pat. No. 6,616,943, issued Sep. 9, 2003. The contents of these preceding applications are hereby incorporated in their entireties by reference into this application.
Throughout this application, various publications are referenced. Disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.


FIELD OF THE INVENTION
This invention relates to extracts from a plant called Wenguanguo (Xanthoceras Sorbifolia Bunge), their usages and functions, and methods of their preparation.
BACKGROUND OF THE INVENTION
Wenguanguo is a species of the sapindaceae family. Its scientific name is Xanthoceras sorbifolia Bunge. Wenguanguo is the common Chinese name; others are Wenguanngguo, Wenguanmu, Wenguanhua, Xilacedeng and xilasendeng. Goldenhorn and Yellowhorn are its common English names. Wenguanguo is grown in Liaoning, Jilin, Hebei, Shandong, Jiangsu, Henan, Shanxi, Shaanxi, Gansu, Ningxia and Inner Mongolia, China. Its seeds, leaves and flowers are edible and its seeds have been used as a folk medicine to treat enuresis for centuries. Its branches and woods are also used as a folk medicine.
Chinese patent applications CN 1092991A and CN 1092992A discussed the methods for producing a medicine from Wenguanguo kernel powder for curing enuresis and enhancing cerebral functions. Chinese patent CN 1052636C discussed a method for producing a medicine with ethanol extract from the Wenguanguo kernel powder for curing enuresis and enhancing cerebral functions. Journal of Shenyang University of Pharmacy (2001), 18(1), 53-56 disclosed the n-butanol extract from the wood of Wenguanguo, which has anti-inflammatory effect.
United States Patent Application Publication No. 20030096030 discussed the extracts from the husks of Wenguanguo which are Bunkankasaponin A. B. C. D and two sterols for preventing cerebral aging, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence, and increasing the body's ability to resist the activity of glycosuria.
United States Patent Application Publication No. 20030082293 disclosed the extracts Bunkankasaponin A. B. C. D, crude fats, crude protein and sugars from the shell of Wenguanguo.
U.S. Pat. No. 6,616,943, issued on Sep. 9, 2003, discussed the composition comprising Wenguanguo combined extracts and the methods for preparing them and uses thereof. The methods for preparing the combined extract from the husks comprise the following steps: extracting Wenguanguo husks with an organic solvent (e.g. ethanol) to form an organic (e.g. ethanol) extract; removing the organic solvent (e.g. ethanol) from the organic (e.g. ethanol) extract to form aqueous extracts; and drying and sterilizing the aqueous extracts to form the combined extracts. The combined extracts contain saponins, saccharides, proteins and others. The extracts can be used for producing medicines or health foods for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunction. The medicines or health foods further comprise Vitamin B, Vitamin D, K, anti-oxidant, Cordyceps or its extracts, gingko or its extracts, Echinacea or its extracts, Huperzine A, folic acid, amino acids, creatine, fiber supplement or a combination thereof.
Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(4)1387-1394 (1985) described a study on the constituent of Xanthoceras sorbifolia Bunge. See Section V. Saponins from the Fruits of Xanthoceras sorbifolia. Four new saponins were isolated from the fruits of Xanthoceras sorbifolia Bunge. The structures of these saponins are bunkankasaponins A, B, C and D:

    (1) 22-O-acetyl-21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin
    (2) 22-O-acetyl-21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin
    (3) 28-O-acetyl-21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin
    (4) 28-O-acetyl-21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin


Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(3)1043-1048 (1985) described studies on the constituent of Xanthoceras sorbifolia Bunge. See Section IV. Structures of the Miner Prosapogenin. The prosapogenins from the partial hydrilyzate of fruit saponin of Xanthoceras sorbifolia were examinated, and are characterized as:

    
    
        16-O-acetyl-21-O-(3,4-di-O-angeloyl-β-D-fucopyranosyl) protoaecigenin
        22-O-acetyl-21-O-(3,4-di-O-angeloyl-β-D-fucopyranosyl) protoaecigenin 3-O-β-D-glucuronopyranoside
    
    


Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(1)127-134 (1985) describe studies on the constituent of Xanthoceras sorbifolia Bunge. See Section III. Minor Prosapogenins aponins from the Fruits of Xanthoceras sorbifolia Bunge. The structure of 3 minor prosapogenins, obtained by acid hydrolysis of the crude saponin faction, were characterized as:

    
    
        21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyltheasapogenol B
        21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyltheasapogenol B
        21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-22-O-acetylprotoaescigenin
    
    


Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(4)1387-1394 (1985) described a study on the constituent of Xanthoceras sorbifolia Bunge. See Section II. Major Sapogenol and prosapogenin from the Fruits of Xanthoceras sorbifolia. 
Laurence Voutquenne, Cecile Kokougan. Catherine Lavaud, Isabelle Pouny, Marc Litaudon. Triterpenoid saponins and Acylated prosapogenins from Harpullia austro-calcdonica. Phytochemistry 59 (2002) 825-832
Zhong Jaing, Jean-francois Gallard, Marie-Therese Adeline, Vincent Dumontet, Mai Van Tri, Thierry Sevenet, and Mary Pais Six Triterpennoid Saponins from Maesa laxiflora. J. Nat. Prod. 1999, 62, 873-876
Young Seo, John M. Berger, Jennine Hoch, Kim M Neddermann, Isia Bursuker, Steven W. Mamber and David G. Kingston. A new Triterpene Saponin from Pittosporum viridiflorum from the Madagascar Rainforest. J. Nat. Prod. 2002, 65, 65-68
Xiu-Wei Yang, Jing Zhao, Xue-Hui Lui, Chao-Mei Ma, Masao Hattori, and Li He Zhang Anti-HIV-1 Protease Triterpenoid Saponins from the Seeds of Aesculus chinensis. J. Nat. Prod. 1999 62, 1510-1513
Yi Lu, Tatsuya Umeda, Akihito Yagi, Kanzo Sakata, Tirthankar Chaudhuri, D.K. Ganguly, Secion Sarma. Triterpenoid Saponins from the roots of the tea plant (Camellia sinensis var. Assamica). Phytochchemistry 53 (2000) 941-946
Sandra Apers, Tess E. De Bruyne, Magda Claeys, Arnold J. Viletinck, Luc A.C. Pieters. New acylated triterpenoid saponins from Maesa laceceolata. Phytochemistry 52 (1999) 1121-1131
Ilaria D'Acquarica, Maria Cristina, Di Giovanni, Francesco Gasparrini, Domenico Misiti, Claudio D'Arrigo, Nicolina Fagnano, Decimo Guarnieri, Giovanni Iacono, Giuseppe Bifulco and Raffaele Riccio. Isolation and structure elucidation of four new triterpenoid estersaponins from fruits of the Pittosporumtobira AIT.
Tetrahedron 58 (2002) 10127-10136
Cancer cells are defined by two heritable properties: (1) they reproduce in defiance of normal restraints on cell division; and (2) they invade and colonize territories normally reserved for other cells.
Cancers require mutations of many genes to develop, and they are classified according to the tissue and cell type from which they arise. Cancers arising from epithelial cells are named carcinomas; those arising from connective tissue or muscle cells are named sarcomas. In addition, there are cancers called leukemias, which are derived from hemopaietic cells; and cancers derived from cells of the nervous system.
Cancers originating from different types of cells are, in general, very different diseases. Each cancer has characteristics that reflect its origin. Even when a cancer has metastasized and proliferated out of control, its origins can be traced back to a single, primary tumor. Therefore it is important to develop drugs against target cells with a specified character.
Ovarian cancer is the 5th leading cause of cancer death in women and the leading cause of death from gynecologic. In the United States, females have a 1.4 to 2.5% (1 out of 40-60 women) lifelong chance of developing ovarian cancer. Older women are at highest risk. More than half of the deaths from ovarian cancer occur in women between 55 and 74 years of age and approximately one quarter of ovarian cancer deaths occur in women between 35 and 54 years of age. (See http://www.nim.nih.gov/medlineplus/ency/article/000889.htm)
Ovarian cancer is disproportionately deadly for a number of reasons. First, symptoms are vague and non-specific, so women and their physicians frequently attribute them to more common conditions. By the time the cancer is diagnosed, the tumor has often spread beyond the ovaries. Also, ovarian cancers shed malignant cells that frequently implant on the uterus, bladder, bowel, and lining of the bowel wall (omentum). These cells can begin forming new tumor growths before cancer is even suspected. Second, because no cost-effective screening test for ovarian cancer exists, more than 50 percent of women with ovarian cancer are diagnosed in the advanced stages of the disease.
This invention provides compounds or compositions extracted from Xanthoceras Sorbifolia or synthesized which have substantial potency against ovarian cancer.
SUMMARY OF THE INVENTION
In accordance with these and other objects of the invention, a brief summary of the present invention is presented. Some simplifications and omission may be made in the following summary, which is intended to highlight and introduce some aspects of the present invention, but not to limit its scope. Detailed descriptions of a preferred exemplary embodiment adequate to allow those of ordinary skill in the art to make and use the invention concepts will follow in later sections.
The invention provides a compound comprising the following structure, with the formula of C57H88O23 and the name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β,22α,28-hexahydroxyolean-12-ene, also known as Xanifolia-Y This compound was isolated from Xanthoceras sorbifolia.




This invention provides a compound comprising the following structure, with the formula of C65H100O27 and the name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene, also known as Xanifolia-Y1




The above compounds (Y and Y1) have anti-cancer effect. They inhibit the growth of human ovarian cancer (FIG. 3, 4).
These compounds (Y and Y1) are two of the active components identified from extracts of Xanthoceras sorbifolia by methods of FPLC and HPLC as shown in FIG. 5, 6, 7.
The compound Y is purified with procedure as described in this application (FIG. 7A). The purified compound Y shows 10 times higher potency (IC50=1.5 ug/ml) than the original extract (IC50=25 ug/ml) (FIG. 8). The compound Y has a high selectivity toward ovarian cancer (FIG. 9).
The compound Y shows inhibitory activity toward the following human cancer cells (eleven human cancer cell lines were tested in this study) with a higher potency toward ovarian carcinoma (comparison of activities is presented in FIG. 10 and Table 3.1).
This invention provides the extract of Xanthoceras Sorbifolia against cancer growth. The cancer includes, but is not limited to ovary cancer, bladder cancer, prostate cancer, leukocytes cancer, and bone cancer.
The compounds can be isolated from the plant called Xanthoceras Sorbifolia or can be synthesized chemically, or extracted from other biological sources.
This invention is related to the use of extracts of Wenguanguo. Extracts from husks, leaves, branches or stems, and fruit-stems, roots and barks of the Wenguanguo can be combined and this invention discloses methods of their preparation.
The extracts contain saponins, saccharides, proteins, glycosides, flavonoids, curmarin extracts, alkaloid extracts, organic acid extracts, tannin and others.
This invention provides the extract of Xanthoceras Sorbifolia for preventing cerebral aging; for improving memory; for improving cerebral functions; for curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence, Alzheimer's disease, brain trauma, or other diseases caused by cerebral dysfunctions.
Wenguanguo extracts may be used for accelerating the growth of bladder, for suppressing deep sleep, for increasing alertness in a sleeping subject, for modulating the release, breakdown and uptake of Antidieuretic hormone (ADH) and its receptors, for modulating the secretion, breakdown and uptake of Adrenocorticotropic hormone (ACTH) and its receptors, for modulating the release, breakdown and uptake of 5-hydroxytryptamine and its receptors, for modulating the release, breakdown and uptake of Acetycholine (Ach) and its receptors, for modulating the release, breakdown and uptake of Adrenaline (AD) and its receptors, for modulating the release, breakdown and uptake of Dopamine (DA) and its receptors, for modulating the release, breakdown and uptake of Norepinephrine (NE) and its receptors, for preventing sleep paralysis, for modulating the formation, release, breakdown and activity of neuropeptides and their receptors, for curing cancer, including but not limited to breast cancer, leukocyte cancer, liver cancer, ovary cancer, bladder cancer, prostate cancer and brain cancer, and for improving the functions of the lung and the bladder.
This invention provides a compound comprising a sugar, a triterpene or Sapogenin, and side chain at Carbon 21 and/or 22 including Angeloyl groups, operatively linked to form a biologically active compound. In an embodiment, the compound comprises one or more sugars.
This invention provides a salt of the above-described compounds.
This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier(s).
This invention provides a method for isolating compounds from Xanthoceras Sorbifolia comprising steps of: extracting Xanthoceras Sorbifolia powder with an appropriate amount of one or more organic solvents for an appropriate amount of time to form an organic extract; collecting the organic extract; refluxing the organic extract to form a second extract; removing the organic solvent from the second extract; drying and sterilizing the second extract to form a Xanthoceras Sorbifolia extract powder; fractionating the extract powder to obtain one or more components of the extract powder; identifying the bioactive components of the extract powder; purifying one or more bioactive components of the extract powder with FPLC to obtain one or more fraction of the bioactive component; and isolating the pure compound with preparative HPLC.
This invention provides a compound having a Structure verified by NMR spectral data derived from proton NMR, Carbon NMR, 2D NMR of the Heteronuclear Multiple Quantum Correlation (HMQC), Heteronuclear Multiple Bond Correlation (HMBC), and COSY, and Mass spectral data derived from WADLI-TOF and ESI-MS.
This invention provides the chemical features of a compound and its derivatives which are effective against cancer. The compounds or compositions of the present invention regulate the receptors or components of cell such as G-protein receptor, Fas protein, receptor Tyrosine Kinases, Mitogen, mitogen receptor. The compound inhibits cellular pathways include TGF Beta-smad, FGF, TGF-beta and TGF-alphaa, ras-GTPase-MAP kinase, jun-fos, Src-fyn, Jak-Jnk-STAT, BMP, Wnt, myc-cell proliferation, etc. The mutation of cancer cell causes the cell-death program to become inactive, allowing cells to divide indefinitely. The Xanthoceras Sorbifolia derived compound and/or composition regulates the components and receptors and re-activates the cell death program.
Abnormal changes in components' activities in pathways cause the cells to fail to stop proliferating so as to form cancer. The pathways include TGF Beta-smad, FGF, TGF-beta and TGF-alphaa, ras-GTPase-MAP kinase, jun-fos, Src-fyn, Jak-Jnk-STAT, BMP, Wnt, myc-cell proliferation, etc. The mutation of cancer cell causes the cell-death program to become inactive, allowing cells to divide indefinitely. The Xanthoceras Sorbifolia derived compound and/or composition regulates the components and receptors and re-activates the cell death program.
In accordance with these and other objects of the invention, a brief summary of the present invention is presented. Some simplifications and omission may be made in the following summary, which is intended to highlight and introduce some aspects of the present invention, but not to limit its scope. Detailed descriptions of a preferred exemplary embodiment adequate to allow those of ordinary skill in the art to make and use the inventive concepts will follow in later sections.



DETAILED DESCRIPTION OF THE FIGURES
FIG. 1 shows the structure Compound Y with the formula of C57H88O23 and the chemical name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene.
FIG. 2 shows the structure of Compound Y1 with the formula of C65H100O27 and the chemical name of 3-O-[β-D-galactopyranosyl(12)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene.
FIG. 3 shows the anticancer activity of purified Compound Y. The periment was performed on ovarian cancer cells (OCAR-3) and the inhibition activity was determined by MTT assay (for details, refer to Experiment 3). Abscissa: Concentration (ug/ml). Ordinate: % Cell Growth. The IC50 is approximately 1 ug/ml. A: Point scale. B: Linear scale.
FIG. 4 shows the inhibition of the purified Compound Y1 and Compound Y2 on Ovarian cancer cells' growth.
FIG. 5 shows the results of the screening of cell growth activity of fractions obtained after FPLC chromatography. The assay was conducted with bladder cells. The fractions obtained from FPLC as shown in FIG. 20 were used. As shown in this figure, that different components of Xanthoceras Sorbifolia extracts cause either growth or inhibition effects on cells. Only fraction 5962 (Fraction Y) causes cell inhibition. Fractions 610 and 1116 cause minor stimulation of cell growth. Abscissa: concentration (ug/ml). Ordinate: % Cell Growth (determined by MTT assay).
FIG. 6 shows the separation of the components of Xanthoceras Sorbifolia extract by HPLC with a μbondapak C18 column. Details of experiment was presented in Experiment 2.
FIG. 7 shows HPLC profile of Fraction Y with 45% Acetonitrile isocratic elution in a preparative C18 column (Delta Pak C18). Under these conditions, fractions Y, Y1 and Y2 are well separated from each other and they are collected individually.
FIG. 7A shows the purity of the collected Compound Y by HPLC using 45% acetonitrile isocratic elution in a preparative C18 column.
FIG. 8 shows the a growth curve of ovarian cancer cells after treatment with the crude extract of Xanthoceras Sorbifolia as determined by MTT assay. This is a preliminary study on the sensitivity of extract of Xanthoceras Sorbifolia on cancer cell. Cell lines from 11 different human organs were employed. With the crude extract, this figure shows the most sensitive cancer cells are Ovary cancer cells. Activities on other cancer cells were represented in FIGS. 10A-D.
FIG. 9 shows the comparison of potency of Compound Y between ovarian cancer cells and cervical cancer cells. Ovarian cancer cells are much more sensitive to Compound Y than the cervical cancer cells. The IC50 for Compound Y in ovary cells is about 1.5. This result confirms that the activity of compound Y is more selective toward ovary cancer.
FIGS. 10A-D show the growth curves of cancer cells derived from different human organs as determined by MTT assay. After treatment with the extract of Xanthoceras Sorbifolia, growth curves of different cell lines were presented and their sensitivities (base on IC50 values) were determined.

    
    
        10A: Sensitive: bladder and bone.
        10B: Semi-sensitive: leukocyte and liver.
        10C: Marginal sensitive: prostate, breast and brain.
        10D: Least sensitive: colon, cervix and lung.
    
    


FIG. 11 shows the spectrum of proton NMR of Compound Y.
FIG. 12 shows 2D NMR (HMQC) results of Compound Y. Also see Table 5.2 for the listed chemical shift data.
FIG. 13 shows 2D NMR (HMBC) results of Compound Y. Also see Table 5.3 for the listed chemical shift data.
FIG. 14 shows the Mass spectrum of compound Y with MALDI-TOF (high mass): Y+Matrix (CHCA)+Angiotensin 1 “two point calibration”.
FIG. 15 shows the Mass spectrum of compound Y with ESI-MS.
FIG. 16 shows the Proton NMR spectrum of Compound Y1.
FIG. 17 shows the 2D NMR (HMQC) results of Compound Y1. Also see the chemical shift data from Table 6.2.
FIG. 18 shows the 2D NMR (HMBC) results of Y1. Also see the chemical shift data from Table 6.3.
FIG. 19 shows COSY-NMR profile of Y1 with chemical shift data from Table 6.4.
FIG. 20 shows the elution profile of an extract of Xanthoceras Sorbifolia in FPLC with 10-80% gradient. Ordinate: Optical density (at 245 nm). Abscissa: Fractions (5 ml/fraction).
FIG. 21 shows the Proton-NMR spectra of compound R1.
FIG. 22 shows the 2D NMR (HMQC) spectra of compound R1.
FIG. 23 shows the 2D NMR (HMBC) spectra of compound R1.
FIG. 24 shows the 2D NMR (COSY) spectra of compound R1.
FIG. 25 shows the C13 NMR spectra of compound R1.
FIG. 26 shows the chemical structure and the chemical name of Compound R1.
FIG. 27 shows the chemical structure and the chemical name of Compound O54.
FIG. 28 shows the Proton-NMR spectra of compound O54.
FIG. 29 shows the 2D NMR (HMQC) spectra of compound O54.
FIG. 30 shows the 2D NMR (HMBC) spectra of compound O54.
FIG. 31 shows one of the four possible chemical structures of Y1. A: structure Y1-1.
FIG. 32 shows one of the four possible chemical structures of Y1. B: structure Y1-2.
FIG. 33 shows one of the four possible chemical structures of Y1. C: structure Y1-3.
FIG. 34 shows one of the four possible chemical structure of Y1, D: Structure Y1-4. Where R1=A or B or C; R2=A or B or C; R3=A or B or C
















R1
R2
R3




















1
A
A
A



2
A
A
B



3
A
A
C



4
A
B
A



5
A
B
B



6
A
B
C



7
A
C
A



8
A
C
B



9
A
C
C



10
B
A
A



11
B
A
B



12
B
A
C



13
B
B
A



14
B
B
B



15
B
B
C



16
B
C
A



17
B
C
B



18
B
C
C



19
C
A
A



20
C
A
B



21
C
A
C



22
C
B
A



23
C
B
B



24
C
B
C



25
C
C 
A



26
C
C
B



27
C
C
C









FIG. 35 shows the chemical structure of Y-a. R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C; Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.
FIG. 36 shows the chemical structure of Y-b. R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C; Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.
FIG. 37 shows the chemical structure of Y-c.
FIG. 38 shows the chemical structure of Y1-a. R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C; Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.
FIG. 39 shows the chemical structure of Y1-b. R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C; Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.
FIG. 40 shows the chemical structure of Y1-c. R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C; Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.
FIG. 41 shows the absorption spectrum of Xanthoceras Sorbifolia extract. Abscissa: Wavelength in nm. Ordinate: Optical Density. The extract has three absorption maximum at 207 nm, 278 nm and 500 nm.
FIG. 42 shows elution profile of Fraction 5962 with 64% acetonitrile isocratic elution. Two major FPLC fractions X and Y are separated. Ordinate: optical density (254 nm). Abscissa: fraction Number (1 ml/fraction).
FIG. 43 shows the comparison of inhibition activity in bladder cells by Fractions X (2021) and Y (2728). Only Fraction Y has inhibition activity.
FIG. 44 shows HPLC profile of Fraction Y with 45% Acetonitrile isocratic elution. Two major and 2-3 minor compounds were identified.
FIG. 45 shows purification of Fraction R from with FPLC.
FIG. 46 shows the HPLC analysis of fractions #9, #10 and #11 obtained from FPLC.
FIG. 47 shows purification of component-R with HPLC (Delta-Pak C18). A: Extract from fraction #10 of FPLC (iso-30) was further separated by HPLC. B: Rechromatogram of the major component under same condition as described in A.
FIG. 48. Fractionation of Fraction-O from FPLC with HPLC with 20% acetonitrile isocratic elution (iso-20).
FIG. 49. Rechromatography of O28 and O34 (from iso-20).
FIG. 50. Rechromatography of O54 (from iso-20).
FIG. 51 shows the proton NMR spectrum of Y2.
FIG. 52 shows the 2D NMR spectrum of Y2 (HMQC).
FIG. 53 shows the proton NMR spectrum of Y4.
FIG. 54 shows the 2D NMR (HMQC) spectrum of Y4.
FIG. 55 shows the proton NMR spectrum of O28.
FIG. 56 shows the 2D NMR (HMQC).
FIG. 57 shows the proton NMR spectrum of O34.
FIG. 58 shows the 2D NMR (HMQC) spectrum of O34.
FIG. 59 shows the effects of the extract X and Y on the quantity of urine in mice after 10 days of administration of X and Y.
FIGS. 60(a) and (b) show the water maze learning effect of plant extract administration of aging mice for 9 days.
FIGS. 61 (a) and (b) show the result of water maze learning of 3 days injected pentobarbital.
FIG. 62 shows the sleep cycle of a typical person.
FIG. 63. Table 15A-1 shows results of urine volume with water load after administration extract for 25 days. FIG. 63A shows the urine volume with water load after administration of FS(X) and FS(Y) extract for 25 days.
FIG. 64. Table 15A-2 shows results of discharging urine speed with water load after administration extract for 25 days. FIG. 64A shows the discharging urine speed with water load after administration of FS(X) and FS(Y) extract for 25 days.
FIG. 65. Table 15A-3 shows results of urine specific gravity and pH with water load after administration extract for 25 days.
FIG. 66. Table 15A-4 shows concentration of Na+, K+ and Cl− in urine with water load after administration extract for 25 days.
FIG. 67 shows the structure of the compound.
FIG. 68 shows the structure of the compound.
FIG. 69 shows the structure of the compound.
FIG. 70 shows the structure of the compound.
FIG. 71 shows the structure of the compound.



DETAILED DESCRIPTION OF THE INVENTION
The invention provides a compound comprising the following structure, with the formula of C57H88O23 and the name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabnofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene, also known as Xanifolia-Y This compound was isolated from Xanthoceras sorbifolia




This compound belongs to saponins consist of a triterpene, sugar moiety and angeloyl groups links to the backbone. The angeloyl groups linked to the C21 and C22 positions. This compound has the anti-cancer activity.
The assignment of this structure is supported by the spectral data (1D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS). Accordingly, this compound has the characteristic property as shown in FIGS. 11-15 or Table 5.1.
This invention provides another compound comprising the following structure, with the formula of C65H100O27 and the name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene, also known as Xanifolia-Y1




This compound belongs to saponins consist of a triterpene, sugar moiety connected to the backbone. A sugar that linked to the C21 position has two angeloyl groups attached. This compound has anti-cancer activity.
The assignment of this structure is supported by the spectral data (1D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS). Accordingly, this compound has the characteristic property as shown in FIGS. 16-19 or Table 6.1.
This invention provides evidence to show that the extract of Xanthoceras Sorbifolia contains anticancer activity. The experiments for determining the anti-cancer activity employed human cells lines derived from eleven human organs (HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary)). Among the 11 cell lines studies, their sensitivity toward Xanthoceras Sorbifolia extract can be divided into four groups: (A) most sensitive: Ovary (FIG. 8); (B) Sensitive: bladder, bone, prostate, and leukocyte, (C) marginal sensitive: liver, breast, and brain; and (D) lease sensitive: colon, cervix, and lung. (FIG. 10A-D). Their IC50 values are listed in Table 3.1.







TABLE 3.1







IC50 values of Xanthoceras Sorbifolia Extract Determined in Different


Cancer Cells









IC50 determined by MTT assay


Cancer cells from different organs
(ug/ml)





Ovary (most sensitive)
15-15


Bladder (sensitive)
45-50


Bone
40-55


Prostate
40-50


Leukocyte
45-50


Liver (marginal sensitive)
45-65


Breast
 65


Brain
70-85


Colon (least sensitive)
 90


Cervix
115


Lung
110









In order to identify the active compounds of Xanthoceras Sorbifolia, the extract from Xanthoceras Sorbifolia were separated by chromatography comprising FPLC (Fast Protein Liquid Chromatography) and HPLC (High Preferment Liquid Chromatography). Multiple fractions were obtained by FPLC procedures (FIG. 20) and HPLC (FIG. 6). Analysis of the components of Xanthoceras Sorbifolia by HPLC shows that the extract comprises 26 identifiable fractions (named a to z) as shown in FIG. 6.
Anti-cancer activities of these fractions were determined by the MTT assay. Only fraction Ys has the anti-cancer activity (FIG. 5). Fraction Ys were further separated into 4 components (FIG. 7). The compounds Y and Y1 are the active components currently isolated from Xanthoceras Sorbifolia as shown in FIG. 3-4.
The invention tested the inhibition effects of ovarian cancer cells with the MTT assay, and the compound Y shows 10 times higher potency (IC50=1.5 ug/ml) (FIG. 3) than the original crude extract as shown in FIG. 8 (IC50=25 ug/ml).
The selectivity of compound Y was tested, and it has been found that compound Y has a much higher potency toward ovarian cancer cells as compared to the cervical cancer cells (FIG. 9).
The compounds Y1 and Y2 have anti-cancer activity as shown in FIG. 4.
This invention provides the detail isolation procedures for the active compounds of the present invention.
This invention provides the spectral data evidence (1D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, ESI-MS) in supporting the assigned structures.
This invention provides a salt of the above-described compounds.
This invention provides a composition comprising the above-described compounds and a suitable carrier.
This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier.
This invention provides an anti-ovarian cancer agents and composition comprising the above-described composition.
This invention provides the compositions against cancer growth. The cancer includes, but is not limited to bladder cancer, bone cancer, and ovary cancer.
This invention provides a composition comprising the above compounds and their derivatives for inhibition of tumour growth.
The following methods and materials were used in the examples and/or experiments described in this application.
Cells. Human cancer cell lines were obtained from American Type Culture
Collection: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary). Cells were grown in culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI-1640; HCT-116, U2OS in McCoy-5A) supplemented with 10% fetal calf serum, glutamine and antibiotics in a 5% CO2 humidified incubator at 37° C.
MTT Assay. The procedure for MTT assay followed the method described in (Carmichael et al., 1987) with only minor modifications. Cells were seeded into a 96-wells plate at concentrations of 10,000/well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2, U2OS), or 40,000/well (K562), for 24 hours before drug-treatment. Cells were then exposed to drugs for 48 hours (72 hours for HepG2, U2OS, and 96 hours for MCF-7). After the drug-treatment, MTT (0.5 mg/ml) was added to cultures for an hour. The formation of formazan (product of the reduction of tetrazolium by viable cells) was dissolved with DMSO and the O.D. at 490 nm was measured by an ELISA reader. The MTT level of cells before drug-treatment was also measured (T0). The % cell-growth (% G) is calculated as:

% G=(TD−T0/TC−T0)×100  (1),

where TC or TD represent O.D. readings of control or drug-treated cells. When T0>TD, then the cytotoxicity (LC) expressed as % of the control is calculated as:

% LC=(TD−T0/T0)×100  (2).

In addition to the compounds Y and Y1, other compounds from the extract including R1 and O54, were also purified and the structure were determined by 1D H-NMR, C13-NMR, 2D NMR (HMQC, HMBC, COSY); MS (MALDI-TOF).
The Structure of Compound R1 shown below and in FIG. 26, has a chemical formula of C65H106O29 and chemical name of
3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene, also known as Xanifolia-R1.




The assignment of this structure is supported by the spectral data (1D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS). Accordingly, this compound has the characteristic property as shown in FIGS. 21-25 or Table 8.1
Compound-O54
This invention provides a compound O54 with formula of C60H100O28 and the structure was determined by 1D NMR, 2D NMR, MS).
The Structure of Compound O54 (also shown in FIG. 27):




The name of Compound O54 is The chemical name of compound O54 is:
3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene
The assignment of this structure is supported by the spectral data (1D H-NMR, 2D NMR (HMBC, HMQC). Accordingly, this compound has the characteristic property as shown in FIGS. 28-30 and table 9.1.
In other embodiments, the structures of the compounds are as follows:
Structure 1 as shown in FIG. 31
Structure 2 as shown in FIG. 32
Structure 3 as shown in FIG. 33
Structure 4 as shown in FIG. 34
Structure Y-a as shown in FIG. 35
Structure Y-b as shown in FIG. 36
Structure Y-c as shown in FIG. 37
Structure Y1-a as shown in FIG. 38
Structure Y1-b as shown in FIG. 39
Structure Y1-c as shown in FIG. 40
This invention provides a compound comprising a sugar, a triterpene or Sapogenin, and a side chain at Carbon 21 and 22 or Angeloyl groups. In an embodiment, the compound comprises two or more sugars.
This invention provides a salt of the above-described compounds.
This invention provides a composition comprising the above-described compounds and a suitable carrier.
This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier.
This invention provides an anti-ovarian cancer agents and composition comprising the above-described composition.
This invention provides the compositions against cancer growth. The cancer includes, but is not limited to bladder cancer, bone cancer, and ovary cancer.
This invention provides composition comprising the above compounds and their derivatives to inhibit tumour growth.
This invention provides composition comprising the above compounds and their derivatives to cure human immunodeficiency virus (HIV) or Severe Acute Respiratory Syndrome (SARS) or flux disease or inhibit virus activities.
This invention provides medicine or health food for improving the sensory stretch receptor in the bladder wall, inhibiting AChE or use as an anti-inflammatory agent.
This invention provides a method for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation.
This invention provide methods for inhibiting tumor cell growth or to treat patients with HIV or SARS, or inhibit virus activities, or for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation. comprising contacting an amount of the compound is a triterpene or sapongenin with any two of angeloyl group or tigloyl group or senecioyl group or their combinations attach to carbon 21 and 22, or any two of angeloyl group or tigloyl group or senecioyl group or their combinations attached to a sugar moiety which bonds to carbon 21 or 22.
Wenguanguo is a species of the sapindaceae family. Its scientific name is Xanthoceras sorbifolia Bunge. Wenguanguo is the common Chinese name; others are Wenguannguo, Wenguanmu, Wenguanhua, and Xilacedeng. This plant can grow up to 8 meters in height. It features odd pinnately compound leaf, eraceme with white flowers, capsules with thick and woody husks. Wenguanguo is grown in Liaoning, Jilin, Hebei, Shandong, Jiangsu, Henan, Shanxi, Shaanxi, Gansu, Ningxia and Inner Mongolia, China. Its seeds are edible and have been used as a folk medicine to treat enuresis for centuries. Its branches and woods are also used as a folk medicine.
This invention is a further description of the extracts from Wenguanguo, their uses and methods for preparation. This invention provides the extracts that can prevent enuresis by improving patients' cerebral functions so that patients can be more aware of the signals sent from the bladder and wake up from deep sleep. When the bladder is full of urine, the smooth muscle of the bladder is extended, which produces a signal up to the cerebral cortex and cerebellum through the pelvic nerve and the sacral spinal cord. The response of the cerebral cortex and cerebellum to the signal is to make the bladder sustain contracted but the sphincter relaxed. The urine is then discharged. When the bladder is filled with urine via the urethra during sleep, the detrusor stretches, allowing the bladder to expand. As the bladder starts to accumulate urine, it will stimulate the stretch receptors in the bladder that will generate signals continually to the brain according to the amount of urine accumulated in the bladder. When the bladder is full enough with urine, then the intra-vesicle has accumulated enough pressure for the brain to recognize and wake the person to urinate. If the signal is not strong enough to wake the sleeping person or blocked due to impairment of cerebral function, then enuresis occurs. This particular plant extract can cure enuresis by improving cerebral functions.
The sensory stretch receptors are located within the bladder wall and help with assessing the degree of bladder fullness. This information is transmitted up to the spinal cord and then via the spinothalamic tracts to the central nervous system. The extracts of Wenguanguo make the central nervous system more aware of the signal.
When the bladder becomes contracted under stress and nervousness, the capacity of the urinary bladder will be reduced and then the frequent micturition occurs. The extracts of Wenguanguo can relax the bladder for storing more urine.
The capacity of the urinary bladder is reduced because of aging, and this may even happen to middle-aged people. They suffer from experience of early detrusor contraction due to a sense of urgency to empty the bladder at low urine level. The extracts of Wenguanguo can help relax the detrusor and therefore the bladder capacity increases and urinary frequency decreases.
Patients with detrusor overactivity, detrusor instability, detrusor hyper-reflexia or uninhibited bladder have early, forceful detrusor contractions before the bladder is full. This creates urgency and frequency urinary discharge. The extract of Wenguanguo relaxes the patient's detrusor. The bladder becomes stable and can store a full amount of urine.
The smooth muscle of the urinary bladder has two functions: When the bladder is relaxed, the urine is stored. When it is contracted, the urine will be discharged. The sensory stretch receptors are located within the bladder wall to assess the bladder's fullness. This information is transmitted up the spinal cord via the spinothalamic tracts to the nervous system. The brain generates inhibitory signals when detrusor relaxation is desired. But the brain generates excitatory signal when detrusor contraction is desired. The extracts of Wenguanguo can relax the bladder tissue by inhibiting Acetylcholinesterase, AchE. The inhibiting effect can be maintained for a long period of time. The extracts of Wenguanguo are a good AChE inhibitor that can cure the diseases caused by deficiency of Acetylcholine, ACh.
Antidiuretic hormone (ADH) is stored in the posterior pituitary gland in the brain. It is the primary regulator of body water. ADH acts on the kidneys to increase or decrease total body water. This has an effect on the volume of urine generated by the kidney. The release of ADH is controlled by the cells of osmoreceptors and baroreceptors. Osmoreceptors are the specialized cell hypothalamus. These cells sense the concentration of particles in the blood. When the concentration of particles is higher, more ADH will be released by the pituitary. This stimulates retention of water to dilute body fluids. When the concentration is lower, less ADH will be released by the pituitary. Baroreceptors are located in the right atria and great veins and carotid sinus the specialized area in the heart that sense blood volume and blood pressure. The heart will generate signals to the hypothalamus and pituitary to release more ADH when blood volume or blood pressure is low and vice versa. The extracts of Wenguanguo can regulate the release of ADH which will reduce the volume of urine produced by the body.
This invention relates to the flavone extracts from Wenguanguo husks and fruit-stems, and methods of their preparation. The methods for preparing the extracts from Wenguanguo husks and fruit-stems comprise the following steps: extracting Wenguanguo powder made from husk and fruit-stem with ethanol 3-4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extracts; drying the aqueous extracts to form the flavone extracts that is yellow powder.
This invention provides a composition comprising extracts from husks and fruit-stems which are flavonols, flavanols, dihydroflavonols, phenoloids, and others.
This invention relates to the crude flavone extracts from Wenguanguo leaves that includes a water-soluble flavone extracts and a water-insoluble flavone extract and methods of their preparation. The methods for preparing the extracts from Wenguanguo leaves comprise the following steps: extracting Wenguanguo powder made from the leaves with ethanol for 3 times to form an ethanol extract; concentrating the ethanol extract to form a concentrated condensed extracts; extracting the concentrated extract with hot water to from an aqueous extracts and a water-insoluble extract; drying the aqueous extracts and the water-insoluble extract to form a water-soluble flavone extracts and a water-insoluble flavone extract. This invention provides a composition comprising the crude extracts from leaf which are flavonols, flavanols, dihydroflavonols, phenoloids and others.
This invention relates to the flavone extracts from Wenguanguo branches or stems and methods of their preparation. The methods for preparing the extract from branches or stem comprise the following steps: extracting Wenguanguo powder made from the branches or stems with ethanol for 4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extracts; drying the aqueous extracts to form flavone extracts which is a yellowish powder.
This invention provides a composition comprising extracts from Wenguanguo branches and stems which are flavonols, flavanols, dihydroflavonols, phenoloids and others.
This invention relates to the flavone extracts from Wenguanguo kernels and methods of their preparation. The methods for preparing the extract from kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with ethanol to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extract; drying the aqueous extracts to form a flavone extracts that is a yellow powder.
This invention provides a composition comprising extracts from kernel which are flavonols, flavanols, dihydroflavonols, proteins, phenoloids, and others.
This invention relates to the flavone extract from Wenguanguo root, and methods of their preparation. The methods for preparing the flavone extract from Wenguanguo root comprise the following steps: extracting Wenguanguo powder made from root with ethanol 3-4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extract; drying the aqueous extracts to form the flavone extracts which is a yellow powder.
This invention provides a composition comprising extracts from roots of Wenguanguo which are flavonols, flavanols, dihydroflavonols, phenoloids and others.
This invention relates to the flavone extracts from Wenguanguo barks, and methods of their preparation. The methods for preparing the bark extracts from Wenguanguo barks comprise the following steps: extracting Wenguanguo powder made from the barks with ethanol 3-4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extract; drying the aqueous extracts to form the flavone extracts which is a yellowish powder.
This invention provides an extract composition from Wenguanguo barks comprising flavonols, flavanols, dihydroflavonols, phenoloids and others.
This invention is related to the combined extracts from Wenguanguo husks or fruit-stems and method of their preparation. The methods for preparing the extract from the husks or fruit-stems comprise the following steps: extracting Wenguanguo powder made from the husks or fruit-stems with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the organic extract to from an aqueous extracts; drying and sterilizing the aqueous extracts to form the combined extracts.
This invention provides a composition comprising the combined extracts from the husks or fruit-stems of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.
This invention is related to the combined extracts from Wenguanguo leaves and method of their preparation. The methods for preparing the extracts from the leaves comprise the following steps: extracting Wenguanguo powder made from leaves with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the second extract to an aqueous extract; extracting the aqueous extract with ether and water to form an second aqueous extract; extracting the second aqueous extract with n-butanol to form a n-butanol extract; removing the n-butanol from the n-butanol extract to form a third aqueous extract; drying and sterilizing the third aqueous extract to form the combined extracts.
This invention provides a composition comprising the organic extracts from the leaves of the Wenguanguo. The organic extracts comprise saponins, saccharides, proteins and others.
This invention is related to the combined extracts from Wenguanguo branches or stems and method of their preparation. The methods for preparing the extracts from the branches or stems comprise the following steps: extracting Wenguanguo powder made from the branches or stems with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the second extract to an aqueous extract; drying and sterilizing the aqueous extracts to form the combined extracts.
This invention provides a composition comprising the organic extracts from the branches, and stems and of the Wenguanguo. The organic extracts comprise saponins, saccharides, proteins and others.
This invention is related to the combined extracts from Wenguanguo kernels and method of their preparation. The methods for preparing the extracts from Wenguanguo kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the second extract to an aqueous extract; drying and sterilizing the aqueous extracts to form the combined extracts.
This invention provides a composition comprising the organic extracts from the kernels of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.
This invention is related to the combined extracts from Wenguanguo roots and method of their preparation. The methods for preparing the extracts from Wenguanguo roots comprise the following steps: extracting Wenguanguo powder made from the roots with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the organic extract to from an aqueous extracts; drying and sterilizing the aqueous extracts to form the combined extracts.
This invention provides a composition comprising the combined extracts from the roots of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.
This invention is related to the combined extracts from Wenguanguo barks and method of their preparation. The methods for preparing the extracts from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder made from the barks with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the organic extract to from an aqueous extract; drying and sterilizing the aqueous extracts to form the combined extracts.
This invention provides a composition comprising the combined extracts from the barks of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.
This invention provides the crude saponins from the husks or fruit-stems or seed's shell of Wenguanguo. The methods for preparing the crude saponins from Wenguanguo husks or fruit-stems comprise the following steps: extracting Wenguanguo powder of the husks or fruit-stems with an organic solvent (ethanol, methanol and others) at ratio of 1:2 for 4-5 times, 20-35 hours for each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.
This invention provides the crude saponins from the leaves of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the leaves comprise the following steps: extracting Wenguanguo powder of the leaves with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form a second extract; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.
This invention provides the crude saponins from the branches and stems of Wenguanguo. The methods for preparing the crude saponins from the branches or stems comprise the following steps: extracting Wenguanguo powder of the branches or stems with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.
This invention provides the crude saponins from the kernels of Wenguanguo. The methods for preparing the crude saponins from Wenguanguo kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract for 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; Extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.
This invention provides the crude saponins from the roots of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from Wenguanguo roots comprise the following steps: extracting Wenguanguo powder of the roots with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts contain saponins.
This invention provides the crude saponins from the barks of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the barks comprise the following steps: extracting Wenguanguo powder of the barks with an organic solvent (ethanol, methanol and others) at a ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.
This invention provides a process of producing a coumarin extract from the husks or fruit-stems of Wenguanguo and their applications. The methods for preparing the coumarin extracts from husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks or fruit-stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.
This invention provides a composition comprising the coumarin extracts from the husks or fruit-stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.
This invention provides a process of producing a coumarin extract from the leaves of Wenguanguo and their applications. The methods for preparing the coumarin extracts from leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.
This invention provides a composition comprising the coumarin extracts from the leaves of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.
This invention provides a process of producing a coumarin extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the coumarin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder branches or stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the extract comprising crude coumarins.
This invention provides a composition comprising the coumarin extracts from the branches and stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides, saccharides, proteins and others.
This invention provides the crude saponins from the leaves of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the leaves comprise the following steps: extracting Wenguanguo powder of the leaves with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form a second extract; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.
This invention provides the crude saponins from the branches and stems of Wenguanguo. The methods for preparing the crude saponins from the branches or stems comprise the following steps: extracting Wenguanguo powder of the branches or stems with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.
This invention provides the crude saponins from the kernels of Wenguanguo. The methods for preparing the crude saponins from Wenguanguo kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract for 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; Extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.
This invention provides the crude saponins from the roots of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from Wenguanguo roots comprise the following steps: extracting Wenguanguo powder of the roots with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts contain saponins.
This invention provides the crude saponins from the barks of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the barks comprise the following steps: extracting Wenguanguo powder of the barks with an organic solvent (ethanol, methanol and others) at a ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.
This invention provides a process of producing a coumarin extract from the husks or fruit-stems of Wenguanguo and their applications. The methods for preparing the coumarin extracts from husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks or fruit-stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.
This invention provides a composition comprising the coumarin extracts from the husks or fruit-stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.
This invention provides a process of producing a coumarin extract from the leaves of Wenguanguo and their applications. The methods for preparing the coumarin extracts from leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.
This invention provides a composition comprising the coumarin extracts from the leaves of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.
This invention provides a process of producing a coumarin extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the coumarin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder branches or stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the extract comprising crude coumarins.
This invention provides a composition comprising the coumarin extracts from the branches and stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides, saccharides, proteins and others.
This invention provides a process of producing a coumarin extract from the kernels of Wenguanguo and their applications. The methods for preparing the coumarin extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form an ether extract; neutralizing the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.
This invention provides a composition comprising the coumarin extracts from the kernels of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.
This invention provides a process of producing a coumarin extract from the roots of Wenguanguo and their applications. The methods for preparing the coumarin extract from roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the root with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.
This invention provides a composition comprising the coumarin extracts from the roots of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.
This invention provides a process of producing a coumarin extract from the barks of Wenguanguo and their applications. The methods for preparing the coumarin extract from barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the bark with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.
This invention provides a composition comprising the coumarin extract from the barks of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.
This invention provides a process of producing an aqueous extract from the husks or fruit-stems of Wenguanguo and their applications. The method for preparing the water extracts from the husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husk or fruit-stem with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extract from the husks or fruit-stems of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an aqueous extract from the leaves of Wenguanguo and their applications. The method for preparing the water extracts from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extract from leaves of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an aqueous extract from the branches or stems of Wenguanguo and their applications. The method for preparing the water extracts from branches or stems of Wenguanguo comprise the following steps: extracting the Wenguanguo powder of the branches or stems with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extract from the branches or stems of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an aqueous extract from the kernels of Wenguanguo and their applications. The method for preparing the water extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extract from kernels of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an aqueous extract from the roots of Wenguanguo and their applications. The method for preparing the water extracts from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the roots with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extract from the roots of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an aqueous extract from the barks of Wenguanguo and their applications. The method for preparing the water extracts from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extracts from the barks of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an alkaloid extract from the husks of Wenguanguo and their applications. The methods for preparing the alkaloid extracts from the husks and fruit-stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the husks or fruit-stems with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.
This invention provides a composition comprising the alkaloid extract from the husks or fruit-stems of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing an alkaloid extract from the leaves of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.
This invention provides a composition comprising the alkaloid extract from the leaves of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing an alkaloid extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the extracts containing alkaloids from branches or stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the branches or stems with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.
This invention provides a composition comprising the extract containing crude alkaloids from the branches or stems of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing an alkaloid extract from the kernels of Wenguanguo and their applications. The methods for preparing the alkaloid extract from kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at ratio of 1:6 for 3-4 times, 10-15 hours for each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.
This invention provides a composition comprising the alkaloid extract from the kernels of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing an alkaloid extract from the roots of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the Wenguanguo roots with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.
This invention provides a composition comprising the alkaloid extract from the roots of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing an alkaloid extract from the barks of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.
This invention provides a composition comprising the alkaloid extract from the barks of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing extract containing organic acids from husks and fruit-stems and their applications. The methods for preparing the extracts containing organic acids from the husks or fruit-stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the husks and or fruit-stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising crude organic acids from the husks of Wenguanguo. The extract comprising aromatic organic acids, fatty organic acids, terpenoid organic acids and others
This invention provides a process of producing extract contains organic acids from leaf and their applications. The methods for preparing the extracts containing organic acids from the leaves of Wenguanguo comprise the following steps: extract Wenguanguo powder of the leaves with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising the extract comprising crude organic acids extract from the leaves of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.
This invention provides a process of producing extract contains organic acids from branches and stems and their applications. The methods for preparing the extracts comprising organic acids from the branches or stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the branches or stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising the crude organic acids extract from the branches and stems of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.
This invention provides a process of producing extract comprise organic acids from kernels and their applications. The methods for preparing the extracts comprising organic acids from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising crude organic acids extract from the kernels of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.
This invention provides a process of producing extract contains organic acids from the roots of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the roots of Wenguanguo comprise the following steps: extract Wenguanguo powder of the roots with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising the extract comprising crude organic acids from the roots of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.
This invention provides a process of producing extract comprising organic acids from barks of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the barks of Wenguanguo comprise the following steps: extract Wenguanguo powder of the bark with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising the extract comprising crude organic acids from the barks of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.
This invention provides two methods of producing a tannin extract from Wenguanguo husks and fruit-stems and its usage. The first method for preparing the tannin extract from the husks or fruit-stems of Wenguanguo comprises the following steps: extracting Wenguanguo powder of husks and or fruit-stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract. The second method for preparing the tannin extracts from the husks and or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks and or fruit-stems with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.
This invention provides a composition comprising the tannin extracts from the husks or fruit-stems of Wenguanguo. The extracts are comprised of tannins and others.
This invention provides two methods of producing a tannin extract from Wenguanguo leaves and its usage. The first method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.
The second method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.
This invention provides a composition comprising the tannin extract from the leaves of Wenguanguo. The extract comprises tannins and others.
This invention provides two methods of producing tannin extract from Wenguanguo branches and stems and its usage. The first method for preparing the extracts comprising tannins from branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of branches or stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.
The second method for preparing the tannin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the branches or stems and with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins. This invention provides a composition comprising the tannin extract from the branch or stem of Wenguanguo. The extract comprises tannins and others.
This invention provides two methods of producing tannin extract from Wenguanguo kernels and its usage. The first method for preparing the tannin extract from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the extract comprising tannins.
The second method for preparing the extracts containing tannins from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.
This invention provides a composition comprising the tannin extract from kernels of Wenguanguo. The extract comprises tannins and others.
This invention provides two methods of producing tannin extract from Wenguanguo roots and its usage. The first method for preparing the tannin extract from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of roots with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The method-2 for preparing the tannin extract from the root of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the root with a solvent of acetone-water at a ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.
This invention provides a composition comprising the tannin extracts from the roots of Wenguanguo. The extracts comprise tannins and others.
This invention provides two methods of producing tannin extract from Wenguanguo barks and its usage. The method-1 for preparing the tannin extract from the barks of Wenguanguo comprises the following steps: extracting Wenguanguo powder of barks with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The second method for preparing the tannin extract from the barks of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the barks with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.
This invention provides a composition comprising the tannin extracts from the barks of Wenguanguo. The extracts comprise tannins and others.
This invention provides a method for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation.
According to the theory of traditional Chinese medicine, enuresis, frequent micturition and urinary incontinence are caused by “deficiency in kidney (“shen”)”. Therefore, they are treated by using Chinese herbs which can tone the kidney such as Ginseng Bajitian, Roucongrong Duzhong and Cordyceps. These tonifying herbs can strengthen function of the kidney and regulate water metabolism of human's body through the “kidney pathway” that will help with curing the enuresis, frequent micturition and urinary incontinence.
The Wenguanguo extracts of the present invention can also be used to treat the enuresis, frequent micturition and urinary incontinence. However, the Wenguanguo extracts cure the enuresis, frequent micturition and urinary incontinence through the “bladderpathway” to regulate water metabolism of human's body and urination. The Wenguanguo extracts of the present invention stimulate the growth of the bladder. See FIG. 10A. The Wenguanguo extracts of the present invention increase the capacity of bladder and function of bladder controlling the urination. See Experiment 15 and Experiment 15A. In another aspect of the present invention, Wenguanguo extracts, when used with the “kidney pathway” herbs to treat the enuresis, frequent micturition and urinary incontinence, will strengthen both the pathways of kidney and bladder, and then will produce better treatment results.
This invention provides the medicines or health foods which further comprise Vitamin B, Vitamin D, Vitamin K, grape seed extract and other antioxidants, Cordyceps or its extract, gingko or its extract, Panax ginseng and P. quinquefolium or their extracts, Huangpi (Clausena lansium) or its extracts, Echinacea or its extract, St John's Wort (Hypericum perforatum) or its extract, Gegen (Pueraria lobata) or its extract, Tianma (Gastrodia elata) or its extract, Armillariella mellea or its extract, Danshen (Salvia miltiorrhiza) or its extract, Sanqi (Panax notoginsen) or its extract, Monascus or Honqu (Red yeast rice), Huanqi (Hedysarum polybotrys) or its extract, D ihuang (Rehmannia glutinosa) or its extract, Danggui (Angelica sinensis), Yuanzhi (Polygala tenuifoila) or its extract, Lingzhi (Ganoderma spp.) or its extracts, Fuling (Poria cocos) or its extract, enokitake (Flammulina velutipes) or its extract, Gan Cao (Glycyrrhiza uralensis Fisch) or its extract, Huperzine A, Lacithin, Metrifonate, Nocetile, folic acid, amino acids, creatine, fiber supplement, or any combination thereof.
This invention provides a process of producing a coumarin extract from the kernels of Wenguanguo and their applications. The methods for preparing the coumarin extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form an ether extract; neutralizing the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.
This invention provides a composition comprising the coumarin extracts from the kernels of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.
This invention provides a process of producing a coumarin extract from the roots of Wenguanguo and their applications. The methods for preparing the coumarin extract from roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the root with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.
This invention provides a composition comprising the coumarin extracts from the roots of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.
This invention provides a process of producing a coumarin extract from the barks of Wenguanguo and their applications. The methods for preparing the coumarin extract from barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the bark with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.
This invention provides a composition comprising the coumarin extract from the barks of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.
This invention provides a process of producing an aqueous extract from the husks or fruit-stems of Wenguanguo and their applications. The method for preparing the water extracts from the husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husk or fruit-stem with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extract from the husks or fruit-stems of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an aqueous extract from the leaves of Wenguanguo and their applications. The method for preparing the water extracts from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extract from leaves of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an aqueous extract from the branches or stems of Wenguanguo and their applications. The method for preparing the water extracts from branches or stems of Wenguanguo comprise the following steps: extracting the Wenguanguo powder of the branches or stems with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extract from the branches or stems of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an aqueous extract from the kernels of Wenguanguo and their applications. The method for preparing the water extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extract from kernels of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an aqueous extract from the roots of Wenguanguo and their applications. The method for preparing the water extracts from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the roots with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extract from the roots of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an aqueous extract from the barks of Wenguanguo and their applications. The method for preparing the water extracts from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.
This invention provides a composition comprising the aqueous extracts from the barks of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.
This invention provides a process of producing an alkaloid extract from the husks of Wenguanguo and their applications. The methods for preparing the alkaloid extracts from the husks and fruit-stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the husks or fruit-stems with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.
This invention provides a composition comprising the alkaloid extract from the husks or fruit-stems of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing an alkaloid extract from the leaves of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.
This invention provides a composition comprising the alkaloid extract from the leaves of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing an alkaloid extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the extracts containing alkaloids from branches or stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the branches or stems with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.
This invention provides a composition comprising the extract containing crude alkaloids from the branches or stems of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing an alkaloid extract from the kernels of Wenguanguo and their applications. The methods for preparing the alkaloid extract from kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at ratio of 1:6 for 3-4 times, 10-15 hours for each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.
This invention provides a composition comprising the alkaloid extract from the kernels of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing an alkaloid extract from the roots of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the Wenguanguo roots with water at a ratio of 1:6, 3-4 times, 10-hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.
This invention provides a composition comprising the alkaloid extract from the roots of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing an alkaloid extract from the barks of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.
This invention provides a composition comprising the alkaloid extract from the barks of Wenguanguo. The extract comprises alkaloids and others.
This invention provides a process of producing extract containing organic acids from husks and fruit-stems and their applications. The methods for preparing the extracts containing organic acids from the husks or fruit-stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the husks and or fruit-stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising crude organic acids from the husks of Wenguanguo. The extract comprising aromatic organic acids, fatty organic acids, terpenoid organic acids and others
This invention provides a process of producing extract contains organic acids from leaf and their applications. The methods for preparing the extracts containing organic acids from the leaves of Wenguanguo comprise the following steps: extract Wenguanguo powder of the leaves with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising the extract comprising crude organic acids extract from the leaves of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.
This invention provides a process of producing extract contains organic acids from branches and stems and their applications. The methods for preparing the extracts comprising organic acids from the branches or stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the branches or stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising the crude organic acids extract from the branches and stems of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.
This invention provides a process of producing extract comprise organic acids from kernels and their applications. The methods for preparing the extracts comprising organic acids from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising crude organic acids extract from the kernels of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.
This invention provides a process of producing extract contains organic acids from the roots of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the roots of Wenguanguo comprise the following steps: extract Wenguanguo powder of the roots with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising the extract comprising crude organic acids from the roots of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.
This invention provides a process of producing extract comprising organic acids from barks of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the barks of Wenguanguo comprise the following steps: extract Wenguanguo powder of the bark with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.
This invention provides a composition comprising the extract comprising crude organic acids from the barks of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.
This invention provides two methods of producing a tannin extract from Wenguanguo husks and fruit-stems and its usage. The first method for preparing the tannin extract from the husks or fruit-stems of Wenguanguo comprises the following steps: extracting Wenguanguo powder of husks and or fruit-stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract. The second method for preparing the tannin extracts from the husks and or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks and or fruit-stems with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.
This invention provides a composition comprising the tannin extracts from the husks or fruit-stems of Wenguanguo. The extracts are comprised of tannins and others.
This invention provides two methods of producing a tannin extract from Wenguanguo leaves and its usage. The first method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.
The second method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.
This invention provides a composition comprising the tannin extract from the leaves of Wenguanguo. The extract comprises tannins and others.
This invention provides two methods of producing tannin extract from Wenguanguo branches and stems and its usage. The first method for preparing the extracts comprising tannins from branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of branches or stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.
The second method for preparing the tannin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the branches or stems and with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins. This invention provides a composition comprising the tannin extract from the branch or stem of Wenguanguo. The extract comprises tannins and others.
This invention provides two methods of producing tannin extract from Wenguanguo kernels and its usage. The first method for preparing the tannin extract from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the extract comprising tannins.
The second method for preparing the extracts containing tannins from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.
This invention provides a composition comprising the tannin extract from kernels of Wenguanguo. The extract comprises tannins and others.
This invention provides two methods of producing tannin extract from Wenguanguo roots and its usage. The first method for preparing the tannin extract from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of roots with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The method-2 for preparing the tannin extract from the root of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the root with a solvent of acetone-water at a ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.
This invention provides a composition comprising the tannin extracts from the roots of Wenguanguo. The extracts comprise tannins and others.
This invention provides two methods of producing tannin extract from Wenguanguo barks and its usage. The method-1 for preparing the tannin extract from the barks of Wenguanguo comprises the following steps: extracting Wenguanguo powder of barks with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The second method for preparing the tannin extract from the barks of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the barks with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.
This invention provides a composition comprising the tannin extracts from the barks of Wenguanguo. The extracts comprise tannins and others.
This invention provides a method for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation.
According to the theory of traditional Chinese medicine, enuresis, frequent micturition and urinary incontinence are caused by “deficiency in kidney (“shen”)”. Therefore, they are treated by using Chinese herbs which can tone the kidney such as Ginseng Bajitian, Roucongrong Duzhong and Cordyceps. These tonifying herbs can strengthen function of the kidney and regulate water metabolism of human's body through the “kidney pathway” that will help with curing the enuresis, frequent micturition and urinary incontinence.
The Wenguanguo extracts of the present invention can also be used to treat the enuresis, frequent micturition and urinary incontinence. However, the Wenguanguo extracts cure the enuresis, frequent micturition and urinary incontinence through the “bladder pathway” to regulate water metabolism of human's body and urination. The Wenguanguo extracts of the present invention stimulate the growth of the bladder. See FIG. 10A. The Wenguanguo extracts of the present invention increase the capacity of bladder and function of bladder controlling the urination. See Experiment 15 and Experiment 15A. In another aspect of the present invention, Wenguanguo extracts, when used with the “kidney pathway” herbs to treat the enuresis, frequent micturition and urinary incontinence, will strengthen both the pathways of kidney and bladder, and then will produce better treatment results.
This invention provides the medicines or health foods which further comprise Vitamin B, Vitamin D, Vitamin K, grape seed extract and other antioxidants, Cordyceps or its extract, gingko or its extract, Panax ginseng and P. quinquefolium or their extracts, Huangpi (Clausena lansium) or its extracts, Echinacea or its extract, St John's Wort (Hypericum perforatum) or its extract, Gegen (Pueraria lobata) or its extract, Tianma (Gastrodia elata) or its extract, Armillariella mellea or its extract, Danshen (Salvia miltiorrhiza) or its extract, Sanqi (Panax notoginsen) or its extract, Monascus or Honqu (Red yeast rice), Huanqi (Hedysarum polybotrys) or its extract, D ihuang (Rehmannia glutinosa) or its extract, Danggui (Angelica sinensis), Yuanzhi (Polygala tenuifoila) or its extract, Lingzhi (Ganoderma spp.) or its extracts, Fuling (Poria cocos) or its extract, enokitake (Flammulina velutipes) or its extract, Gan Cao (Glycyrrhiza uralensis Fisch) or its extract, Huperzine A, Lacithin, Metrifonate, Nocetile, folic acid, amino acids, creatine, fiber supplement, or any combination thereof.
There are many different periods of sleep a person goes through. These include Slow-Wave-Sleep 1 (SWS 1), Slow-Wave-Sleep 2 (SWS 2), Slow-Wave-Sleep 3 (SWS 3) Slow-Wave-Sleep 4 (SWS 4) and Rapid Eye Movement (REM). SWS 1 and SWS 2 are both periods of light sleep where it is relatively easy to wake someone up. Light sleep is usually more frequent in the second half of sleep. SWS 3 and SWS 4 are both periods of deep sleep, where it is difficult to wake the sleeper. Deep sleep is more frequent in the first half of sleep and each period will get shorter each time afterward. REM is a period of sleep in which people have their most vivid dreams. The wave patterns are similar to the patterns in which a person is awake. However, it is difficult to wake someone up who is in this state of sleep. The sleep cycle of a typical person can be described as follows:
SWS1, SWS2, SWS3, SWS4, SWS3, SWS2, REM, SWS1, SWS2, SWS3, SWS4, SWS3, SWS2, REM. (See FIG. 62)
However, the above sequence may not be in a fixed order. The sleep status may shift from SWS 4 to SWS1 or wake up because the body turns from one side to the other. It may shift to the SWS2 status after the movement. The interval between two REM is about 90 min. For healthy people, SWS1 will occupy about 5% of sleep, SWS2 will occupy about 50% of sleep, SWS3 will occupy about 10% of sleep, SWS4 will occupy about 10% of sleep and REM will occupy about 25% of sleep. Since a person in SWS1 and SWS2 can easily be woken, a healthy person has enough opportunities to wake up to urinate. However if a person's sleeping status is mostly in SWS4, he has less chance of waking up when the bladder is full. It is difficult for him to break through the barriers of deep sleep. Then enuresis occurs. This invention relates to a plant extract, including Wenguanguo, for preventing enuresis.
This invention provides the extract of Wenguanguo for inhibiting the uptake of 5-hydroxytryptamine (5HT) in a subject.
5-HT controls and modulates a sleep factor that sustains and increases deep sleep. Inhibiting the uptake of 5HT will decrease deep sleep. People who spend too much time in SWS 3 and SWS 4 are unable to awaken from their sleep when their bladder is full because their sleep is too deep. This is the reason that enuresis often occurs during SWS 3 and SWS 4.
This invention provides the extract of Wenguanguo for increasing the activity of Dopamine in a subject thereby making the central nerve system of said subject alert.
This invention provides the extract of Wenguanguo for increasing the secretion of antidiuretic hormone (ADH) in a subject, which reduces urine in said subject.
This invention provides the extract of Wenguanguo for modulating the release, breakdown and uptake of Acetylcholine (Ach) and its receptors in a subject. The said extracts of this invention inhibits the deep sleep created by 5HT and increase REM sleep.
This invention provides the extract of Wenguanguo for preventing sleep paralysis in a subject.
This invention provides the extract of Wenguanguo for providing alertness to a sleeping subject.
This invention provides the extract for helping the growth of the bladder and sphincter.
An immature bladder and sphincter cannot control the process and action of urination. By accelerating the growth of the bladder and the sphincter, this problem will be overcome, and enuresis will not occur.
This invention provides the extract of Wenguanguo against cancer growth. The cancer includes, but is not limited to bladder cancer, cervix cancer, prostate cancer, lung cancer, breast cancer, leukocytes cancer, colon cancer, liver cancer, bone cancer, skin cancer, brain cancer, and ovary cancer
This invention provides the extract of Wenguanguo inhibit tumor activities.
This invention provides the pathways interacted by compounds isolated from Xanthoceras Sorbifolia. In an embodiment, a compound has the formula C57H88O23 and the chemical name 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene, designated herein as “Structure Y”, and derivative compounds which are effective against cancer. In another embodiment, the compounds of the present invention comprise the chemical structures designated herein as “Structure Y1”, “Structure R1”, “Structure 1 to 4”, “Structure Y-a to Y-c” and “Structure Y1-a to Y1-c”, “Structure Y1-1 to Y1-4” and their derivatives. See FIG. 31-40.
They regulate the receptors or components of cells. The compounds can be isolated from the plant called Xanthoceras Sorbifolia or can be synthesized.
The compounds of the present invention have structures as shown below:
Structure Y (Also Shown in FIG. 1)




3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene
Structure 1 as shown in FIG. 31
Structure 2 as shown in FIG. 32
Structure 3 as shown in FIG. 33
Structure 4 as shown in FIG. 34
Structure Y-a as shown in FIG. 35
Structure Y-b as shown in FIG. 36
Structure Y-c as shown in FIG. 37
Structure Y1 (Also Shown in FIG. 2).




3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene
Structure Y1-a as shown in FIG. 38
Structure Y1-b as shown in FIG. 39
Structure Y1-c as shown in FIG. 40.
This invention further provides a compound comprising the following structure:




Structure R1: 3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene
This invention further provides a compound comprising the following structure:
The structure of compound O54 is presented in the following figure.




The chemical name of compound O54 is:
054: 3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene.
There are many components and pathways monitoring cell proliferation.
The Xanthoceras Sorbifolia compound or its derivatives work in the Wnt (Wingless-type MMTV integration site family member) signaling pathway. The Wnt signaling pathway is evolutionarily conserved and controls many events during the embryogenesis. This pathway regulates cell morphology, proliferation, motility and as well as cell apoptosis. It also plays an important role during tumorigenesis. The Wnt pathway has also been observed as inappropriately activated in several different types of cancers in humans.
In the nucleus, the target genes for Wnt signaling are normally kept silent by an inhibitory complex of gene regulatory proteins, e.g. the Groucho corepressor protein bound to the gene regulatory protein LEF-I/TCF. In the absence of a Wnt signal, some β-cartenin is bound to the cytosolic tail of cadherin proteins, and any cytosolic β-cartenin that becomes bound by the APC-axin-GSK-3β will trigger its ubiquitylation and degradation in proteasomes. The result is the decrease of intracellular amount of β-cartenin. However, when the Wnt binding to Frizzled (a seven transmembrane receptor) and LRP (Low density lipoprotein Receptor) activates Dishevelled (a cytoplasmic signaling protein) by a mechanism, this leads to the inactivation of GSK-β3 in the degradation complex by a mechanism which requires casein kinase I, as well as casein kinase II. The activity of the multiprotein complex of β-catenin-axin-adenomatous-polyposis coli (APC)-glycogen synthase kinase (GSK)-3β, which targets β-catenin by phosphorylation for degradation by the proteasome, is then inhibited by Dsh/Dvl (Dishevelled, dsh homolog 1). This then inhibits priming of β-catenin, and indirectly prevents the GSK-3βphosphorylation of β-catenin. When stimulated by Wnt, Dvl recruits the GSK-3 binding protein, GBP, to the multiprotein complex of β-catenin-axin-adenomatous-polyposis coli (APC)-glycogen synthase kinase (GSK)-3β. GBP then titrates GSK-β from axin, and in this way, phosphorylation of β-catenin is inhibited. Then, axin is sequestrated by LRP at the cell membrane. The result of all of this is an accumulation of cytosolic β-catenin. In the nucleus, β-catenin binds to LEF-I/TCF, displaces Groucho, and acts a co-activator to stimulate the transcription of Wnt target genes.
Xanthoceras Sorbifolia compositions regulate the components related to Wnt pathways or its receptors, thereby stopping the proliferation of cancer cells.
The compound or its derivatives work in the Mitogens, Ras and a MAP (Mitogen activation protein) kinase pathway. Mitogens stimulate cell division. The binding of mitogens to cell-surface receptors leads to the activation of Ras and a MAP kinase cascade. One effect of this pathway is the increased production of the gene regulatory protein Myc. Myc increases the transcription of several genes, including the gene encoding cyclin D and a subunit of the SCF ubiquitin ligase. The resulting increase in G1-Cdk and G1/S-Cdk activities promotes Rb phosphyorylation and activation of the gene regulatory protein E2F, resulting in S-phase entry, in which G1-Cdk activity initiates Rb phosphorylation, in turn inactivating Rb and freeing E2F to activate the transcription of S-phase genes including the genes for a G1/S-cyclin (cyclin E) and S-cyclin (cyclin A). The resulting appearance of G1/S-Cdk and S-Cdk further enhances Rb phosphorylation, forming a positive feedback loop, and the E2F acts back to stimulate the transcription of its own gene, forming another positive feedback loop. Myc may also promote E2F activity directly by stimulating the transcription of the E2F gene. The result is the increased transcription of genes entry into S phase. However if this pathway is overactive, it will cause cancer cell growth.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the Ras-MAP kinase cascade so that the pathway is not overactive.
The compound or its derivatives work in Ras-dependent or Myc pathway. Sometimes the mutation of amino acid in Ras causes the protein to become permanently overactive, stimulating the Ras-dependent signal pathways overactive in absence of mitogenic stimulation. Similarly, mutations that cause an overexpression of Myc promote excessive cell growth, which in turn promotes the development of cancer.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components of the Ras-dependent or Myc pathway to make sure it is not overactive.
The compound or its derivatives reactivate the abnormal cell checkpoint mechanism. Inside the cell, there is a checkpoint mechanism which detects abnormal mitogenic stimulation and causes abnormally overactive cells to go into apoptosis. However this mechanism is not active in cancer cells due to mutations in the genes that encode essential components of the checkpoint responses. If the mutation happens in the checkpoint mechanism, the cancer cell will grow and divide endlessly.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia reactivate the checkpoint mechanism to stop the cancer cell growth.
The compound or its derivatives affect the extracellular growth signaling pathways. The extracellular growth factors that stimulate cell growth are bound to receptors on the cell surface and activate intracellular signaling pathways. It activates the enzyme PI3-kinase, which promotes protein synthesis, at least partly through the activation of EIF4e and phosphorylated S6 kinase, resulting in increased mRNA translation and then a stimulation of cell growth.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor relate to extracellular growth. It binds the receptor of ovarian cancer cells so as to stop the cancer cell growth.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components relating to Ras and MAP Kinase, which ceases ovarian cancer cell growth.
The compound or its derivatives affect the intracellular mechanism. Cell division is also controlled by an intracellular mechanism that can limit cell proliferation. In normal cells, the Myc protein acts in the nucleus as a signal for cell proliferation. Large quantities of Myc can cause the cell to proliferate in excess and form a tumor.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of the Myc cell's proliferation to stop the tumor cells from dividing.
The compound or its derivatives affect the TGF-alpha signaling pathway. TGF-alpha is produced by keratincytes, macrophages, hepatocytes, and platelets. Its synthesis is stimulated by the infection by viruses. TGF-Alpha induces the long term proliferation of murine and chicken immature hematopoietic progenitor cell such as BFU-E without causing differentiation. It also induces the terminal differentiation of BFU-Ecell into erythrocytes. TGF-Alpha stimulates the proliferation of cultured endothelial cells. It plays an importance role in the vascularisation of tumor tissues.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of TGF-alpha to suppress ovarian cancer and bladder cancer cell growth.
The compound or its derivative compounds affect the TGF-beta signaling pathway. TGF-beta regulates growth and proliferation of cells, blocking growth of many cell types. There are two TGF-beta receptors: Type 1 and Type 2. They are serine-threonine kinases that signal through the SMAD (Protein named after the first two identified, Sma in C. elegans and Mad in Drosophila) family of transcriptional regulators. The TGF-beta pathway and mutation in SMADs are associated with cancer in humans.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of TGF-beta to suppress the ovarian cancer and bladder cancer cell growth.
The compound or its derivatives reactivate the cell functions which are damaged by DNA viruses. DNA tumor viruses cause cancer by interfering with cell cycle control Rb protein and the p53 protein. Mutation in p53 gene will allow cancer cells to survive and proliferate despite DNA damage. The papillomanius uses the proteins E6 and E7 to release the p53 and Rb respectively. This action activates mutated cells, allowing them to survive and then divide and accumulate. The accumulation of damaged cells can lead to cancer.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the proteins E6 and E7 and release the proteins Rb and p53, which will prevent abnormal cells from dividing. It also regulates or reacts with the protein, causing the cancer cells to die.
The compound or its derivatives affect the p53 signaling pathway. p53 helps multi-cellular organisms cope safely with DNA damage and other stressful cellular events, stopping cell proliferation in circumstances where it would be dangerous. Cancer cells tend to contain large quantities of mutant p53 protein, suggesting that the genetic accidents they undergo or the stresses of growth in an inappropriate environment created the signals that normally activate the p53 protein. Thus, the loss of p53 activity can be extremely dangerous in relation to cancer because it allows mutant cells to continue through the cell cycle. It also allows them to escape apoptosis. So, if their DNA is damaged, some cells will die but the cells which survive will carry on dividing without pausing to repair the damage. This may cause the cells to die, or they could survive and proliferate with a corrupted genome, which could lead to loss of both tumor suppressor genes and the activation of oncogenes, for example by gene amplification. Gene amplification could enable cells to develop resistance against therapeutic drugs.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components and receptor of the p53 pathway, which stops the cancer cells from dividing.
The compound or its derivatives affect the cell suicide signaling pathway. All cells with a nucleus contain various inactive procaspases, awaiting a signal before destroying the cell. Each suicide protease is made as an inactive proenzyme called procaspase. It is usually activated by proteolytic cleavage by another member of the caspase family. Two of the cleaved fragments come together to form the active part of the caspase, and the active enzyme is thought to be a tetramer of two of these two parts. Each activated caspase molecule can cleave many procaspase molecules, which in turn activates more molecules. Through a chain reaction or cascade, this leads to the explosive action of a large number of procaspase molecules. Then, some of the activated procaspases cleave a number of key proteins in the cell, including specific cytosolic proteins and nuclear-lamins leading to the controlled death of the cell.
Activating the death receptor on the outside of the cell can also trigger inactive procaspases. For example, killer lymphocytes can cause apoptosis by producing the protein Fas on the surface of the targeted cell. These clusters of Fas protein then recruit intracellular adaptor proteins that bind and aggregate procaspase-8 molecules. These then cleave and activate one another. The activated caspase-8 molecules then activate downstream procaspases to induce apoptosis.
However in cancer cells, the signal to destroy the cell is blocked, due to gene mutation. This means that the cancer cells continue to divide, thereby causing a tumor.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia unblock the suicide signals, allowing cancer cells to destroy themselves.
Structure showed in FIG. 67.
This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above-described compound, wherein R1, R2, R3, R4 are short aliphatic chain and R5 contains an oxyl group; and a pharmaceutically acceptable carrier effective to inhibit growth of said tumor cells.
This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above-described compounds.
This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the compound comprising: a sugar; a triterpene or Sapogenin; side chain at Carbon 21 and 22 or Angeloyl groups, operatively linked form the compound; and a pharmaceutically acceptable carrier.
Structure showed in FIG. 67.
This invention provides a method for inhibiting tumor cell growth in a subject comprising administering to the above-described subject, wherein R1, R2, R3, R4 are short aliphatic chain and R5 contains an oxyl group; effective to inhibit growth of said tumor cells and a pharmaceutically acceptable carrier.
This invention provides a method of for inhibiting tumor cell wherein R1=R2=R3=R4=CH3 and R5 contains an oxyl bond.
This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above-described compounds.




This invention provides a compound consist of a triterpene or sapongenin, sugar moiety connected to the backbone. A sugar was linked the C21 position where two angeloyl groups were attached. This compound has the anti-cancer activity

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the compound is a triterpene or sapongenin with any two of angeloyl group or tigloyl group or senecioyl group or their combinations attach to carbon 21 and 22, or any two of angloyl group or tigloyl group or senecioyl group or their combinations attached to a sugar moiety which bonds to carbon 21 or 22.
The structures of this invention or its derivative can be synthesis or from biological sources.
This invention will be better understood from the examples which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.
EXPERIMENTAL DETAILS
Experiment 1
Herb Extraction
(a) extracting Xanthoceras Sorbifolia powder of husks or branches or stems or leaves or kernels or roots or barks with organic solvent at ratio of 1:2 for 4-5 times for 20-35 hours for each time to form an organic extract; (b) collecting the organic extract; (c) refluxing the organic extract for 2-3 times at 80° C. to form second extracts; (d) removing the organic solvent from the second extract; and (e) Drying and sterilizing the extract to form a Xanthoceras Sorbifolia extract powder.
Experiment 2
Analysis of Xanthoceras Sorbifolia Extract Components by HPLC Chromatography
Methods
HPLC. A C-18 reverse phase μbondapak column (Water P/N 27324) was equilibrated with 10% acetonitrile, 0.005% Trifluoroacetic acid (equilibration solution). An extract of Xanthoceras Sorbifolia prepared using the methods of the present invention was dissolved in equilibration solution (1 mg/ml) before being applied onto the column. 20 ug of samples was applied into column. Elution conditions: Fractions were eluted (flow rate 0.5 ml/min.) with acetonitrile (concentration gradient from 10% to 80% in 70 min) and then remains at 80% for 10 min (70-80 min). The acetonitrile concentration then decreased to 10% (80-85 min) and remained at 10% for 25 min (85-110 min). The fractions were monitored at 207 nm and recorded in chart with a chart speed of 0.25 cm/min and with a OD full scale of 0.128.
Instruments. Waters Model 510 Solvent Delivery System; Waters 484 tunable Absorbance Detector; Waters 745/745B Data Module
Absorbance analysis. The absorption profile of Xanthoceras Sorbifolia extract at various wavelengths was determined. An extract of Xanthoceras Sorbifolia of the present invention was dissolved in 10% acetonitrile/TFA and scanned at 200-700 nm with a spectrophotometer [Spectronic Ins. Model Gene Sys2].
Results
HPLC. About 60-70 peaks can be accounted for in the profile. Among them four are major peaks, 10 are medium size and the rest are small fractions. The major peaks are labelled with a to z following increased concentration of acetonitrile elution. See FIG. 6.
Absorption maximum. Three absorption maximum were identified for Xanthoceras Sorbifolia plant extract; 207 nm, 278 nm and 500 nm. See FIG. 41.
Experiment 3
Screening of Cytotoxicity of Xanthoceras Sorbifolia Extract with Cancer Cells Derived from Different Human Organs using MTT Assay
Methods and Materials
Cells. Human cancer cell lines were obtained from American Type Culture Collection: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary). Cells were grown in culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI-1640; HCT-116, U2OS in McCoy-5A) supplemented with 10% fetal calf serum, glutamine and antibiotics in a 5% CO2 humidified incubator at 37° C.
MTT assay. The procedure for MTT assay followed the method described in (Carmichael et al., 1987) with only minor modifications. Cells were seeded into a 96-wells plate at concentrations of 10,000/well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2, U2OS), or 40,000/well (K562), for 24 hours before drug-treatment. Cells were then exposed to drugs for 48 hours (72 hours for HepG2, U2OS, and 96 hours for MCF-7). After the drug-treatment, MTT (0.5 mg/ml) was added to cultures for an hour. The formation of formazan (product of the reduction of tetrazolium by viable cells) was dissolved with DMSO and the O.D. at 490 nm was measured by an ELISA reader [Dynatech. Model MR700]. The MTT level of cells before drug-treatment was also measured (T0). The % cell-growth (% G) is calculated as:

% G=(TD−T0/TC−T0)×100  (1)

where TC or TD represent O.D. readings of control or drug-treated cells.
When T0>TD, then the cytotoxicity (LC) expressed as % of the control is calculated as:

% LC=(TD−T0/T0)×100.

Results. Among the 10 cell lines studies, their sensitivity toward Xanthoceras Sorbifolia extract can be divided into four groups (most sensitive: Ovary. Sensitive: bladder, bone, prostate, and leukocyte, marginal sensitive: liver, breast, and brain; and lease sensitive: colon, cervix, and lung) (FIG. 8, 10A-D). Their IC50 values are listed in Table 3.1.







TABLE 3.1







IC50 values of Xanthoceras Sorbifolia Extract Determined in


Different Cancer Cells









IC50 determined by MTT assay


Cancer cells from different organs
(ug/ml)





Ovary (most sensitive)
15-15


Bladder (sensitive)
45-50


Bone
40-55


Prostate
40-50


Leukocyte
45-50


Liver (marginal sensitive)
45-65


Breast
 65


Brain
70-85


Colon (least sensitive)
 90


Cervix
115


Lung
110









Xanthoceras Sorbifolia plant extract stimulate cell growth of bladder, bone and lung cells. See FIGS. 10A, 10D.
To invest Among these cell line studied, it was found that low concentrations of the igate the growth and inhibition components of the Xanthoceras Sorbifolia plant extract, the plant extract was fractionated. FIG. 5 shows the results of the screening of cell growth activity of fractions obtained after FPLC chromatography. The assay was conducted with bladder cells. The fractions obtained from FPLC as shown in FIG. 20 were used. As shown in this figure, that different components of Xanthoceras Sorbifolia extracts cause either growth or inhibition effects on cells. Only fraction 5962 (Fraction Y) causes cell inhibition. Fractions 610 and 1116 cause minor stimulation of cell growth. Abscissa: concentration (ug/ml). Ordinate: % Cell Growth (determined by MTT assay).
Experiment 4
Purification of Inhibition Components in the Xanthoceras Sorbifolia Extract
(A) Fractionation of Xanthoceras sorbifolia Extracts Components with FPLC.
Methods
Column. Octadecyl functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 10% acetonitrile—0.005% TFA.

    
    
        Sample loading: 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA.
        Gradient elution: 10-80% acetonitrile in a total volume of 500 ml.
        Monitor absorption wavelength: at 254 nm.
        Fraction Collector: 5 ml/fractions (collect from 10% to 72% acetonitrile, total 90 fractions)
        Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
    
    


Results. The elution profile shows 4-5 broad fractions. See FIG. 20. These fractions were analyzed with HPLC. Specific components, i.e., a-z as specified in FIG. 6, are then assigned in the FPLC fractions.
FPLC fractions are grouped into 7 pools and analyzed for cell growth activity with bladder cells with MTT assay. It was found only one pool (#5962) contains inhibition activity. See FIG. 5.
(B) Fractionation of Fraction #5962 with FPLC by a C18 Open Column with 64% Acetonitrile Isocratic Elution
Methods
Column. Octadecyl-functionalized silica gel; 50 ml; 2 cm×28 cm; equilibrated with 64% acetonitrile—0.005% TFA.

    
    
        Sample loading: 0.2 ml, with concentration: 1-2 mg/ml in 65% acetonitrile/TFA.
        Elution: 64% acetonitrile isocratic.
        Monitor absorption wavelength: at 254 nm.
        Fraction Collector: 1 ml fraction (collect the first 90 fractions)
        Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
    
    


Results. Fraction 5962 was further separated with an open ODS-C18 column using isocratic 64% acetonitrile elution. Two major fractions, i.e., X and Y, were collected. See FIG. 42. MTT assay showed that only the Y fraction has the inhibition activity. See FIG. 43.
(C) Analysis of Fraction Y with HPLC
Methods
Column. Waters μ-bondapak C18 (3.9 mm×300 cm).

    
    
        Elution: 35% or 45% isocratic elution.
        Flow rate: 0.5 ml/min; monitored at 207 nm with O.D. Scale of 0.128; chart speed: 0.25 cm/min.
    
    


Results. On 45% isocratic analysis, Three fractions of Y were obtained (FIG. 44).
(D) Final Isolation of Active Y Component with Preparative HPLC.
Methods
Column: A preparative HPLC column (Waters Delta Pak C18-300A);

    
    
        Elution: 45% acetonitrile isocratic elution with flow rate of 1 ml/min.
        Monitor at 207 nm;
        Fractions (designated as Y1, Y2, etc.) were collected and lyophilized.
    
    


Results. Final separation of Y fractions was achieved by HPLC with a preparative column (FIG. 7). These fractions (compound Y1, Y2, Y3 and Y4) including the major fraction Y3 (designated as compound Y) were collected. Re-chromatography of the Compound Y showed a single peak in HPLC with a C18 reverse phase column. See FIG. 7A.
(E) Appearance and solubility. The pure Compound Y is amorphous white powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.
(F) Inhibition Analysis of Compound Y with MTT Assay.
Inhibition analysis of Compound Y was determined with MTT assay. The results indicated that (a) Compound Y has activity against ovarian cancer cells (OCAR-3) with IC50 value of 1.5 ug/ml which is 10-15 times more potent than the unpurified extract. See FIG. 3; and FIG. 8.
(b) Compound Y maintains its selectivity against ovarian cancer cells versus cervical cancer cells (HeLa). See FIG. 9.
Experiment 5
Determination of the Chemical Structure of Compound Y of Xanthoceras Sorbifolia Extract
Methods
NMR analysis. The pure compound Y of Xanthoceras Sorbifolia were dissolved in pyridine-D5 with 0.05% v/v TMS. All NMR spectra were acquired using a Bruker Avance 600 MHz NMR spectrometer with a QXI probe (1H/13C/15N/31P) at 298 K. The numbers of scans for 1D 1H spectra were 16 to 128, depending on the sample concentration. 2D HMQC spectra were recorded with spectral widths of 6000×24,000 Hz and data points of 2024×256 for t2 and t1 dimensions, respectively. The numbers of scans were 4 to 128. 2D HMBC were acquired with spectral widths of 6000×30,000 Hz and data points of 2024×512 for t2 and t1 dimensions, respectively. The numbers of scans were 64. The 2D data were zero-filled in t1 dimension to double the data points, multiplied by cosine-square-bell window functions in both t1 and t2 dimensions, and Fourier-transformed using software XWIN-NMR. The final real matrix sizes of these 2D spectra are 2048×256 and 2048×512 data points (F2×F1) for HMQC and HMBC, respectively.
Mass spectral analysis. The mass of samples was analyzed by (A) MALDI-TOF Mass Spectrometry and by (B) ESI-MS Mass spectrometry. (A) Samples for MALDI-TOF were first dissolved in acetonitrile, then mixed with the matrix CHCA (Alpha-cyano-4-hydroxycinnamic acid, 10 mg CHCA/mL in 50:50 water/acetonitrile and 0.1% TFA in final concentration). The molecular weight was determined by the high resolution mass spectroscope analysis with standards. (B) For ESI, the sample was analyzed with LCQ DECA XP Plus machine made by Thermo Finnigan. It is ionized with ESI source and the solvent for the compound is acetonitrile.
Results. The profile of the proton NMR is presented in FIG. 11. The 2D NMR profiles of HMQC and HMBC are shown in FIGS. 12 and 13, respectively.
Table 5.1 summarizes the 2D NMR chemical shift data and shows the assignment of functional groups derived from these chemical shifts. Based on these data and analysis, the structure of compound Y is assigned and shown below.
Structure of Compound Y:




The chemical name of compound Y is: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene.







TABLE 5.1







13C and 1H NMR Data for Compound Y (in Pyridine-d5)a










Position
C
H
Key HMBC correlations













 1
38.7
0.83, 1.40
C-3, C-5, C-9


 2
26.4
1.81, 2.14
—


 3
89.6
3.25, 1H, dd,
C-23, C-24, GlcA C-1′




12.0/4.0 Hz



 4
39.4
—
—


 5
55.3
0.78
—


 6
18.5
1.55, 1.59
C-8, C-10


 7
36.5
2.00, 2.10
C-5, C-9


 8
41.2
—
—


 9
47.0
3.06
C-7, C-8, C-12, C-14, C-





26


10
37.2
—
—


11
23.7
1.74, 1.89
—


12
125.2
5.49, 1H, br s
C-9, C-11, C-14, C-18


13
143.4
—
—


14
47.5
—
—


15
67.3
4.21
C-8, C-27


16
73.6
4.45
C-14, C-15, C-18


17
48.3
—
—


18
40.8
3.07
C-12, C-13, C-14,





C-16, C-19, C-20, C-28,


19
46.8
1.41, 1.69
—


20
36.2
—
—


21
79.3
6.71, 1H, d, 10 Hz
C-20, C-22, C-29, C-30,





21-O-Ang C-1′′′′


22
73.5
6.32, 1H, d, 10 Hz
C-16, C-17, C-21, C-28,





22-O-Ang C-1′′′′


23
27.7
1.26, 3H, s
C-3, C-4, C-5, C-24


24
16.5
1.16, 3H, s
C-3, C-4, C-5, C-23


25
16.0
0.81, 3H, s
C-1, C-5, C-9, C-10


26
17.3
0.99, 3H, s
C-7, C-8, C-9, C-14


27
21.0
1.85, 3H, s
C-8, C-13, C-14, C-15


28
62.9
3.50, 1H, d, 11.0 Hz,
C-16, C-17, C-18, C-22




3.76, 1H, d, 11.0 Hz,



29
29.2
1.09, 3H, s
C-19, C-20, C-21, C-30


30
20.0
1.32, 3H, s
C-19, C-20, C-21, C-29


GlcA





 1′
104.9
4.89, 1H, d, 7.8 Hz
C-3


 2′
79.1
4.38
GlcA C-1′, C-3′, Gal C-1′′


 3′
86.1
4.20
GlcA C-2′, C-4′, Ara C-1′′′


 4′
71.5
4.42
GlcA C-3′, C-5′, C-6′


 5′
78.0
4.52
GlcA C-4′, C-6′


 6′
171.9
—
—


Gal





 1′′
104.6
5.32, 1H, d, 7.7 Hz
GlcA C-2′


 2′′
73.6
4.42
Gal C-1′′, C-3′′


 3′′
74.9
4.10
Gal C-2′′


 4′′
69.5
4.56
Gal C-2′′, C-3′′


 5′′
76.4
3.94
Gal C-4′′, C-6′′


 6′′
61.6
4.43, 4.52
Gal C-4′′, C-5′′


Ara-f





 1′′′
110.6
6.03, 1H, br s
GlcA C-3′, Ara C-2′′′, C-4′′′


 2′′′
83.4
4.94
Ara C-3′′′


 3′′′
78.3
4.78
Ara C-2′′′


 4′′′
85.2
4.82
Ara C-5′′′


 5′′′
62.2
4.12, 4.28
Ara C-3′′′


21-O-Ang





 1′′′′
167.7
—
—


 2′′′′
129.6
—
—


 3′′′′
137.2
5.96, 1H, dq, 7.0/1.5 Hz
Ang C-1′′′′, C-4′′′′, C-5′′′′


 4′′′′
15.5
2.10, 3H, dq, 7.0/1.5 Hz
Ang C-2′′′′, C-3′′′′


 5′′′′
20.8
2.00, 3H, s
Ang C-1′′′′, C-2′′′′, C-3′′′′


22-O-Ang





 1′′′′
167.9
—
—


 2′′′′
129.8
—
—


 3′′′′
136.3
5.78, 1H, dq, 7.0/1.5 Hz
Ang C-1′′′′, C-4′′′′, C-5′′′′


 4′′′′
15.5
1.93, 3H, dq, 7.0/1.5 Hz
Ang C-2′′′′, C-3′′′′


 5′′′′
20.5
1.74, 3H, s
Ang C-1′′′′, C-2′′′′, C-3′′′′





aThe data were assigned based on HMQC and HMBC correlations.






FIG. 14 and 15 show the mass spectrum of Compound Y as determined by MALDI-TOF and ESI-MS techniques. Based on these data, the mass of compound Y is 1140.57 which agrees with the theoretical mass of the compound Y.
Conclusion
The active compound Y isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with three sugars and biangeloyl groups attached to the C21 and C22 positions of the backbone. The formula of Y is C57H88O23, and the chemical name of Compound Y is: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene.
Experiment 6
Determination of the Chemical Structure of Compound Y1 of Xanthoceras Sorbifolia Extract
Methods
The method for NMR and MS analysis for compound Y1 are same as described in Experiment 5.
Results. The profile of the H-NMR of Y1 is presented in FIG. 16. The 2D NMR profiles of HMQC, HMBC and COSY are shown in FIGS. 17, 18 and 19, respectively.
Table 6.1 summarizes the 1D and 2D NMR chemical shift data and shows the assignment of functional groups derived from these chemical shifts.







TABLE 6.1







13C and 1H NMR Data for Compound Y1 (in Pyridine-d5)a











Position
C
H














1
38.6
0.85, 1.33



2
26.3
1.86, 2.10



3
89.7
3.25, 1H, dd



4
39.5
—



5
55.5
0.75



6
18.3
1.40, 1.43



7
33.1
1.20, 1.50



8
40.0
—



9
46.7
1.69



10 
36.5
—



11 
22.5
2.30



12 
123.6
5.36, 1H, br s



13 
143.5
—



14 
41.8
—



15 
34.7
1.53, 1.73



16 
68.5
4.45



17 
48.2
—



18 
39.9
3.04



19 
47.6
1.30, 3.05



20 
36.7
—



21 
85.3
5.05, 1H, d



22 
73.8
6.17, 1H, d



23 
27.7
1.29, 3H, s



24 
16.5
1.16, 3H, s



25 
15.5
0.81, 3H, s



26 
17.1
0.82, 3H, s



27 
20.6
1.83, 3H, s



28 
63.7
3.42, 1H, d, 3.60, 1H, d



29 
29.9
1.42, 3H, s



30 
19.9
1.37, 3H, s



GlcA





1
105.0
4.88, 1H, d



2
79.0
4.37



3
86.0
4.20



4
71.6
4.43



5
78.0
4.50



6
171.8
—



Gal





1
104.5
5.31, 1H, d



2
73.5
4.43



3
74.9
4.10



4
69.5
4.57



5
76.3
3.95



6
61.1
4.44, 4.53



Ara-f





1
110.9
6.04, 1H, br s



2
83.3
4.95



3
78.3
4.78



4
85.2
4.82



5
62.0
4.13, 4.31



21-O-Rha





1
105.1
4.92, 1H, d



2
70.5
4.25



3
74.0
5.59



4
71.5
5.70



5
68.5
3.89



6
17.6
1.18, 3H, d



Rh-3-Ang





1
167.2
—



2
127.9
—



3
138.7
5.92, 1H, q



4
15.7
2.02, 3H, d



5
20.6
1.92, 3H, s



Rh-4-Ang





1
167.2
—



2
128.0
—



3
137.9
5.87, 1H, q



4
15.5
1.96, 3H, d



5
19.8
1.85, 3H, s



22-O-Ac





1
171.4
—



2
21.8
2.31, 3H, s









Based on these data and analysis, the structure of compound Y1 is assigned and shown below.




The chemical name of Y1 is:
3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β, 16α,21β,22α,28-pentahydroxyolean-12-ene.
Conclusion. Based on the chemical shift analysis, the active compound Y1 isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with four sugars and biangeloyl groups attached to the sugar moiety. The formula of Y1 is C65H100O27,
Results of Y2 analysis
The profile of the proton NMR of Y2 is presented in FIG. 51.
The profiles of 2D NMR (HMQC) of Y2 is presented in FIG. 52.
Results of Y4 analysis
The profile of the proton NMR of Y4 is presented in FIG. 53.
The profiles of 2D NMR (HMQC) of Y4 is presented in FIG. 54.
Experiment 7
Acid and Alkaline and Enzyme Hydrolysis of Compound Y
(A) Removal of sugars from compound Y. Acid Hydrolysis of compound Y generates a compound with the following structure, designated herein as Y-c: (FIG. 37)




Methods: 5 mg of compound Y is dissolved in 3 ml of MeOH and then treated with 3 ml of 3N HCl. Hydrolysis of saponins will be conducted under reflux for 4 hr. After hydrolysis, the solution will be neutralized with 5% Na2CO3 and extracted with Ethyl acetate three times to afford an aqueous layer and an organic layer, containing sugars and aglycon, respectively. Aglycon from the organic layer will be further purified on Silica gel chromatography in (CHCl3: MeOH, 1:9) or with C18 ODS HPLC chromatography. About 2 mg of compound with the above structure Y-c can be obtained.
Method reference: Essentials of Carbohydrate Chemistry. By John F. Robyt, (Springer, 1998).
(B) Partial removal of sugars from compound Y. The linkage of oligosaccharide can be cleaved by partial acid hydrolysis and by specific enzyme hydrolysis. For example, the 1→4 linkage of arabinofuranosyl can be removed by α-amylase. Other enzymes such as β-amylase, isoamylase, glucose oxidase, mannanse and pullulanase can be used to cleave individual saccharide in saponins.
Coumpound Structure showed in FIG. 70.
Compound structure showed in FIG. 69.
Compound Structure showed in FIG. 68.
Experiment 8
Purification of component R from Xanthoceras Sorbifolia Extract
(A) Fractionation of Xanthoceras Sorbifolia extracts components with FPLC.
Methods

    
    
        Column: Octadecyl functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 10% acetonitrile—0.005% TFA.
        Sample loading: volume: 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA.
        Gradient elution: 10-80% acetonitrile in a total volume of 500 ml.
        Monitored at 254 nm.
        Fraction Collector: 5 ml/fraction.
        Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
    
    


Results
The elution profile shows 4-5 broad fractions (FIG. 20). These fractions were analyzed with HPLC. By comparison with the profiles of the original sample, specific component, in this case the R component, is identified and then collected for further purification.
(B) Fractionation of R with FPLC with 30% Acetonitrile isocratic Elution
Methods

    
    
        Column: Octadecyl-functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 30% acetonitrile—0.005% TFA.
        Sample loading: 0.2 ml, with concentration: 1-2 mg/ml.
        Elution: 30% acetonitrile isocratic. Monitor absorption wavelength: at 254 nm.
        Fraction Collector: 5 ml/fraction.
        Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
    
    


Results
Fraction No. 39-41 from gradient elution of FPLC were pooled and further purified with an open ODS-C18 column with isocratic 30% acetonitrile elution. Six identifiable fractions in two groups were collected. See FIG. 45. Fractions 6-13 were further characterized with HPLC.
(C) Analysis and Isolation of R with HPLC
Methods

    
    
        Column: Waters μ-bondapak C18 (3.9×300 nm) and Waters DeltaPak C18 (7.8 mm×30 cm).
        Elution: Gradient (10-80%) and 30% isocratic elution.
        Flow rate: 0.5 ml/min; monitored at 207 nm; with attenuation 0.128;

chart speed: 0.25 cm/min.

    
    


Results
On HPLC gradient elution analysis, Fractions #9-11 contain a major component with a few minor components. See FIG. 46. These components were further separated into 4-5 components with the 30% acetonitrile isocratic elution in a DeltaPak column. The fraction designated herein as “R1”, is the major component. See FIG. 47A. The pure R1 was subsequently collected from the column elution. See FIG. 47B.
(D) Appearance and Solubility.
The pure R1 is amorphous white powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.
(E) Determination of the Chemical Structure of R1 Isolated from Xanthoceras Sorbifolia Extract
Methods
The NMR and MS Analysis of R1 are same as those described in Experiment 5.
Results
The proton NMR profile of pure R1 is presented in FIG. 21. The 2D NMR (HMQC) spectra of R1 are presented in FIG. 22. The 2D NMR (HMBC) spectra of R1 are presented in FIG. 23. The 2D COSY spectrum is presented in FIG. 24. The Carbon 13 NMR spectrum is presented in FIG. 25.
Based on all the data presented above, Table 8.1 summarizes the results of the structural analysis and the assignment of the functional groups of compound R1.







TABLE 8.1







13C and 1H NMR Data for R1 (in Pyridine-d5)a










Position
C
H
Key HMBC correlations













1
38.6
1.01, 1.63
C-3, C-25


2
26.4
1.89, 2.33
C-3


3
89.1
3.26, 1H, dd, 12.0/4.2 Hz
C-23, C-24, Glc′ C-1


4
39.2
—
—


5
55.5
0.69, 1H, d, 11.4 Hz
C-4, C-6, C-7, C-9,





C-10, C-23, C-24, C-25


6
18.3
1.30, 1.39
C-5, C-8, C-10


7
32.5
1.41
C-6, C-26


8
40.1
—
—


9
47.7
1.61
C-1, C-5, C-8, C-10, 





C-11, C-14, C-25, C-26


10 
36.7
—
—


11 
23.7
1.90, 2.00
C-8, C-12, C-14


12 
123.5
5.35, 1H, br s
C-9, C-14, C-18


13 
142.9
—
—


14 
41.9
—
—


15 
25.7
1.88, 1.90
—


16 
18.0
1.95, 2.29
C-14, C-17, C-18


17 
43.1
—
—


18 
41.6
2.60, dd, 12.0/2.4 Hz
C-19


19 
46.3
1.28, 2.11
C-18, C-20, C-29, C-30


20 
36.1
—
—


21 
76.5
3.73, 1H, d, 9.6 Hz
C-20, C-22, C-29, C-30


22 
75.1
4.31, 1H, d, 9.6 Hz
C-16, C-17, C-21


23 
27.9
1.20, 3H, s
C-3, C-4, C-5, C-24


24 
16.6
0.95, 3H, s
C-3, C-4, C-5, C-23


25 
15.7
0.95, 3H, s
C-1, C-5, C-9, C-10


26 
16.7
1.07, 3H, s
C-7, C-8, C-9, C-14


27 
26.1
1.25, 3H, s
C-8, C-13, C-14


28 
75.8
4.10, 2H, br s
C-16, C-17, C-18,





C-22, Glc′′′ C-1


29 
30.2
1.22, 3H, s
C-19, C-20, C-21, C-30


30 
19.5
1.26, 3H, s
C-19, C-20, C-21, C-29


3-Glc′





1
106.5
4.84, 1H, d, 7.2 Hz
C-3, Glc′ C-5


2
73.2
3.99
—


3
79.1
5.90, 1H, t, 9.6 Hz
Ang C-1, Glc′ C-2, C-4


4
69.4
4.26
Glc′ C-6


5
76.4
3.95
Glc′ C-1, C-3


6
69.8
4.40, 4.83
Glc′ C-4, Glc′′ C-1


Ang





1
167.9
—
—


2
128.7
—
—


3
136.7
5.80, 1H, ddd,
Ang C-1, C-4, C-5




6.6/0.6 Hz



4
15.7
1.93, 3H, dd, 6.6/0.6 Hz
Ang C-2, C-3


5
20.6
1.81, 3H, s
Ang C-1, C-2, C-3


Glc′′





1
105.4
5.09, 1H, d, 7.8 Hz
Glc′ C-6, Glc′′ C-5


2
74.9
4.05
Glc′′ C-1


3
78.2b
4.22
Glc′′ C-5


4
71.4c
4.24
Glc′′ C-6


5
78.3d
3.90
—


6
62.4e
4.40, 4.51
Glc′′ C-4


28-Glc′′′





1
103.5
4.72, 1H, d, 7.2 Hz
C-28


2
75.3
4.22
Glc′′′ C-1, C-3, Rha C-1


3
79.8
4.25
—


4
71.6
4.20
—


5
76.6
4.13
Glc′′′ C-3


6
70.0
4.67, 2H, d, 10 Hz
Glc′′′′ C-1, Glc′′′ C-4, C-5


Rha





1
100.7
6.52, 1H, br s
Glc′′′ C-2, Rha C-3, C-5


2
72.3f
4.70, d, 3.0 Hz
Rha C-4


3
72.3f
4.63, dd,
Rha C-2


4
74.1
4.35
Rha C-2, C-5


5
69.1
4.79
—


6
18.7
1.82, 3H, d, 6.6 Hz
Rha C-4, C-5


Glc′′′′





1
105.5
5.01, 1H, d, 7.8 Hz
Glc′′′ C-6, Glc′′′′ C-5


2
74.9
4.05
Glc′′′′ C-1


3
78.2b
4.22
Glc′′′′ C-5


4
71.4c
4.24
Glc′′′′ C-6


5
78.3d
3.90
—


6
62.5e
4.40, 4.51
Glc′′′′ C-4





aThe data were assigned based on COSY, HMQC and HMBC correlations.


b,c,d,fThe data with the same labels in each column were overlapped.


eThe data with the same labels in each column may be interchanged.






Conclusion
Based on the chemical shift analysis, the compound R1 isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with five sugars and one angeloyl group attached to the sugar moiety. The chemical structure of R1 is:




The formula of compound R1 is C65H106O29, and the chemical name of R1 is: 3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene
Experiment 9
Purification of Component-O from Xanthoceras sorbifolia Extract
(A) Fractionation of Xanthoceras Sorbifolia Extracts Components with FPLC
Methods:

    
    
        Column: Octadecyl functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 10% acetonitrile—0.005% TFA.
        Sample loading: 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA.
        Gradient elution: 10-80% acetonitrile in a total volume of 500 ml.
        Monitor absorption wavelength: at 254 nm.
        Fraction Collector: 5 ml/fraction.
        Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
    
    


Results.
The elution profile shows 4-5 broad fractions (FIG. 20). These fractions were analyzed with HPLC. By comparison with the profiles of the original sample, specific component, in this case the component-O, is identified (#28-30) and were collected for further purification.
(B) Purification of Component-O with HPLC with 20% Acetonitrile Isocratic Elution.
Methods.

    
    
        Column: A preparative HPLC column (Waters Delta Pak C18-300A);
        Samples: Fraction #28-30 from the gradient elution of FPLC were pooled and applied into the HPLC.
        Elution: 20% acetonitrile isocratic with flow rate of 1 ml/min. Fractions were collected.
        Monitored at 207 nm;
        Fractions of interested were collected and lyophilized.
    
    


Results.
Sixteen identifiable fractions were observed in the elution profiles (FIG. 48). Fractions 28, 34 and 54 were further characterized with HPLC using same condition (FIGS. 49 and 50). As show in these figures, a single peak elution of fractions 28, 34 and 54 was obtained, indicating that they are homogeneous fractions (pure). These purified components are named as Compound O28, O34 and O54, respectively.
Appearance and solubility: The purified O-23 and O-34 are light yellow amorphous powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine. The purified O-54 is white amorphous powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.
(C) Structure analysis of Compound-O54.
Methods: The NMR and MS analysis of O54 are same as those described in Experiment 5.
Results
The profile of the proton NMR of compound-O54 is presented in FIG. 28. The 2D NMR (HMQC) spectra of O54 is presented in FIG. 29. The 2D NMR (HMBC) spectra of O54 are presented in FIG. 30.
Based on all the data presented above, Table 9.1 summarizes the results of the structural analysis and the assignment of the functional groups of compound-O54.







TABLE 9.1







13C and 1H NMR data for 054 (in Pyridine-d5)a










Position
C
H
Key HMBC correlations













1
38.6
1.03, 1.60
C-3, C-25


2
26.3
1.90, 2.40
C-3


3
89.1
3.30, 1H, dd, 12.0/4.8 Hz
C-23, C-24, Glc′ C-1


4
39.3
—
—


5
55.6
0.70, 1H, d, 12.0 Hz
C-4, C-6, C-7, C-9,





C-10, C-23, C-24, C-25


6
18.5
1.30, 1.40
C-5, C-8, C-10


7
32.5
1.40
C-6, C-26


8
40.2
—
—


9
47.8
1.59
C-1, C-5, C-8, C-10, C-11,





C-14, C-25, C-26


10 
36.6
—
—


11 
23.6
1.90, 1.95
C-8, C-12, C-14


12 
123.5
5.34, 1H, br s
C-9, C-14, C-18


13 
142.8
—
—


14 
41.9
—
—


15 
25.8
1.90
—


16 
18.1
1.90, 2.31
C-14, C-17, C-18


17 
43.2
—
—


18 
41.7
2.58, dd, 13.2/2.0 Hz
C-19


19 
46.5
1.28, 2.08 (t, 1H, t, 13.2 Hz)
C-18, C-20, C-29, C-30


20 
36.2
—
—


21 
76.6
3.73, 1H, d, 10.2 Hz
C-20, C-22, C-29, C-30


22 
75.1
4.31, 1H, d, 10.2 Hz
C-16, C-17, C-21


23 
27.9
1.22, 3H, s
C-3, C-4, C-5, C-24


24 
16.8
0.98, 3H, s
C-3, C-4, C-5, C-23


25 
15.8
0.95, 3H, s
C-1, C-5, C-9, C-10


26 
16.9
1.07, 3H, s
C-7, C-8, C-9, C-14


27 
26.1
1.26, 3H, s
C-8, C-13, C-14


28 
75.9
4.10, 2H, br s
C-16, C-17, C-18,





C-22, Glc′′′ C-1


29 
30.1
1.24, 3H, s
C-19, C-20, C-21, C-30


30 
19.6
1.26, 3H, s
C-19, C-20, C-21, C-29


3-Glc′





1
106.5
4.86, 1H, d, 7.8 Hz
C-3, Glc′ C-5


2
74.9
3.99
—


3
78.2b
4.20
—


4
70.8
4.36
—


5
76.5
3.95
—


6
70.5
4.40, 4.88 (d, 1H, 9.6 Hz)
Glc′′ C-1


Glc′′





1
105.5
5.12, 1H, d, 7.8 Hz
Glc′ C-6, Glc′′ C-5


2
75.0c
4.03
Glc′′ C-1


3
78.2b
4.20
—


4
71.5d
4.20
—


5
78.3e
3.91
—


6
62.4f
4.40, 4.48
Glc′′ C-4, C-5


28-Glc′′′





1
103.6
4.72, 1H, d, 7.2 Hz
C-28


2
75.3
4.22
Glc′′′ C-1, C-3, Rha C-1


3
79.8
4.21
—


4
71.6
4.19
—


5
76.8
4.14
Glc′′′ C-3


6
70.3
4.67, 2H, d, 10.2 Hz
Glc′′′′ C-1, Glc′′′ C-4, C-5


Rha





1
100.6
6.51, 1H, br s
Glc′′′ C-2, Rha C-3, C-5


2
72.3g
4.72, d, 3.0 Hz
Rha C-4


3
72.3g
4.61, dd, 10.8/3.6 Hz,
Rha C-2


4
74.1
4.36
Rha C-2, C-5


5
69.2
4.77
—


6
18.6
1.80, 3H, d, 6.0 Hz
Rha C-4, C-5


Glc′′′′





1
105.6
5.00, 1H, d, 7.8 Hz
Glc′′′ C-6, Glc′′′′ C-5


2
75.0c
4.03
Glc′′′′ C-1


3
78.2b
4.20
—


4
71.5d
4.20
—


5
78.3e
3.91
—


6
62.5f
4.40, 4.48
Glc′′′′ C-4, C-5





aThe data were assigned based on COSY, HMQC and HMBC correlations.


b,c,d,e,gThe data with the same labels in each column were overlapped.


fThe data with the same labels in each column may be interchanged.






Conclusion
Based on the chemical shift analysis, the compound O54 isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with five sugars. The chemical structure of Compound O54 is:
The structure of compound O54 is presented in the following figure.




The formula of compound O54 is C60H100O28, and the chemical name of O54 is: The chemical name of compound O54 is: 3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β, 21β,22α,28-tetrahydroxyolean-12-ene.
Results of O28 Analysis
The profile of the proton NMR of O28 is presented in FIG. 55.
The profiles of 2D NMR (HMQC) of O28 is presented in FIG. 56.
Results of O34 Analysis
The profile of the proton NMR of O34 is presented in FIG. 57.
The profiles of 2D NMR (HMQC) of O34 is presented in FIG. 58.
Experiment 11
Extracts X and Y from the Wenguanguo Plant (Xanthoceras Sorbifolia) Improve Learning in Normal Aging Mice
Extracts X and Y are Different Wenguanguo Extracts
Aging male mice at 16 months of age weighing 35-55 gm were trained in a SMG-2 filled with water 11 cm deep (25-26° C.). The SMG-2 had a start point, 4 blind terminals, the escape platform and their routes. The mice were trained to find the escape platform, and the escape latencies from the water and error frequencies were recorded. After training 3 times, the mice which escaped from the water in 2 minutes were selected for the test. The selected aging mice were divided into 9 groups of 11:1) control: receiving normal saline (NS); 2) Positive control: receiving Xi-en-kai 0.9 g/kg; 3) X-I group: receiving 100 mg/kg4; 4) X-II group: receiving 200 mg/kg; 5) X-III group: receiving 400 mg/kg; 6) Y-I group: receiving 125 mg/kg; 7) Y-II group: receiving 250 mg/kg; 8) Y-III group: receiving 500 mg/kg and 9) model group.
All the drugs were received through oral administration, 20 ml/kg, 3, 6 and 9 days before the water maze test. The escape latencies (EL) from the water and error frequencies were recorded. All data were analyzed with t-test.
11.1. After 3 days of administration of extracts X and Y from Wenguanguo Plant, hereinafter as “X” and “Y”, respectively, the escape latency in the water maze by the group 9 mice receiving 500 mg/kg of Y decreased significantly compared with the control (P<0.05). The other dosage treatments showed improvement too but not significant ones. See Table 11.1.







TABLE 11.1







The Learning Effects of Plant Extracts after Administration in Aging Mice for 3 days














Blind
Blind
Blind
Blind
Error



Group
Terminal 1
Terminal 2
Terminal 3
Terminal 4
Frequency
EL (sec)





control
2.12 ± 2.27
3.62 ± 1.63
1.37 ± 2.41
2.68 ± 2.52
9.81 ± 3.42
64.12 ± 24.8 


positive
1.09 ± 1.57
 2.0 ± 0.89
 2.0 ± 1.26
 2.0 ± 2.64
7.09 ± 2.30
39.45 ± 16.32


X I
1.18 ± 1.4 
3.36 ± 2.65
2.27 ± 2.14
0.81 ± 1.07
7.63 ± 4.47
51.72 ± 17.23


X II
 2.2 ± 1.61
 2.5 ± 1.51
 2.0 ± 1.69
 2.8 ± 1.03
 9.5 ± 4.03
 50.3 ± 20.84


X III
1.45 ± 2.33
2.72 ± 2.64
2.09 ± 2.11
1.81 ± 1.94
8.09 ± 4.67
46.91 ± 19.18


Y I
1.36 ± 1.91
 3.0 ± 1.94
3.45 ± 3.2 
1.09 ± 1.44
8.18 ± 3.78
46.36 ± 22.33


Y II
1.63 ± 1.80
3.81 ± 1.94
2.36 ± 1.12
 .36 ± 1.50
10.18 ± 3.02 
48.36 ± 20.61


Y III
2.18 ± 3.34
1.63 ± 1.21
1.54 ± 1.29
1.81 ± 1.40
7.18 ± 4.30
 41.45 ± 16.48*





*P < 0.05






11.2. After 6 days of administration of X and Y, the error frequency in the water maze in the mice with all dosage treatments decreased significantly (P<0.05, P<0.01). The escape latency in the water maze by the group 9 mice receiving 500 mg/kg of Y decreased significantly (P<0.05). See Table 11.2.







TABLE 11.2







The Learning Effects of Plant Extract after Administration in aging mice for 6 days














Blind
Blind
Blind
Blind
Error



Group
Terminal
Terminal
Terminal
Terminal
Frequency
EL (sec)





Control
2.74 ± 2.98
 4.0 ± 2.67
3.26 ± 3.02
 3.0 ± 2.67
12.9 ± 4.20
60.58 ± 24.6 


positive
1.64 ± 2.06
3.73 ± 2.05
1.18 ± 1.47
 2.0 ± 1.90
 8.55 ± 4.61*
38.64 ± 13.68


X I
 1.7 ± 1.49
 3.1 ± 2.02
 1.8 ± 2.10
 1.9 ± 1.66
 8.5 ± 1.65*
 47.7 ± 11.13


X II
1.18 ± 1.66
 3.5 ± 2.02
 2.0 ± 1.73
1.73 ± 2.05
 8.45 ± 3.14*
 49.0 ± 13.29


X III
1.09 ± 1.30
3.55 ± 2.07
1.91 ± 1.58
1.55 ± 1.44
 8.0 ± 2.32*
46.36 ± 13.31


Y I
 1.0 ± 1.67
3.09 ± 1.58
2.64 ± 1.8 
1.54 ± 2.02
 8.36 ± 3.07*
57.27 ± 19.88


Y II
1.36 ± 1.50
3.36 ± 2.06
 2.0 ± 1.67
1.18 ± 1.17
 7.91 ± 3.05**
47.55 ± 22.93


Y III
 1.2 ± 1.14
 4.1 ± 1.79
 2.5 ± 2.84
 1.4 ± 1.90
 9.0 ± 3.23*
 39.9 ± 8.56*





*P < 0.05


**P < 0.01






11.3. After 9 days of administration of X and Y, the error frequency in the water maze in the mice with all dosage treatments decreased significantly (P<0.05, P<0.01). The escape latency in the water maze by the group 9 mice receiving 500 mg/kg of Y decreased significantly (P<0.05). See Table 11.3. FIG. 60a, 60b.







TABLE 11.3







The Learning Effects of Plant Extracts after Administration in aging mice for 9 days














Blind
Blind
Blind
Blind
Error



Group
Terminal 1
Terminal 2
Terminal 3
Terminal 4
Frequency
EL (sec)





Control
2.36 ± 1.65
2.64 ± 1.69
2.86 ± 2.54
 1.5 ± 2.18
9.71 ± 3.52
59.71 ± 29.42


positive
1.64 ± 1.80
2.18 ± 1.54
1.55 ± 1.37
1.64 ± 2.20
 7.0 ± 2.19*
33.36 ± 10.87


X I
 1.1 ± 1.29
 2.5 ± 2.37
 1.9 ± 0.74
 1.0 ± 1.15
 6.5 ± 3.27*
40.8 ± 20.4


X II
1.18 ± 1.17
2.18 ± 1.60
1.36 ± 1.36
2.18 ± 1.25
 6.91 ± 3.27*
42.73 ± 15.82


X III
 1.0 ± 1.25
 1.9 ± 1.79
 1.3 ± 1.25
 2.2 ± 1.16
 6.4 ± 2.84*
 35.1 ± 11.76*


Y I
1.82 ± 1.33
1.64 ± 1.69
1.82 ± 1.33
1.82 ± 1.47
 7.09 ± 2.47*
42.09 ± 20.93


Y II
 1.2 ± 1.32
 1.9 ± 1.37
 2.6 ± 1.58
 1.2 ± 1.32
 7.1 ± 1.52*
 34.4 ± 13.47*


Y III
 0.8 ± 1.03
 2.5 ± 1.43
 1.8 ± 1.40
 2.0 ± 1.70
 7.0 ± 1.41*
 31.9 ± 9.87**





*P < 0.05


**P < 0.01






The results indicated that the extracts X and Y had positive effects on improving acquisition and retention of the tested aging mice. In addition, the effects increased with the period of receiving the extracts of X and Y prolonged
Experiment 12
Effects of Extracts X and Y on Improving Impairment Induced by Pentobarbital Sodium in Water Maze Learning
12.1 After 10 days of administration of the extracts X and Y, the administrated mice were injected with pentobarbital sodium to induce amnesia.
After 1 day administration of pentobarbital sodium, the results of water maze learning showed that the time spent searching the terminal platform in the water maze by the mice receiving 100 mg/kg of X, and 125 mg, 250 mg/kg and 500 mg/kg of Y decreased significantly (P<0.05).
Error frequency made in the water maze by the mice receiving 500 mg/kg of Y decreased significantly (P<0.05). See Table 12.1.







TABLE 12.1







Results of Water Maze Learning (First Day after Injection with Pentobarbital)














Blind
Blind
Blind
Blind
Error



Group
Terminal 1
Terminal 2
Terminal 3
Terminal 4
Frequency
EL (sec)





control
0.92 ± 0.76
1.46 ± 0.97
1.85 ± 1.07
1.23 ± 0.73
5.38 ± 2.33
 63.0 ± 25.31


positive
0.69 ± 0.70
 1.3 ± 0.95
 1.1 ± 0.74
 1.1 ± 0.74
 4.1 ± 1.85
  36.5 ± 15.76**


X I
 0.5 ± 0.53
 1.7 ± 0.82
 1.2 ± 0.92
 0.9 ± 0.32
 4.2 ± 1.62
 42.2 ± 18.83*


X II
 0.9 ± 0.88
 1.4 ± 0.70
 1.6 ± 1.35
 1.1 ± 0.88
 5.0 ± 2.49
 53.8 ± 16.10


X III
 0.9 ± 0.74
 1.7 ± 0.82
 1.8 ± 0.42
 0.9 ± 0.57
 5.4 ± 1.58
 58.1 ± 16.11


Y I
 1.0 ± 0.89
1.36 ± 0.81
1.27 ± 1.01
0.73 ± 0.65
4.09 ± 2.02
 42.73 ± 16.17*


Y II
 0.9 ± 0.74
 1.7 ± 0.82*
 1.0 ± 0.82*
 0.6 ± 0.70*
 4.2 ± 1.87
  38.4 ± 15.19**


Y III
 0.6 ± 0.70
 0.8 ± 0.63
 1.4 ± 1.35
 0.8 ± 0.63
 3.6 ± 1.26*
  38.5 ± 13.81**





*P < 0.05


**P < 0.01






12.2. After two days of injected of pentobarbital sodium, the time spent searching the terminal platform and the error frequency made in the water maze by all groups of mice receiving X and Y decreased significantly (P<0.05). See Table 12.2.







TABLE 12.2







Results of Water Maze Learning (Second Day after Injection with Pentobarbital)














Blind
Blind
Blind
Blind
Error



Group
Terminal 1
Terminal 2
Terminal 3
Terminal 4
Frequency
EL (sec)





control
1.36 ± 0.50
1.86 ± 0.53
1.29 ± 1.07
1.14 ± 0.66
5.64 ± 1.91
 59.5 ± 34.95


positive
 0.8 ± 0.63
 1.8 ± 0.42
 1.1 ± 0.74
 0.6 ± 0.70
 4.3 ± 0.95*
 34.7 ± 11.45*


X I
 0.9 ± 0.57*
  1.0 ± 0.67**
 1.2 ± 0.79
 0.8 ± 0.63
 3.9 ± 1.73*
 34.5 ± 12.67*


X II
 0.8 ± 0.79*
 1.5 ± 0.71
 1.1 ± 0.88
 0.5 ± 0.53*
 3.8 ± 1.40*
 35.9 ± 12.70*


X III
 1.0 ± 1.05
 1.3 ± 0.48*
 1.3 ± 0.82
 0.5 ± 0.53*
 4.1 ± 1.79*
 36.1 ± 11.10*


Y I
1.09 ± 0.94
1.45 ± 0.52
0.91 ± 0.83
0.73 ± 0.65
 4.18 ± 1.08*
 36.64 ± 14.38*


Y II
 0.9 ± 0.57*
 1.3 ± 0.82
 1.1 ± 0.88
 0.8 ± 0.63
 4.1 ± 1.45*
 35.5 ± 14.27*


Y III
 0.8 ± 0.63*
  0.9 ± 0.74**
 0.9 ± 0.57
 0.9 ± 0.57
  3.4 ± 1.43**
 32.1 ± 13.12*





*P < 0.05


**P < 0.01






12.3. After three days of administration of pentobarbital sodium, the time spent searching the terminal platform in the water maze by all groups of mice receiving X and Y decreased significantly (P<0.05). The error frequency made in the water maze by the mice receiving 250 mg/kg and 500 mg/kg of Y decreased significantly (P<0.05). See Table 12.3, FIG. 61a and FIG. 61b







TABLE 12.3







Results of Water Maze Learning (Third day after Injection with Pentobarbital)














Blind
Blind
Blind
Blind
Error



Group
Terminal 1
Terminal 2
Terminal 3
Terminal 4
Frequency
EL (sec)





control
0.85 ± 0.99 
1.0 ± 0.58
1.46 ± 1.05 
0.62 ± 0.65 
3.92 ± 1.75 
48.92 ± 23.49


positive
0.4 ± 0.52
0.7 ± 0.48
0.9 ± 0.74
0.6 ± 0.70
 2.6 ± 0.97*
 28.4 ± 13.78


X I
0.6 ± 0.70
0.8 ± 0.63
 0.6 ± 0.70*
0.8 ± 0.92
2.8 ± 1.69
 28.0 ± 17.10*


X II
0.4 ± 0.52
1.0 ± 0.47
1.0 ± 0.82
0.6 ± 0.84
3.0 ± 1.25
 32.0 ± 12.36*


X III
0.5 ± 0.71
0.8 ± 0.63
1.0 ± 0.82
0.9 ± 0.74
3.2 ± 1.23
 31.8 ± 12.48*


Y I
0.5 ± 0.53
0.7 ± 0.48
1.0 ± 0.67
0.6 ± 0.52
2.8 ± 0.63
 31.0 ± 9.66*


Y II
0.5 ± 0.53
0.6 ± 0.52
0.8 ± 0.42
0.6 ± 0.52
 2.5 ± 0.53*
 29.0 ± 7.80*


Y III
0.3 ± 0.48
1.0 ± 0.47
0.9 ± 0.57
0.4 ± 0.52
 2.6 ± 0.84*
 30.2 ± 7.54*





*P < 0.05






The results indicated that the extracts X and Y had distinct positive effects on improving spatial learning and retention impairment induced by pentobarbital sodium.
Experiment 13
Effects of Extracts X and Y on Improving Impairment Induced by Scopolamine Hydrobromide in Passive Avoidance
ICR mice weighing 16-20 gm were trained in a STT-2. A mouse was placed on the platform and the SDL were recorded. When the mouse stepped down and all four feet were on the grid, it received electric shock (36 V) immediately, and the EL was recorded. The mice with SDL and EL within 2-60 seconds were selected for the test. The selected aging mice were divided into 9 groups. Each group had 5 male and 5 female mice. All the drugs were received with oral administration, 20 ml/kg, 3, 6 and 9 days before the platform training. The SDL, El and error frequency (the times of receiving electric shocks) made in 5 minutes were recorded. After 10 days of administration all the groups of mice received Scopolamine hydrobromide by injection, 3 mg/kg. After 30 minutes of administration of Scopolamine hydrobromide the mice were trained on the platform and the training was repeated the next day. The performances of the mice in the training were recorded. The SDL, EL and error frequency were recorded. All data were analyzed with t-test.
13.1. After 9 days of administration of X and Y, the results of passive avoidance in aging mice showed that the EL and the error frequency made by the mice received the X and Y in all doses reduced. See Table 13.1.







TABLE 13.1







Passive Avoidance in Aging Mice after 9 days of Administration










Group
EL (sec)
SDL (min)
Error Frequency (in 5 min)





control
6.8 ± 5.93
131.5 ± 106.50
2.5 ± 1.47


positive
5.3 ± 3.74
148.6 ± 81.26 
1.7 ± 1.25


X I
7.0 ± 5.46
143.3 ± 19.77 
1.8 ± 0.92


X II
6.2 ± 5.71
141.1 ± 109.89
2.0 ± 1.25


X III
4.9 ± 3.70
145.7 ± 107.0 
1.9 ± 1.52


Y I
5.1 ± 3.95
153.0 ± 123.52
1.6 ± 1.33


Y II
3.9 ± 2.77
162.7 ± 108.92
1.8 ± 1.48


Y III
5.7 ± 3.12
159.4 ± 83.20 
1.9 ± 0.93









13.2. After 10 days of administration of the X and Y, the tested mice were administrated with scopolamine. The results of passive avoidance in aging mice showed that the error frequency made in the passive avoidance by the mice receiving 400 mg/kg of X, 250 mg/kg and 500 mg/kg of Y reduced significantly (P<0.05, P<0.01). See Table 13.2.







TABLE 13.2







Effects of plant extract X and Y on improving


impairment induced by Scopolamine











Group
EL (sec)
Error Frequency (5 min)






control
3.4 ± 3.03
0.9 ± 1.29



model
4.4 ± 4.09
1.6 ± 1.35



positive
3.1 ± 1.20
 0.4 ± 0.52*



X I
3.1 ± 2.08
0.8 ± 1.03



X II
3.7 ± 3.06
0.9 ± 1.52



X III
2.8 ± 1.48
 0.3 ± 0.48**



Y I
3.2 ± 2.49
0.7 ± 1.06



Y II
2.5 ± 0.97
 0.5 ± 0.71*



Y III
2.5 ± 0.71
 0.5 ± 0.71*





*p < 0.05


**p < 0.01






The results of passive avoidance test impaired mice by scopolamine showed that the error frequency made by the mice receiving X and Y in all doses reduced significantly (P<0.05). The SDL prolonged significantly in mice receiving 250 mg/kg of Y.
The results indicated that the extracts X and Y had distinct positive effects on improving learning and retention impairment induced by scopolamine. See Table 3.3.







TABLE 13.3







Effects of plant extract X and Y on improving


impairment induced by Scopolamine.











Group
SDL (sec)
Error Frequency (5 min)






control
230.4 ± 96.61 
0.7 ± 1.06 



model
216.2 ± 100.77
1.5 ± 1.35 



positive
286.0 ± 34.38*
0.4 ± 0.70*



X I
245.7 ± 114.48
0.4 ± 0.84*



X II
260.4 ± 87.14 
0.4 ± 0.84*



X III
266.8 ± 65.64 
0.5 ± 0.71*



Y I
252.7 ± 101.11
0.4 ± 0.84*



Y II
285.8 ± 29.21*
0.4 ± 0.70*



Y III
277.4 ± 47.62 
0.4 ± 0.70*





*p < 0.05






The results indicated that the extracts X and Y had distinct positive effects on improving learning and retention impairment induced by scopolamine hydrobromide.
Experiment 14
Effects of Extracts X and Y on Improving Impairment Induced by NaNO2 in Water Maze Learning
ICR male mice weighing 16-19 gm were trained in a SMG-2 filled with water 11 cm deep (25-26° C.). The SMG-2 has a start point, 4 blind terminals, the escape platform and their routes. The mice were trained to find the escape platform, and the escape latencies from the water and error frequencies were recorded. After training, the mice which escaped from the water within 1 minute were selected for the test. The selected aging mice were divided into 9 groups of 11 mice. All the drugs were received with oral administration, 20 ml/kg, 3, 6 and 9 days before the water maze test. After 10 days of administration all the groups of mice were received with NaNO2 by injection, 120 mg/kg. After 24 hours of administration of NaNO2 the mice were trained to find the escape platform, and the escape latencies from the water and error frequencies made in 2 minutes were recorded. The administration of pentobarbital sodium continued for 3 days and the performances of the mice in water maze test were recorded. The escape latencies (EL) from the water and errors frequencies were recorded. All data were analyzed with t-test.
14.1. After 3 days of administration of X and Y the escape latency from the water maze and error frequency by the mice receiving X and Y reduced, although not significantly. See Table 14.1.







TABLE 14.1







Effects of Extracts X and Y on Water Maze Learning in Mice after 3


Days of Administration














Blind
Blind
Blind
Blind
Error



Group
Terminal 1
Terminal 2
Terminal 3
Terminal 4
Frequency
EL (sec)





control
1.05 ± 0.49
1.82 ± 0.66
1.09 ± 0.75
0.86 ± 0.47
4.82 ± 0.96
48.27 ± 21.47


positive
0.91 ± 0.30
1.27 ± 0.65
0.82 ± 0.60
0.64 ± 0.50
 3.64 ± 0.92**
36.27 ± 11.83


X I
1.36 ± 0.81
1.73 ± 0.79
1.09 ± 0.83
0.55 ± 0.52
4.73 ± 2.05
37.82 ± 14.24


X II
0.91 ± 0.30
1.82 ± 0.40
1.09 ± 0.94
1.00 ± 0.89
4.91 ± 1.51
36.46 ± 11.97


X III
1.09 ± 0.54
1.45 ± 0.52
0.91 ± 0.70
0.45 ± 0.52
3.91 ± 0.70
36.46 ± 11.78


Y I
1.55 ± 0.52
1.82 ± 0.40
 1.0 ± 0.89
0.45 ± 0.69
4.82 ± 1.33
41.46 ± 16.37


Y II
0.91 ± 0.30
1.18 ± 0.60
1.27 ± 1.10
0.73 ± 0.79
4.09 ± 2.21
36.82 ± 20.61


Y III
0.91 ± 0.30
1.55 ± 0.82
0.45 ± 0.52
0.82 ± 0.40
3.73 ± 1.27
37.55 ± 13.85





*P < 0.05


**P < 0.01






14.2. After 6 days of administration of X and Y the escape latency from the water maze by the mice receiving 400 mg/kg of X and 500 mg/kg of Y reduced significantly compared with the control (P<0.01). See Table 14.2.







TABLE 14.2







Effects of Extracts X and Y on Water Maze Learning in Mice after 6


Days of Administration














Blind
Blind
Blind
Blind
Error



Group
Terminal 1
Terminal 2
Terminal 3
Terminal 4
Frequency
EL (sec)





control
0.95 ± 0.38
1.09 ± 0.43
0.77 ± 0.61
0.91 ± 0.53
3.82 ± 0.80
42.96 ± 13.48


positive
0.73 ± 0.65
1.09 ± 0.30
0.55 ± 0.52
0.82 ± 0.40
 3.18 ± 0.75*
32.91 ± 7.15*


X I
0.73 ± 0.65
1.27 ± 0.47
0.73 ± 0.65
0.73 ± 0.65
3.45 ± 1.21
37.18 ± 7.65 


X II
0.91 ± 0.30
1.00 ± 0.45
0.64 ± 0.92
0.91 ± 0.54
3.45 ± 1.21
37.73 ± 13.26


X III
0.91 ± 0.30
1.09 ± 0.54
0.91 ± 0.83
0.82 ± 0.75
3.73 ± 1.19
 31.09 ± 8.15**


Y I
0.91 ± 0.30
 1.0 ± 0.45
0.82 ± 0.40
0.55 ± 0.52
3.36 ± 0.67
35.82 ± 9.93 


Y II
0.91 ± 0.54
1.09 ± 0.54
1.00 ± 0.77
0.64 ± 0.67
3.64 ± 1.12
35.09 ± 12.13


Y III
0.82 ± 0.40
1.09 ± 0.54
0.82 ± 0.60
0.55 ± 0.52
3.27 ± 1.10
 31.73 ± 8.36**





*P < 0.05


**P < 0.01






4.3. After 9 days of administration of X and Y, the escape latency from the water maze by the mice receiving 250 mg/kg of X, 250 mg and 500 mg/kg of Y reduced significantly compared with the control (P<0.05). See Table 14.3.







TABLET 14.3







Effects of Extracts X and Y on Water Maze Learning in Mice after 9 Days of Administration.














Blind
Blind
Blind
Blind
Error



Group
Terminal 1
Terminal 2
Terminal 3
Terminal 4
Frequency
EL (sec)





control
0.59 ± 0.73
1.14 ± 0.64
0.55 ± 0.67
 1.0 ± 0.76
3.27 ± 1.32
39.27 ± 15.52


positive
0.55 ± 0.52
1.00 ± 0.00
0.27 ± 0.65
0.91 ± 0.54
2.73 ± 0.65
27.64 ± 6.96*


X I
0.45 ± 0.52
1.27 ± 0.47
0.73 ± 0.65
0.55 ± 0.52
3.00 ± 0.77
33.55 ± 9.59 


X II
0.45 ± 0.52
0.91 ± 0.70
0.55 ± 0.69
0.82 ± 0.89
2.73 ± 0.90
28.00 ± 9.53*


X III
0.45 ± 0.52
1.09 ± 0.70
0.82 ± 0.75
0.45 ± 0.52
2.82 ± 1.25
29.45 ± 8.49 


Y I
0.91 ± 0.70
0.91 ± 0.54
0.45 ± 0.52
0.55 ± 0.52
2.82 ± 0.98
32.00 ± 9.49 


Y II
0.64 ± 0.50
0.82 ± 0.75
0.64 ± 0.67
0.82 ± 0.60
2.91 ± 1.30
26.36 ± 9.82*


Y III
0.73 ± 1.01
0.91 ± 0.30
0.45 ± 0.69
0.55 ± 0.69
2.64 ± 1.12
28.09 ± 9.26*





*P < 0.05






14.4. After 10 days of administration of X and Y, the error frequency made in the water maze by the mice receiving 250 mg and 500 mg/kg of Y, the escape latency from the water maze by the mice receiving 500 mg/kg of Y reduced significantly compared with the control (P<0.05, P<0.01). See Table 14.4.







TABLE 14.4







Effects of Extracts X and Y on Water Maze Learning in Mice after


10 Days of Administration














Blind
Blind
Blind
Blind
Error



Group
Terminal 1
Terminal 2
Terminal 3
Terminal 4
Frequency
EL (sec)





control
0.64 ± 0.73
1.14 ± 0.56
0.64 ± 0.66
0.91 ± 0.68
3.32 ± 0.95
36.73 ± 13.02


positive
0.45 ± 0.69
0.82 ± 0.40
0.55 ± 0.52
0.82 ± 0.40
 2.64 ± 0.81*
 29.0 ± 10.10


X I
0.36 ± 0.67
0.91 ± 0.70
0.91 ± 0.83
0.64 ± 0.81
2.82 ± 1.08
33.09 ± 13.96


X II
0.73 ± 0.79
0.82 ± 0.40
0.55 ± 0.69
0.73 ± 0.47
2.82 ± 0.75
28.91 ± 13.34


X III
0.91 ± 0.70
0.91 ± 0.54
0.73 ± 0.65
0.36 ± 0.67
2.91 ± 0.94
32.45 ± 13.57


Y I
0.73 ± 0.79
0.91 ± 0.30
0.36 ± 0.50
0.73 ± 0.65
2.73 ± 0.90
29.55 ± 13.87


Y II
0.55 ± 0.52
0.64 ± 0.50
0.45 ± 0.52
0.64 ± 0.67
 2.27 ± 0.79**
30.36 ± 12.30


Y III
0.45 ± 0.69
1.09 ± 0.54
0.27 ± 0.65
0.55 ± 0.52
 2.36 ± 1.21*
 25.64 ± 11.02*





*P < 0.05


**P < 0.01






The results indicated that the extracts X and Y had distinct positive effects on improving the learning and retention in mice in a water maze. In addition, the effects increased with the period of receiving the extracts of X and Y prolonged.
14.5. After 10 days of receiving X and Y, the mice were administrated with Na NO2 after the test. The results of treatment with X and Y to prevent impairments induced by Na NO2 in water maze learning in aging mice showed that error frequency made by the mice receiving 100 mg/ka and 200 mg/kg of X, and Y of all doses reduced significantly (P<0.05). See Table 14.5. It indicated the extracts X and Y had distinct positive effects on preventing the impairments induced by Na NO2. See Table 14.5.







TABLE 14.5







Effects of Extract X and Y on preventing Impairments Induced by NaNO2














Blind
Blind
Blind
Blind




Group
Terminal 1
Terminal 2
Terminal 3
Terminal 4
Error Frequency
EL (sec)





Control
0.27 ± 0.47
0.91 ± 0.54
0.55 ± 0.52
1.09 ± 0.54
2.82 ± 0.75
30.91 ± 12.36


Model
1.18 ± 0.75
0.91 ± 0.30
0.45 ± 0.52
0.73 ± 0.65
3.27 ± 1.01
36.45 ± 16.89


Positive
0.45 ± 0.52
0.91 ± 0.30
0.55 ± 0.52
0.45 ± 0.52
 2.36 ± 0.81*
32.00 ± 15.83


X I
0.55 ± 0.52
0.82 ± 0.60
0.18 ± 0.40
0.82 ± 0.60
 2.36 ± 0.81*
29.09 ± 13.80


X II
0.18 ± 0.40
0.82 ± 0.60
0.55 ± 0.52
0.82 ± 0.75
 2.36 ± 0.92*
25.82 ± 10.82


X III
0.45 ± 0.52
0.64 ± 0.50
0.82 ± 0.40
0.91 ± 0.70
2.82 ± 1.33
31.09 ± 11.76


Y I
0.27 ± 0.47
0.91 ± 0.30
0.36 ± 0.50
0.73 ± 0.47
 2.27 ± 0.79*
27.00 ± 10.73


Y II
0.45 ± 0.52
0.64 ± 0.50
0.36 ± 0.50
0.91 ± 0.30
 2.36 ± 0.81*
25.82 ± 11.43


Y III
0.64 ± 0.50
0.82 ± 0.40
0.36 ± 0.50
0.64 ± 0.50
 2.45 ± 0.82*
25.09 ± 9.67 





*P < 0.05


**P < 0.01






Experiment 15
Effects of Wenguanguo (Xanthoceras sorbifolia) Extracts on Urination in Mice
As used herein, Extracts (or Fractions) X and Y are different extracts of Wenguanguo plant or Xanthoceras sorbifolia. 
Methods of Experiment. One hundred twelve male ICR mice weighing 18-22 gm were divided into 8 groups of 14: 1, control: receiving normal saline (NS); 2, DCT group: receiving DCT 33.4 mg/kg; 3, X-I group: receiving 100 mg/kg4; 4, X-II group: receiving 200 mg/kg; 5, X-III receiving 400 mg/kg; 6, Y-I group: receiving 125 mg/kg; 7, Y-II group: receiving 250 mg/kg and 8, Y-III group: receiving 500 mg/kg.
All the drugs were received with oral administration, 20 ml/kg, once a day for 3 days. After the last administration, the mouse was placed on a filter paper. The filter paper was on the bottom of a 500 ml beak. The quantity of urine was measured at 30, 60, 120, 180, 240, 300, and 360 minutes by weighing the filter paper with the electronic analytical scale. All data were analyzed with t-test.
Results. After 3 days of administration of X and Y the quantity of urine discharged at 30 minute by the mice receiving 400 mg/kg of X decreased significantly (P<0.01) compared with the mice receiving normal saline. The quantity of urine discharged at 60 minute by the mice receiving 600 mg/kg of Y decreased significantly compared with the mice receiving normal saline (P<0.01).
The quantity of urine discharged at 180 minutes by the mice receiving 200 mg/kg of X, 125 mg and 500 mg/kg of Y decreased significantly compared with the mice receiving normal saline (P<0.01). See Table 15.1.







TABLE 15.1





Effects of Extracts X and Y on the Quantity of Urine in Mice after


3 Days of Administration




















Group
30 min
60 min
120 min






Control
0.267 ± 0.105
0.367 ± 0.162
0.382 ± 0.109



Positive
0.348 ± 0.06*
 0.471 ± 0.169**
0.574 ± 0.249



X I
0.304 ± 0.072
0.274 ± 0.076
0.323 ± 0.173



X II
0.341 ± 0.107
0.323 ± 0.102
0.404 ± 0.138



X III
 0.155 ± 0.056**
 0.200 ± 0.140**
0.455 ± 0.211



Y I
0.216 ± 0.130
0.309 ± 0.093
0.341 ± 0.061



Y II
0.278 ± 0.063
0.278 ± 0.119
0.437 ± 0.112



Y III
0.227 ± 0.058
 0.235 ± 0.035**
0.425 ± 0.133










Compared with the control group: P < 0.05* P < 0.01**











Group
180 min
240 min
300 min
360 min





Control
0.191 ± 0.080
0.161 ± 0.083
0.116 ± 0.06 
0.103 ± 0.057


Positive
0.272 ± 0.131
0.182 ± 0.096
0.110 ± 0.051
0.085 ± 0.031


X I
0.184 ± 0.105
0.154 ± 0.093
0.124 ± 0.091
0.102 ± 0.064


X II
 0.336 ± 0.103**
0.163 ± 0.10 
0.107 ± 0.076
0.106 ± 0.075


X III
0.207 ± 0.112
0.204 ± 0.088
0.150 ± 0.066
0.116 ± 0.077


Y I
 0.367 ± 0.104**
0.171 ± 0.085
 0.173 ± 0.068*
0.093 ± 0.053


Y II
0.275 ± 0.206
0.145 ± 0.029
0.109 ± 0.036
0.106 ± 0.045


Y III
 0.319 ± 0.086**
 0.264 ± 0.114**
0.152 ± 0.084
0.135 ± 0.051





Compared with the control P < 0.05* and P < 0.01**






The results indicated that the extract X and Y can regulate the quantity of urine after 3 days of administration of X and Y.
After 5 days of administration of X and Y, the quantity of urine discharged at 30 minute by the mice receiving 400 mg/kg of X and 500 mg/kg of Y decreased, but not significantly compared with the mice receiving normal saline. The quantity of urine discharged at 4 hour by the mice receiving 400 mg/kg of X and Y in all doses increased significantly compared with the mice receiving normal saline (P<0.05, P<0.01). See Table 15.2.







TABLE 15.2





Effects of Extracts X and Y on the Quantity of Urine in Mice after 5


Days of Administration




















Group
30 min
60 min
120 min






Control
0.327 ± 0.148
0.330 ± 0.194
0.291 ± 0.146



Positive
 0.524 ± 0.206**
 0.478 ± 0.185*
 0.472 ± 0.292*



X I
0.382 ± 0.138
0.251 ± 0.071
0.265 ± 0.172



X II
0.348 ± 0.144
0.324 ± 0.113
0.277 ± 0.131



X III
0.245 ± 0.167
0.236 ± 0.129
0.251 ± 0.142



Y I
0.331 ± 0.098
0.340 ± 0.133
0.291 ± 0.081



Y II
0.357 ± 0.130
0.290 ± 0.145
0.327 ± 0.157



Y III
0.230 ± 0.121
0.307 ± 0.082
0.363 ± 0.100














Group
180 min
240 min
300 min
360 min





Control
0.186 ± 0.086
0.117 ± 0.069
0.105 ± 0.06 
0.104 ± 0.08 


Positive
0.214 ± 0.151
0.110 ± 0.045
0.126 ± 0.056
0.112 ± 0.065


X I
0.188 ± 0.097
0.175 ± 0.088
 0.177 ± 0.102*
0.133 ± 0.092


X II
0.258 ± 0.143
0.150 ± 0.077
 0.167 ± 0.097*
0.130 ± 0.094


X III
0.226 ± 0.107
 0.233 ± 0.132**
0.120 ± 0.059
0.125 ± 0.048


Y I
0.273 ± 0.156
 0.215 ± 0.095**
0.166 ± 0.151
0.116 ± 0.068


Y II
0.181 ± 0.088
 0.181 ± 0.089*
0.151 ± 0.104
0.101 ± 0.042


Y III
0.193 ± 0.09 
 0.217 ± 0.092**
0.112 ± 0.056
0.117 ± 0.043





Compared with the control P < 0.05* and P < 0.01**






The results indicated that the extract X and Y can regulate the quantity of urine after 5 days of administration of X and Y.
After 7 days of administration of X and Y, the quantity of urine was measured at 30, 60, 120, 180, 240, 300, and 360 minutes. The quantity of urine discharged at 30 minute by the mice receiving 200, 400 mg/kg of X and 250, 500 mg/kg of Y decreased significantly (P<0.05) but increased at 240 minutes compared with the mice receiving normal saline. See Table 15.3.







TABLE 15.3





Effects of Extracts X and Y on the Quantity of Urine in Mice


after 7 Days of Administration




















Group
30 min
60 min
120 min






Control
0.252 ± 0.142
0.347 ± 0.159
0.430 ± 0.192



Positive
 0.434 ± 0.230*
 0.606 ± 0.214**
0.590 ± 0.333



X I
0.301 ± 0.152
0.314 ± 0.149
0.342 ± 0.186



X II
0.291 ± 0.161
0.332 ± 0.135
 0.285 ± 0.173*



X III
0.212 ± 0.113
0.260 ± 0.103
 0.309 ± 0.117*



Y I
0.254 ± 0.175
0.283 ± 0.137
0.313 ± 0.178



Y II
0.261 ± 0.189
0.292 ± 0.129
 0.300 ± 0.128*



Y III
0.246 ± 0.170
0.268 ± 0.240
 0.281 ± 0.146*














Group
180 min
240 min
300 min
360 min





Control
0.285 ± 0.136
0.155 ± 0.119
0.122 ± 0.071
0.111 ± 0.061


Positive
0.314 ± 0.119
 0.279 ± 0.192*
0.163 ± 0.087
0.148 ± 0.071


X I
0.267 ± 0.179
0.200 ± 0.114
0.176 ± 0.147
0.157 ± 0.077


X II
0.250 ± 0.116
0.203 ± 0.134
 0.180 ± 0.079*
0.129 ± 0.085


X III
0.293 ± 0.142
 0.250 ± 0.116*
 0.194 ± 0.104*
0.151 ± 0.076


Y I
0.310 ± 0.168
0.248 ± 0.178
0.155 ± 0.108
0.113 ± 0.05 


Y II
0.334 ± 0.208
0.259 ± 0.205
 0.205 ± 0.109*
 0.188 ± 0.113*


Y III
0.267 ± 0.133
0.212 ± 0.125
 0.205 ± 0.119*
 0.169 ± 0.073*





Compared with the control P < 0.05* and P < 0.01**






The results indicated that the extract X and Y can regulate the quantity of urine after 7 days of administration of X and Y.
After 10 days of administration of X and Y, the quantity of urine discharged at 120 minutes by the mice receiving 200, 400 mg/kg of X and 250, 500 mg/kg of Y decreased significantly (P<0.05) compared with the mice receiving normal saline. See FIG. 59.







TABLE 15.4





Effects of Extracts X and Y on the Quantity of Urine in Mice


after 10 Days of Administration




















Group
30 min
60 min
120 min






Control
0.292 ± 0.184
0.323 ± 0.158
0.418 ± 0.221



Positive
0.374 ± 0.159
 0.432 ± 0.163*
 0.643 ± 0.181**



X I
0.306 ± 0.124
0.317 ± 0.088
0.339 ± 0.145



X II
0.292 ± 0.082
0.343 ± 0.120
 0.279 ± 0.118*



X III
0.266 ± 0.116
0.348 ± 0.161
 0.274 ± 0.111*



Y I
0.273 ± 0.117
0.331 ± 0.103
0.406 ± 0.175



Y II
0.289 ± 0.126
0.344 ± 0.147
 0.254 ± 0.102*



Y III
0.227 ± 0.129
0.322 ± 0.162
 0.255 ± 0.124*














Group
180 min
240 min
300 min
360 min





Control
0.203 ± 0.087
0.144 ± 0.098
0.108 ± 0.074
0.091 ± 0.060


Positive
0.253 ± 0.116
0.147 ± 0.067
0.095 ± 0.094
0.068 ± 0.049


X I
0.249 ± 0.094
0.172 ± 0.093
0.120 ± 0.058
0.093 ± 0.050


X II
0.225 ± 0.074
0.163 ± 0.051
0.116 ± 0.052
0.093 ± 0.051


X III
0.247 ± 0.104
0.186 ± 0.102
0.121 ± 0.053
0.098 ± 0.065


Y I
0.243 ± 0.101
0.171 ± 0.098
0.126 ± 0.086
0.098 ± 0.058


Y II
0.229 ± 0.097
0.164 ± 0.091
0.124 ± 0.094
0.111 ± 0.067


Y III
0.213 ± 0.102
0.170 ± 0.081
0.121 ± 0.059
0.095 ± 0.045





Compared with the control P < 0.05* and P < 0.01**






The results indicated that the extract X and Y can regulate the quantity of urine after 10 days of administration of X and Y.
Conclusion. The results indicated that the extract X and Y can regulate the quantity of urine after 3-10 days of administration of X and Y.
Experiment 15A
Antidiuresis Test of Rat in Metabolism Cage
The antidiuresis tests using Xanthoceras Sorbifolia Extract FS(X) and Xanthoceras Sorbifolia saponin extract FS(Y) were carried out in rats. Note: Extracts FS(X) and FS(Y) similar to Extracts X and Y respectively. FS(Y) and FS(X) are crude saponin extracts and the “FS(Y)” and “FS(X)” are the extract names that were used in the experiments.
SD rats were treated with FS(X) by oral administration, daily at the doses of 100, 200, 400 mg/kg for 25 days. The experiment included a negative control group and positive control. Collecting urine in Standard metabolism cage and observation indicators, including urine volume, urine Na+, K+, Cl− content, pH and osmotic pressure. The results obtained were as follows: (1) Extract FS(X) has dose-dependant antidiuresis effect. There is significant antidiuresis at all the time phases for 200 and 400 mg/kg. However, by urination rate, the 400 mg/kg dosage group has a more significant effect in that it can delay urination output by two hours. In addition, there is no change in the impact on total urine volume output. (2) The extract FS(X) increased the concentration of ions in urine after drug treatment. However, there is no apparent dose dependency. (3) FS (X) and FS (Y) have no significant impact on the pH value and osmotic pressure, but have slight impact on the specific gravity.
Objective. Standard metabolism cage method was carried out in rats to research the antidiuresis of FS(X) and FS(Y).
Test Drug. FS(X) is compositions of Xanthoceras Sorbifolia extract. FS(Y) is compositions of Xanthoceras Sorbifolia extract. The test drug is a suspension formulated with 0.5% sodium carboxymethyl cellulose (0.5% CMC-Na).
Test Animals. Male SD rats, initial weight 150 ̃200 g, 100 individuals. The test animals are raised in cages (volume: 20×30×45 cm), and each cage has 5 animals. The basal feed is the full-rate pellet feed for experimental rat, self-made by the Experimental Animal Center. The cage bottom padding is wood shaving and chaff, dried before use. After the padding replacement that is made every other day on average, the cages are disinfected before reuse. Laboratory temperature 23±2° C., humidity 40 ̃70%, with air-conditioning, exhaust and ventilation equipment, natural lighting and a light-shade cycle of about 12 hours.
Animal Screening. The animals are placed individually in the cage for adaptation once a day for 6 ̃10 hours per day for 2 days. Before placing them into the case, press the lower abdomen gently to discharge the remaining urine, inject 38° C. distilled water into the stomach at the volume of 25 ml/kg as the water load. Collect the urine in the metabolism cage within 2 hours after stomach injection; collect the remaining urine by pressing the lower abdomen gently immediately before taking them out of the cage. Animals whose urine volume attains above 40% of the injection volume will be qualified ones.
Test Instruments. Standard metabolism cage, Automatic urine analyzer (Miditron Junior II), Urine osmotic pressure tester, and Urine ion tester (EL-ISE, Beckman).
Methods
Test groups. There are 3 groups for the FS (X), i.e., for 100, 200, 400 mg/kg/day, respectively. There is a dosage group for FS (Y): 400 mg/kg/day; once a day, fed at 30 min after administration, for 25 days. Control group were fed with 0.5% CMC-Na into the stomach every day. The positive control drug is pituitrin, injected into the abdominal cavity at the rate of 0.25 u/kg before the animals are placed in the cage. Each group includes 10 animals.
Urine collection and observation indicators. For the test groups, the animals that have experienced the administration period are on diet for 18 hours before urine collection without prohibiting drinking. After pressing their lower abdomen gently to discharge the remaining urine, apply the water load of 38° C. distilled water at the volume of 50 ml/kg, and place them in the cage for urine collection. Collect the urine volume and times at 0.25 hr, 0.5 hr, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr and 6 hr after water feeding, when the animals are taken out of the cage at 6 hr, press their lower abdomen gently to collect the remaining urine in the bladder. Conduct the routine urine examination (pH, erythrocyte, leucocyte, protein, etc); measure urine Na+, K+, Cl− concentrations and urine osmotic pressure for the urine samples.
Data processing. Urination speed, Relative urine volume, Urine Na+, K+, Cl− content data are shown in X±Sd, and are compared with the control group and subject to a student t-test.
Results
Impact on urine volume. FS (X) has dose-dependant antidiuresis effects. There is a significant antidiuresis effect at all the time phases using dosages of 200 and 400 mg/kg. See Table 15A-1, FIGS. 63 and 63A. However, by urination discharge rate, the 400 mg/kg group has a more significant effect in reducing the urine output during the first 2 hrs as compared with the control. See Table 15A-2, FIGS. 64 and 64A. The FS (Y) 400 mg/kg group has an equivalent drug efficacy to FX (X) 400 mg/kg during the first 2 hours of experiment. In addition, there is no apparent dose dependency in the impact on total urine volume during the whole experiment. The 400 mg/kg groups of FS(X) can significantly reduce the urine volume during the first 6 hours after the drug-treatment which is more effective than the positive control drug (pituitrin).
Impact on Na+, K+, Cl− ions in urine. By reducing the urine volume, the test drug has increased the concentration of ions in urine to different extent. However, there is no apparent dose dependency. By ionic, there is almost no impact on Na+ and Cl− ions, there is a certain discharge promotion action on K+ ions. The concentration and discharge amount of all ions in urine from the positive drug have risen apparently.
Impact on urine pH and osmotic pressure. FS (X) and FS (Y) have no significant impact on the pH value and osmotic pressure, but have slight impact on the specific gravity.
FIGS. 63 and 63A, Table 15A-1, shows results of the urine volume with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days. FIGS. 64 and 64A, Table 15A-2, shows results of discharging urine speed with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days. FIG. 65, Table 15A-3, shows results of urine specific gravity and pH with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days. FIG. 66, Table 15A-4, Concentration of Na+, K+ and Cl− in urine with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days.
Although the present invention has been described in detail with particular reference to preferred embodiments thereof, it should be understood that the invention is capable of other different embodiments, and its details are capable of modifications in various obvious aspects. As is readily apparent to those skilled in the art, variations and modifications can be affected while remaining within the spirit and scope of the invention. Accordingly, the foregoing disclosure, description, and figures are for illustrative purpose only, and do not in any way limit the invention which is defined only by the claims.







TABLE 5.2







Chemical Shift Data of 2D NMR chemical shift of HMQC analysis of 


compound Y


DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan, 


EXPNO = 5, PROCNO = 1


F1PLO = 144.360 ppm, F1PHI = 10.797 ppm, F2PLO = 7.966 ppm, 


F2PHI = 0.417 ppm


MI = 1.00 cm, MAXI = 10000.00 cm, PC = 1.400











ADDRESS
FREQUENCY













row
[Hz]F1
[PPM]F1



#
col
[Hz]F2
[PPM]F2
INTENSITY














1
148
20698.986
137.1608




384
3574.377
5.9557
2.00


2
152
20564.195
136.2676




401
3471.337
5.7840
2.67


3
157
20465.209
135.6117




220
4533.779
7.5543
45.24


4
223
18893.424
125.1963




431
3295.261
5.4906
6.22


5
234
18649.311
123.5787




258
4311.820
7.1845
100.00


6
315
16736.119
110.9011




376
3620.289
6.0322
7.49


7
353
15834.069
104.9237




493
2934.550
4.8896
3.98


8
355
15778.398
104.5548




449
3192.387
5.3192
2.99


9
355
15778.398
104.5548




492
2936.414
4.8927
1.18


10
451
13524.788
89.6213




660
1951.827
3.2522
2.41


11
451
13524.788
89.6213




663
1934.401
3.2231
2.60


12
473
12994.274
86.1059




563
2520.987
4.2005
1.93


13
479
12861.933
85.2290




500
2891.933
4.8186
3.96


14
491
12583.008
83.3807




487
2967.012
4.9437
6.95


15
523
11826.204
78.3658




307
4025.464
6.7073
3.92


16
523
11826.204
78.3658




309
4011.801
6.6846
4.78


17
523
11826.204
78.3658




545
2627.492
4.3780
1.99


18
529
11690.020
77.4633




504
2866.164
4.7757
4.60


19
532
11624.016
77.0260




530
2713.960
4.5221
2.32


20
532
11624.016
77.0260




532
2703.563
4.5047
2.51


21
535
11536.339
76.4450




590
2363.678
3.9384
3.34


22
545
11299.475
74.8754




573
2461.387
4.1012
2.35


23
545
11299.475
74.8754




576
2447.179
4.0775
2.10


24
555
11063.554
73.3121




541
2653.693
4.4216
6.62


25
567
10795.113
71.5333




537
2673.042
4.4539
1.84


26
567
10795.113
71.5333




539
2662.683
4.4366
2.82


27
567
10795.113
71.5333




541
2650.933
4.4170
1.72


28
579
10495.725
69.5494




527
2734.037
4.5555
5.06


29
594
10156.363
67.3006




563
2523.651
4.2050
7.18


30
622
9486.037
62.8588




608
2256.690
3.7601
2.91


31
622
9486.037
62.8588




611
2241.336
3.7346
2.67


32
622
9486.037
62.8588




635
2100.199
3.4994
2.96


33
622
9486.037
62.8588




637
2086.756
3.4770
3.42


34
627
9381.439
62.1656




552
2586.967
4.3105
3.58


35
627
9381.439
62.1656




555
2568.334
4.2794
3.42


36
627
9381.439
62.1656




568
2494.546
4.1565
2.54


37
627
9381.439
62.1656




571
2474.559
4.1232
2.51


38
630
9297.809
61.6115




531
2709.734
4.5150
2.61


39
630
9297.809
61.6115




539
2660.734
4.4334
2.66


40
670
8349.676
55.3287




911
480.283
0.8003
2.14


41
670
8349.676
55.3287




913
465.738
0.7760
2.18


42
726
7038.860
46.6427




679
1839.659
3.0653
2.51


43
726
7038.860
46.6427




819
1016.627
1.6939
1.42


44
726
7038.860
46.6427




848
848.511
1.4138
2.49


45
764
6151.769
40.7644




679
1841.145
3.0678
6.40


46
764
6151.769
40.7644




682
1821.592
3.0352
1.04


47
777
5836.727
38.6768




850
837.869
1.3961
2.15


48
777
5836.727
38.6768




853
818.861
1.3644
2.05


49
777
5836.727
38.6768




907
500.256
0.8335
1.64


50
791
5512.022
36.5251




775
1277.340
2.1283
2.38


51
791
5512.022
36.5251




778
1258.929
2.0977
1.38


52
791
5512.022
36.5251




785
1218.892
2.0309
1.38


53
791
5512.022
36.5251




788
1201.847
2.0025
1.09


54
837
4417.038
29.2693




881
655.973
1.0930
23.74


55
848
4174.679
27.6633




864
757.206
1.2617
20.53


56
848
4174.679
27.6633




872
709.060
1.1815
3.17


57
856
3984.149
26.4008




774
1281.339
2.1350
1.66


58
856
3984.149
26.4008




778
1259.751
2.0990
1.59


59
856
3984.149
26.4008




804
1107.062
1.8446
1.02


60
856
3984.149
26.4008




807
1089.219
1.8149
1.21


61
873
3578.068
23.7099




799
1138.137
1.8964
1.68


62
873
3578.068
23.7099




814
1046.185
1.7432
1.26


63
891
3142.837
20.8259




788
1200.655
2.0006
13.95


64
891
3142.837
20.8259




804
1107.046
1.8446
28.24


65
894
3086.147
20.4502




788
1200.275
1.9999
3.08


66
894
3086.147
20.4502




804
1106.803
1.8442
1.17


67
894
3086.147
20.4502




815
1041.758
1.7358
27.19


68
894
3086.147
20.4502




858
789.804
1.3160
1.16


69
897
3015.337
19.9810




858
790.292
1.3168
33.47


70
906
2802.854
18.5730




830
953.652
1.5890
1.35


71
906
2802.854
18.5730




834
931.208
1.5516
1.89


72
914
2613.995
17.3215




892
592.663
0.9875
12.71


73
919
2490.082
16.5004




875
693.445
1.1554
8.90


74
919
2490.082
16.5004




880
660.343
1.1003
1.76


75
925
2342.840
15.5247




778
1258.345
2.0967
5.45


76
925
2342.840
15.5247




782
1237.122
2.0613
5.31


77
925
2342.840
15.5247




791
1183.410
1.9718
2.33


78
925
2342.840
15.5247




795
1159.732
1.9324
2.38


79
925
2342.840
15.5247




907
503.260
0.8385
6.27


80
925
2342.840
15.5247




912
474.699
0.7910
7.15
















TABLE 5.3







Chemical Shift Data of 2D NMR chemical shift


of HMBC analysis of Y


DU = C:/Bruker/XWIN-NMR, USER = guest,


NAME = chan, EXPNO = 6, PROCNO = 1


F1PLO = 178.339 ppm, F1PHI = 10.721 ppm,


F2PLO = 6.881 ppm, F2PHI = 0.573 ppm


MI = 1.00 cm, MAXI = 10000.00 cm, PC = 1.400











ADDRESS
FREQUENCY













row
[Hz]F1
[PPM]F1



#
col
[Hz]F2
[PPM]F2
INTENSITY














1
123
26590.750
176.2058




895
573.276
0.9552
3.11


2
145
25939.373
171.8894




531
2710.686
4.5166
2.92


3
145
25939.373
171.8894




539
2662.033
4.4355
2.54


4
166
25312.006
167.7321




308
4016.437
6.6923
3.45


5
166
25312.006
167.7321




346
3793.847
6.3214
14.90


6
166
25312.006
167.7321




348
3785.322
6.3072
12.47


7
166
25312.006
167.7321




385
3564.443
5.9392
1.47


8
166
25312.006
167.7321




400
3477.948
5.7950
4.16


9
166
25312.006
167.7321




403
3458.552
5.7627
3.96


10
166
25312.006
167.7321




781
1243.983
2.0728
1.17


11
166
25312.006
167.7321




788
1200.447
2.0002
10.44


12
166
25312.006
167.7321




793
1171.992
1.9528
3.35


13
166
25312.006
167.7321




815
1041.336
1.7351
33.24


14
290
21640.068
143.3997




679
1839.970
3.0658
5.67


15
290
21640.068
143.3997




788
1199.609
1.9988
1.03


16
290
21640.068
143.3997




804
1107.222
1.8449
33.90


17
290
21640.068
143.3997




813
1053.918
1.7561
2.30


18
290
21640.068
143.3997




848
848.155
1.4132
1.25


19
322
20697.354
137.1527




780
1246.505
2.0770
31.46


20
322
20697.354
137.1527




788
1200.075
1.9996
44.60


21
322
20697.354
137.1527




793
1170.377
1.9501
3.79


22
322
20697.354
137.1527




815
1040.860
1.7343
4.31


23
327
20566.367
136.2847




780
1246.629
2.0772
3.06


24
327
20566.367
136.2847




788
1201.192
2.0015
5.78


25
327
20566.367
136.2847




793
1170.748
1.9507
47.25


26
327
20566.367
136.2847




815
1041.067
1.7346
58.19


27
365
19434.006
128.7811




780
1245.861
2.0759
16.02


28
365
19434.006
128.7811




788
1200.442
2.0002
33.73


29
365
19434.006
128.7811




793
1171.204
1.9515
46.70


30
365
19434.006
128.7811




815
1040.940
1.7344
80.60


31
384
18893.113
125.1968




679
1839.547
3.0651
9.80


32
384
18893.113
125.1968




788
1200.729
2.0007
2.34


33
384
18893.113
125.1968




801
1124.942
1.8744
1.93


34
384
18893.113
125.1968




803
1113.960
1.8561
1.36


35
384
18893.113
125.1968




809
1077.556
1.7954
3.32


36
384
18893.113
125.1968




813
1051.694
1.7524
3.14


37
384
18893.113
125.1968




848
847.499
1.4121
1.45


38
457
16738.236
110.9173




361
3707.331
6.1772
6.44


39
457
16738.236
110.9173




390
3534.169
5.8887
6.75


40
457
16738.236
110.9173




486
2972.803
4.9534
1.24


41
457
16738.236
110.9173




488
2962.223
4.9357
1.40


42
457
16738.236
110.9173




563
2520.559
4.1998
12.21


43
488
15822.760
104.8508




531
2712.055
4.5189
1.39


44
488
15822.760
104.8508




538
2668.887
4.4470
4.68


45
488
15822.760
104.8508




545
2627.709
4.3783
14.61


46
488
15822.760
104.8508




660
1952.474
3.2533
2.34


47
488
15822.760
104.8508




662
1941.101
3.2343
3.52


48
488
15822.760
104.8508




664
1928.930
3.2140
2.41


49
489
15777.470
104.5507




538
2669.101
4.4473
12.69


50
489
15777.470
104.5507




546
2622.941
4.3704
6.06


51
489
15777.470
104.5507




590
2363.695
3.9384
3.08


52
489
15777.470
104.5507




660
1953.443
3.2549
2.02


53
489
15777.470
104.5507




662
1940.913
3.2340
1.47


54
566
13527.920
89.6439




492
2938.198
4.8957
11.15


55
566
13527.920
89.6439




805
1101.852
1.8359
1.93


56
566
13527.920
89.6439




851
830.552
1.3839
5.09


57
566
13527.920
89.6439




864
757.374
1.2620
53.64


58
566
13527.920
89.6439




874
696.110
1.1599
39.08


59
566
13527.920
89.6439




880
658.836
1.0978
4.62


60
566
13527.920
89.6439




911
480.432
0.8005
1.38


61
566
13527.920
89.6439




913
466.506
0.7773
1.40


62
584
12992.842
86.0981




376
3621.487
6.0342
5.29


63
584
12992.842
86.0981




531
2708.564
4.5131
1.17


64
584
12992.842
86.0981




539
2662.648
4.4366
4.18


65
584
12992.842
86.0981




545
2625.947
4.3754
6.92


66
584
12992.842
86.0981




864
757.040
1.2614
1.36


67
588
12860.941
85.2241




370
3655.044
6.0901
2.76


68
588
12860.941
85.2241




376
3621.609
6.0344
10.88


69
588
12860.941
85.2241




554
2574.294
4.2893
1.21


70
588
12860.941
85.2241




864
756.227
1.2600
1.05


71
598
12585.614
83.3996




376
3621.268
6.0338
1.92


72
598
12585.614
83.3996




475
3038.901
5.0635
1.23


73
598
12585.614
83.3996




500
2890.984
4.8170
1.60


74
598
12585.614
83.3996




505
2863.233
4.7708
3.39


75
598
12585.614
83.3996




881
655.156
1.0916
1.11


76
623
11822.784
78.3447




295
4093.818
6.8212
2.30


77
623
11822.784
78.3447




321
3940.803
6.5663
2.25


78
623
11822.784
78.3447




348
3785.120
6.3069
18.28


79
623
11822.784
78.3447




434
3277.040
5.4603
1.13


80
623
11822.784
78.3447




448
3194.918
5.3234
7.26


81
623
11822.784
78.3447




563
2521.171
4.2008
7.36


82
623
11822.784
78.3447




845
867.271
1.4451
1.26


83
623
11822.784
78.3447




849
844.669
1.4074
10.09


84
623
11822.784
78.3447




858
790.448
1.3171
26.63


85
623
11822.784
78.3447




881
656.434
1.0938
51.16


86
630
11619.674
76.9987




348
3785.085
6.3068
1.21


87
630
11619.674
76.9987




539
2661.936
4.4354
6.49


88
630
11619.674
76.9987




552
2584.607
4.3065
1.49


89
630
11619.674
76.9987




858
790.978
1.3179
2.74


90
630
11619.674
76.9987




881
655.977
1.0930
5.21


91
641
11304.499
74.9102




307
4021.328
6.7004
1.03


92
641
11304.499
74.9102




526
2736.013
4.5588
4.14


93
641
11304.499
74.9102




538
2670.608
4.4498
9.80


94
641
11304.499
74.9102




881
656.537
1.0939
1.61


95
650
11028.925
73.0841




308
4015.948
6.6915
16.79


96
650
11028.925
73.0841




346
3793.459
6.3207
13.94


97
650
11028.925
73.0841




526
2736.761
4.5601
7.46


98
650
11028.925
73.0841




574
2454.410
4.0896
4.70


99
650
11028.925
73.0841




610
2245.856
3.7421
11.08


100
650
11028.925
73.0841




637
2088.895
3.4806
5.03


101
650
11028.925
73.0841




679
1839.510
3.0650
8.83


102
650
11028.925
73.0841




848
850.226
1.4167
1.52


103
650
11028.925
73.0841




881
656.986
1.0947
1.80


104
658
10790.329
71.5030




308
4016.477
6.6923
1.26


105
658
10790.329
71.5030




531
2708.840
4.5135
2.58


106
658
10790.329
71.5030




564
2518.203
4.1959
5.49


107
658
10790.329
71.5030




609
2253.841
3.7554
1.07


108
658
10790.329
71.5030




881
655.677
1.0925
1.26


109
668
10496.015
69.5527




530
2715.370
4.5244
1.73


110
668
10496.015
69.5527




533
2699.760
4.4984
1.85


111
668
10496.015
69.5527




540
2658.708
4.4300
3.19


112
668
10496.015
69.5527




590
2363.392
3.9379
4.52


113
668
10496.015
69.5527




804
1107.602
1.8455
1.00


114
668
10496.015
69.5527




881
656.508
1.0939
1.01


115
680
10155.364
67.2954




541
2651.054
4.4172
2.15


116
680
10155.364
67.2954




804
1107.228
1.8449
32.23


117
680
10155.364
67.2954




881
656.545
1.0939
1.00


118
703
9486.470
62.8629




346
3793.169
6.3203
9.40


119
703
9486.470
62.8629




348
3784.896
6.3065
9.63


120
703
9486.470
62.8629




679
1839.346
3.0648
3.08


121
703
9486.470
62.8629




863
758.265
1.2634
1.07


122
706
9376.976
62.1373




347
3790.748
6.3162
1.61


123
706
9376.976
62.1373




505
2862.456
4.7695
2.79


124
706
9376.976
62.1373




881
655.399
1.0920
1.01


125
709
9296.042
61.6010




590
2363.684
3.9384
7.53


126
741
8346.307
55.3075




777
1264.802
2.1074
3.77


127
741
8346.307
55.3075




852
825.908
1.3761
3.30


128
741
8346.307
55.3075




864
757.401
1.2620
35.15


129
741
8346.307
55.3075




874
696.341
1.1603
25.71


130
741
8346.307
55.3075




881
658.199
1.0967
3.92


131
741
8346.307
55.3075




891
595.647
0.9925
1.15


132
741
8346.307
55.3075




909
489.518
0.8156
23.33


133
781
7163.191
47.4675




346
3793.181
6.3203
1.69


134
781
7163.191
47.4675




431
3294.477
5.4893
5.30


135
781
7163.191
47.4675




539
2661.476
4.4346
3.10


136
781
7163.191
47.4675




541
2647.966
4.4121
3.42


137
781
7163.191
47.4675




564
2517.984
4.1955
1.14


138
781
7163.191
47.4675




679
1839.442
3.0649
3.64


139
781
7163.191
47.4675




804
1107.164
1.8448
37.94


140
781
7163.191
47.4675




822
1002.093
1.6697
1.62


141
781
7163.191
47.4675




849
845.058
1.4081
2.98


142
781
7163.191
47.4675




858
790.203
1.3167
4.12


143
781
7163.191
47.4675




874
695.441
1.1588
1.07


144
781
7163.191
47.4675




881
656.256
1.0935
6.01


145
781
7163.191
47.4675




891
594.695
0.9909
39.60


146
781
7163.191
47.4675




909
489.613
0.8158
3.90


147
786
7041.051
46.6581




432
3290.762
5.4831
2.39


148
786
7041.051
46.6581




669
1898.191
3.1628
1.21


149
786
7041.051
46.6581




677
1850.653
3.0836
2.37


150
786
7041.051
46.6581




680
1835.948
3.0591
2.84


151
786
7041.051
46.6581




682
1821.518
3.0351
2.66


152
786
7041.051
46.6581




689
1782.432
2.9699
2.02


153
786
7041.051
46.6581




776
1274.214
2.1231
2.05


154
786
7041.051
46.6581




800
1129.576
1.8821
1.51


155
786
7041.051
46.6581




804
1106.939
1.8444
6.33


156
786
7041.051
46.6581




808
1082.787
1.8042
3.27


157
786
7041.051
46.6581




811
1065.178
1.7748
3.51


158
786
7041.051
46.6581




829
959.508
1.5988
3.10


159
786
7041.051
46.6581




838
906.837
1.5110
1.42


160
786
7041.051
46.6581




848
847.280
1.4118
1.15


161
786
7041.051
46.6581




858
790.530
1.3172
49.82


162
786
7041.051
46.6581




874
696.770
1.1610
1.14


163
786
7041.051
46.6581




881
656.443
1.0938
70.13


164
786
7041.051
46.6581




891
594.532
0.9906
18.99


165
786
7041.051
46.6581




909
489.363
0.8154
21.10


166
814
6218.377
41.2066




431
3293.885
5.4883
1.45


167
814
6218.377
41.2066




561
2531.574
4.2182
3.03


168
814
6218.377
41.2066




564
2517.867
4.1953
3.26


169
814
6218.377
41.2066




635
2096.944
3.4940
2.64


170
814
6218.377
41.2066




678
1846.079
3.0760
2.51


171
814
6218.377
41.2066




776
1274.484
2.1236
1.75


172
814
6218.377
41.2066




783
1228.144
2.0464
1.41


173
814
6218.377
41.2066




786
1211.726
2.0190
1.51


174
814
6218.377
41.2066




799
1136.142
1.8931
1.43


175
814
6218.377
41.2066




804
1107.204
1.8448
67.13


176
814
6218.377
41.2066




819
1018.301
1.6967
4.37


177
814
6218.377
41.2066




822
1001.489
1.6687
4.96


178
814
6218.377
41.2066




833
939.695
1.5657
3.66


179
814
6218.377
41.2066




864
756.271
1.2601
2.02


180
814
6218.377
41.2066




874
695.963
1.1596
2.98


181
814
6218.377
41.2066




880
659.072
1.0982
1.66


182
814
6218.377
41.2066




891
594.287
0.9902
49.75


183
814
6218.377
41.2066




895
572.724
0.9543
2.16


184
814
6218.377
41.2066




909
489.811
0.8161
1.62


185
823
5950.421
39.4310




662
1940.805
3.2338
1.07


186
823
5950.421
39.4310




776
1271.011
2.1178
1.62


187
823
5950.421
39.4310




804
1107.587
1.8455
2.29


188
823
5950.421
39.4310




831
947.726
1.5791
1.04


189
823
5950.421
39.4310




858
790.520
1.3172
1.45


190
823
5950.421
39.4310




864
757.090
1.2615
54.14


191
823
5950.421
39.4310




874
696.048
1.1598
72.55


192
823
5950.421
39.4310




880
658.503
1.0972
9.62


193
823
5950.421
39.4310




891
594.530
0.9906
2.75


194
823
5950.421
39.4310




911
479.432
0.7988
7.32


195
823
5950.421
39.4310




913
465.776
0.7761
7.03


196
827
5834.797
38.6648




804
1106.687
1.8440
2.69


197
827
5834.797
38.6648




819
1017.803
1.6959
1.66


198
827
5834.797
38.6648




822
1001.708
1.6691
1.68


199
827
5834.797
38.6648




841
889.985
1.4829
1.01


200
827
5834.797
38.6648




858
789.621
1.3157
1.33


201
827
5834.797
38.6648




864
758.164
1.2633
3.94


202
827
5834.797
38.6648




874
696.137
1.1599
6.09


203
827
5834.797
38.6648




881
656.905
1.0945
2.43


204
827
5834.797
38.6648




892
593.606
0.9891
1.90


205
827
5834.797
38.6648




909
489.245
0.8152
51.13


206
830
5743.329
38.0586




804
1108.031
1.8462
1.40


207
830
5743.329
38.0586




858
790.551
1.3172
1.95


208
830
5743.329
38.0586




864
757.091
1.2615
2.85


209
830
5743.329
38.0586




874
696.052
1.1598
3.54


210
830
5743.329
38.0586




881
655.620
1.0924
2.41


211
830
5743.329
38.0586




891
594.762
0.9910
3.06


212
830
5743.329
38.0586




909
489.474
0.8156
10.76


213
834
5623.896
37.2672




804
1107.433
1.8452
1.25


214
834
5623.896
37.2672




822
1002.039
1.6696
1.43


215
834
5623.896
37.2672




833
939.620
1.5656
1.01


216
834
5623.896
37.2672




858
790.603
1.3173
4.03


217
834
5623.896
37.2672




864
756.768
1.2609
1.89


218
834
5623.896
37.2672




874
696.444
1.1604
2.15


219
834
5623.896
37.2672




881
656.011
1.0931
4.39


220
834
5623.896
37.2672




891
594.776
0.9910
4.19


221
834
5623.896
37.2672




909
489.272
0.8152
10.53


222
837
5533.835
36.6704




777
1268.681
2.1139
1.72


223
837
5533.835
36.6704




804
1108.282
1.8466
1.01


224
837
5533.835
36.6704




811
1067.120
1.7781
1.70


225
837
5533.835
36.6704




819
1017.407
1.6952
4.37


226
837
5533.835
36.6704




822
1001.730
1.6691
4.67


227
837
5533.835
36.6704




832
943.762
1.5725
2.78


228
837
5533.835
36.6704




849
841.934
1.4028
2.09


229
837
5533.835
36.6704




858
790.630
1.3174
8.57


230
837
5533.835
36.6704




864
757.963
1.2629
2.10


231
837
5533.835
36.6704




874
695.245
1.1584
1.83


232
837
5533.835
36.6704




881
656.351
1.0936
11.03


233
837
5533.835
36.6704




891
594.692
0.9909
25.74


234
837
5533.835
36.6704




909
489.139
0.8150
53.53


235
837
5533.835
36.6704




913
467.893
0.7796
6.55


236
840
5450.419
36.1177




307
4023.448
6.7040
5.35


237
840
5450.419
36.1177




309
4013.866
6.6880
5.05


238
840
5450.419
36.1177




346
3794.546
6.3226
1.25


239
840
5450.419
36.1177




679
1839.242
3.0646
3.88


240
840
5450.419
36.1177




849
842.830
1.4043
6.34


241
840
5450.419
36.1177




858
790.237
1.3167
88.69


242
840
5450.419
36.1177




862
767.125
1.2782
1.79


243
840
5450.419
36.1177




864
755.041
1.2581
1.99


244
840
5450.419
36.1177




874
695.441
1.1588
2.24


245
840
5450.419
36.1177




881
656.060
1.0931
84.89


246
840
5450.419
36.1177




891
594.675
0.9909
7.64


247
840
5450.419
36.1177




909
489.300
0.8153
8.88


248
875
4418.238
29.2778




307
4022.778
6.7028
8.21


249
875
4418.238
29.2778




309
4014.385
6.6889
7.83


250
875
4418.238
29.2778




679
1841.576
3.0685
1.85


251
875
4418.238
29.2778




849
844.669
1.4074
1.65


252
875
4418.238
29.2778




858
790.492
1.3171
100.00


253
875
4418.238
29.2778




863
763.811
1.2727
3.72


254
875
4418.238
29.2778




870
718.885
1.1978
22.51


255
875
4418.238
29.2778




875
691.903
1.1529
4.90


256
875
4418.238
29.2778




878
670.147
1.1166
1.92


257
875
4418.238
29.2778




882
648.470
1.0805
1.77


258
875
4418.238
29.2778




892
593.301
0.9886
19.66


259
883
4176.082
27.6732




662
1941.300
3.2346
1.71


260
883
4176.082
27.6732




853
819.336
1.3652
15.64


261
883
4176.082
27.6732




857
794.801
1.3243
4.59


262
883
4176.082
27.6732




863
763.374
1.2720
1.70


263
883
4176.082
27.6732




874
696.058
1.1598
89.76


264
883
4176.082
27.6732




878
671.861
1.1195
2.96


265
883
4176.082
27.6732




881
657.198
1.0950
7.96


266
883
4176.082
27.6732




911
479.487
0.7989
2.54


267
883
4176.082
27.6732




913
466.645
0.7775
2.27


268
903
3588.775
23.7813




431
3296.117
5.4921
2.85


269
903
3588.775
23.7813




791
1182.756
1.9707
1.05


270
903
3588.775
23.7813




801
1127.886
1.8793
1.45


271
903
3588.775
23.7813




804
1108.540
1.8471
1.43


272
903
3588.775
23.7813




811
1064.128
1.7731
1.37


273
903
3588.775
23.7813




821
1006.100
1.6764
3.82


274
903
3588.775
23.7813




874
696.649
1.1608
2.42


275
903
3588.775
23.7813




881
655.757
1.0926
2.43


276
917
3174.632
21.0370




307
4021.419
6.7006
1.44


277
917
3174.632
21.0370




384
3571.435
5.9508
2.36


278
917
3174.632
21.0370




403
3463.331
5.7707
1.11


279
917
3174.632
21.0370




561
2531.850
4.2186
5.21


280
917
3174.632
21.0370




564
2517.700
4.1950
5.22


281
917
3174.632
21.0370




778
1262.748
2.1040
3.52


282
917
3174.632
21.0370




793
1170.055
1.9496
22.51


283
917
3174.632
21.0370




800
1133.275
1.8883
2.77


284
917
3174.632
21.0370




805
1100.419
1.8335
3.08


285
917
3174.632
21.0370




809
1079.427
1.7986
2.91


286
917
3174.632
21.0370




815
1043.118
1.7381
24.13


287
917
3174.632
21.0370




827
975.020
1.6246
2.05


288
917
3174.632
21.0370




848
851.242
1.4184
1.60


289
917
3174.632
21.0370




864
757.267
1.2618
1.67


290
917
3174.632
21.0370




869
727.306
1.2119
1.80


291
917
3174.632
21.0370




881
656.407
1.0937
6.11


292
922
3013.427
19.9687




309
4014.743
6.6895
13.55


293
922
3013.427
19.9687




401
3472.374
5.7857
2.11


294
922
3013.427
19.9687




679
1841.875
3.0690
7.26


295
922
3013.427
19.9687




777
1263.952
2.1060
1.96


296
922
3013.427
19.9687




794
1165.725
1.9424
1.57


297
922
3013.427
19.9687




799
1137.940
1.8961
2.13


298
922
3013.427
19.9687




804
1104.456
1.8403
3.32


299
922
3013.427
19.9687




815
1043.070
1.7380
1.92


300
922
3013.427
19.9687




826
975.688
1.6257
3.29


301
922
3013.427
19.9687




847
852.676
1.4207
19.79


302
922
3013.427
19.9687




863
759.553
1.2656
3.39


303
922
3013.427
19.9687




869
726.647
1.2108
19.23


304
922
3013.427
19.9687




881
656.158
1.0933
72.09


305
929
2803.190
18.5756




308
4021.188
6.7002
1.12


306
929
2803.190
18.5756




823
997.155
1.6615
1.03


307
929
2803.190
18.5756




847
852.409
1.4203
2.46


308
929
2803.190
18.5756




864
756.417
1.2604
2.78


309
929
2803.190
18.5756




869
728.727
1.2142
2.15


310
929
2803.190
18.5756




881
657.345
1.0953
5.08


311
929
2803.190
18.5756




902
531.583
0.8857
1.25


312
929
2803.190
18.5756




911
478.022
0.7965
4.88


313
929
2803.190
18.5756




913
466.494
0.7773
5.42


314
936
2611.793
17.3073




777
1266.005
2.1094
1.92


315
936
2611.793
17.3073




783
1228.491
2.0469
2.67


316
936
2611.793
17.3073




786
1211.785
2.0191
2.98


317
936
2611.793
17.3073




819
1018.301
1.6967
5.90


318
936
2611.793
17.3073




822
1001.860
1.6693
7.01


319
936
2611.793
17.3073




864
757.418
1.2620
6.44


320
936
2611.793
17.3073




869
725.753
1.2093
1.48


321
936
2611.793
17.3073




872
707.264
1.1785
1.56


322
936
2611.793
17.3073




881
654.782
1.0910
17.83


323
936
2611.793
17.3073




902
531.793
0.8861
19.86


324
936
2611.793
17.3073




909
490.194
0.8168
2.94


325
936
2611.793
17.3073




920
427.649
0.7126
1.52


326
940
2489.586
16.4974




662
1941.819
3.2355
2.46


327
940
2489.586
16.4974




664
1929.172
3.2144
1.76


328
940
2489.586
16.4974




822
1001.010
1.6679
1.20


329
940
2489.586
16.4974




864
757.021
1.2614
66.00


330
940
2489.586
16.4974




871
712.622
1.1874
10.06


331
940
2489.586
16.4974




880
660.618
1.1007
2.75


332
940
2489.586
16.4974




885
632.387
1.0537
18.76


333
940
2489.586
16.4974




891
594.244
0.9901
3.19


334
940
2489.586
16.4974




899
548.142
0.9133
1.83


335
940
2489.586
16.4974




911
478.615
0.7975
8.03


336
940
2489.586
16.4974




913
466.429
0.7772
10.10


337
940
2489.586
16.4974




920
426.462
0.7106
1.57


338
945
2348.374
15.5617




383
3576.612
5.9594
2.25


339
945
2348.374
15.5617




402
3464.839
5.7732
1.77


340
945
2348.374
15.5617




769
1311.156
2.1847
9.92


341
945
2348.374
15.5617




783
1233.208
2.0548
4.90


342
945
2348.374
15.5617




791
1184.039
1.9729
8.41


343
945
2348.374
15.5617




804
1106.429
1.8436
5.06


344
945
2348.374
15.5617




819
1018.365
1.6968
3.91


345
945
2348.374
15.5617




822
1001.834
1.6693
5.15


346
945
2348.374
15.5617




864
756.741
1.2609
6.60


347
945
2348.374
15.5617




872
710.417
1.1837
1.20


348
945
2348.374
15.5617




881
655.039
1.0914
2.79


349
945
2348.374
15.5617




885
632.486
1.0539
2.24


350
945
2348.374
15.5617




899
550.147
0.9167
19.61


351
945
2348.374
15.5617




906
510.518
0.8506
3.78


352
945
2348.374
15.5617




911
480.301
0.8003
8.74


353
945
2348.374
15.5617




914
464.362
0.7737
8.49


354
945
2348.374
15.5617




920
426.538
0.7107
18.76
















TABLE 5.5







Chemical Shift of the Proton NMR of compound Y of Xanthoceras


Sorbifolia Extract










FREQUENCY












#
ADDRESS
[Hz]
[PPM]
INTENSITY














1
5031.9
5309.524
8.8468
0.46


2
5430.0
5222.264
8.7015
134.66


3
5840.9
5132.173
8.5513
0.48


4
8204.9
4613.901
7.6878
0.26


5
8568.1
4534.263
7.5551
88.28


6
8943.7
4451.919
7.4179
0.33


7
9209.7
4393.602
7.3207
0.80


8
9575.2
4313.488
7.1872
180.00


9
9952.1
4230.846
7.0495
0.87


10
10277.2
4159.585
6.9308
1.02


11
10886.8
4025.922
6.7081
5.68


12
10933.6
4015.677
6.6910
6.19


13
11939.8
3795.084
6.3235
6.26


14
11986.7
3784.803
6.3063
5.85


15
12576.0
3655.587
6.0910
1.47


16
12728.0
3622.280
6.0355
6.92


17
12880.5
3588.844
5.9798
1.87


18
12914.2
3581.450
5.9675
4.48


19
12946.5
3574.366
5.9557
4.59


20
12979.5
3567.130
5.9436
2.00


21
13382.6
3478.754
5.7964
2.41


22
13415.6
3471.539
5.7844
5.03


23
13447.3
3464.568
5.7727
5.15


24
13479.1
3457.598
5.7611
2.66


25
14218.8
3295.432
5.4909
14.00


26
14655.9
3199.603
5.3312
28.97


27
14691.2
3191.875
5.3184
27.63


28
15715.3
2967.359
4.9443
5.64


29
15826.3
2943.028
4.9037
4.82


30
15860.6
2935.504
4.8912
5.15


31
16047.0
2894.632
4.8231
4.20


32
16149.6
2872.131
4.7856
3.79


33
16171.9
2867.242
4.7775
4.42


34
16774.0
2735.249
4.5575
5.92


35
16856.4
2717.187
4.5274
4.26


36
16883.5
2711.235
4.5175
4.56


37
16903.5
2706.854
4.5102
5.89


38
16930.6
2700.914
4.5003
4.68


39
17021.2
2681.046
4.4672
2.99


40
17056.9
2673.228
4.4542
8.41


41
17099.1
2663.974
4.4388
8.87


42
17135.6
2655.976
4.4254
8.11


43
17200.4
2641.771
4.4018
1.88


44
17232.6
2634.706
4.3900
2.90


45
17271.0
2626.288
4.3760
4.33


46
17308.8
2618.001
4.3622
2.85


47
17401.8
2597.607
4.3282
0.83


48
17459.1
2585.043
4.3073
3.41


49
17512.6
2573.328
4.2877
4.04


50
17596.4
2554.947
4.2571
0.89


51
17628.2
2547.968
4.2455
0.80


52
17666.8
2539.518
4.2314
1.30


53
17723.0
2527.184
4.2109
6.71


54
17745.9
2522.172
4.2025
7.98


55
17791.2
2512.248
4.1860
2.28


56
17874.1
2494.071
4.1557
2.90


57
17895.0
2489.477
4.1480
3.09


58
17927.1
2482.454
4.1363
2.64


59
17948.8
2477.683
4.1284
2.51


60
18034.1
2458.993
4.0972
3.27


61
18064.6
2452.313
4.0861
2.96


62
18076.5
2449.702
4.0817
2.96


63
18118.3
2440.538
4.0665
0.72


64
18156.4
2432.180
4.0526
0.89


65
18196.3
2423.432
4.0380
0.44


66
18432.1
2371.735
3.9518
2.34


67
18459.7
2365.684
3.9418
4.30


68
18487.4
2359.600
3.9316
2.24


69
18962.4
2255.466
3.7581
3.86


70
19011.0
2244.816
3.7404
4.38


71
19669.3
2100.485
3.4999
4.39


72
19717.6
2089.898
3.4822
4.16


73
20365.2
1947.920
3.2457
2.42


74
20401.1
1940.054
3.2326
2.35


75
20418.9
1936.161
3.2261
2.38


76
20751.3
1863.290
3.1047
0.70


77
20815.8
1849.152
3.0811
3.20


78
20857.9
1839.919
3.0657
11.24


79
20924.8
1825.236
3.0412
1.13


80
23431.3
1275.730
2.1257
3.95


81
23491.3
1262.573
2.1037
5.36


82
23538.7
1252.187
2.0864
15.56


83
23570.5
1245.203
2.0748
15.89


84
23696.1
1217.673
2.0289
2.98


85
23770.1
1201.463
2.0019
27.17


86
23827.6
1188.849
1.9809
1.36


87
23887.4
1175.734
1.9590
15.22


88
23919.5
1168.700
1.9473
15.28


89
24010.2
1148.811
1.9142
0.49


90
24104.2
1128.207
1.8798
1.77


91
24197.7
1107.716
1.8457
28.22


92
24263.8
1093.228
1.8216
3.27


93
24321.8
1080.503
1.8004
2.93


94
24373.5
1069.166
1.7815
2.83


95
24497.0
1042.094
1.7364
26.10


96
24598.4
1019.871
1.6993
3.17


97
24626.1
1013.793
1.6892
3.14


98
24676.1
1002.826
1.6709
1.84


99
24931.2
946.900
1.5777
2.14


100
24983.0
935.533
1.5588
3.02


101
25226.8
882.103
1.4698
0.25


102
25370.4
850.608
1.4173
4.11


103
25412.0
841.492
1.4021
4.88


104
25499.7
822.270
1.3701
4.07


105
25556.8
809.746
1.3492
2.62


106
25639.9
791.527
1.3189
31.95


107
25717.9
774.418
1.2904
2.84


108
25790.4
758.539
1.2639
22.85


109
26011.7
710.018
1.1830
4.66


110
26070.7
697.082
1.1615
24.95


111
26249.2
657.953
1.0963
31.39


112
26536.4
594.981
0.9914
25.26


113
26610.9
578.657
0.9642
0.97


114
26914.0
512.196
0.8534
2.04


115
27012.2
490.676
0.8176
25.88


116
27118.1
467.463
0.7789
3.69


117
27226.4
443.715
0.7393
1.07


118
28513.4
161.554
0.2692
0.89


119
28539.8
155.777
0.2596
1.54
















TABLE 6.2







HMQC of Y1 peaklist












ADDRESS

FREQUENCY













row
[Hz] F1
[PPM] F1



#
col
[Hz] F2
[PPM] F2
INTENSITY














1
315
16740.193
110.9281




375
3624.445
6.0391
30.72


2
352
15855.943
105.0686



490
2950.420
4.9161
20.34


3
450
13537.575
89.7061



490
2950.420
4.9161
20.34


4
479
12863.844
85.2416



476
3032.063
5.0521
12.42


5
479
12863.844
85.2416



500
2892.360
4.8193
17.76


6
491
12577.806
83.3462



487
2969.087
4.9472
25.72


7
522
11857.070
78.5703



545
2627.024
4.3772
13.55


8
529
11685.553
77.4337



504
2870.087
4.7822
16.93


9
535
11537.104
76.4500



589
2370.150
3.9492
15.06


10
550
11191.633
74.1608



421
3355.320
5.5907
12.94


11
550
11191.633
74.1608



423
3342.807
5.5699
15.14


12
569
10733.316
71.1238



410
3421.295
5.7006
24.93


13
579
10497.793
69.5631



526
2740.480
4.5662
26.50


14
586
10332.951
68.4708



537
2674.056
4.4556
14.53


15
586
10332.951
68.4708



596
2330.159
3.8826
11.00


16
617
9621.364
63.7555



643
2051.886
3.4189
12.23


17
630
9299.908
61.6254



529
2718.381
4.5294
13.25


18
630
9299.908
61.6254



539
2665.365
4.4411
14.42


19
669
8391.979
55.6090



529
2718.381
4.5294
13.25


20
669
8391.979
55.6090



539
2665.365
4.4411
14.42


21
720
7193.972
47.6705



682
1821.991
3.0358
10.83


22
725
7055.427
46.7524



820
1015.280
1.6917
8.84


23
770
5994.782
39.7241



682
1822.241
3.0363
21.78


24
778
5810.328
38.5019



682
1822.241
3.0363
21.78


25
803
5229.784
34.6549



836
918.974
1.5312
9.20


26
813
4978.483
32.9897



866
743.550
1.2389
9.04


27
833
4517.225
29.9332



847
852.860
1.4211
64.24


28
847
4185.850
27.7373



861
773.417
1.2887
74.68


29
850
4113.891
27.2605



806
1096.582
1.8271
80.21


30
850
4113.891
27.2605



861
773.392
1.2886
31.46


31
885
3291.884
21.8135



756
1389.088
2.3145
100.00


32
893
3110.641
20.6125



796
1153.093
1.9213
89.09


33
893
3110.641
20.6125



803
1110.425
1.8502
50.66


34
897
2998.505
19.8694



803
1111.101
1.8513
45.64


35
897
2998.505
19.8694



853
820.195
1.3666
78.07


36
919
2497.783
16.5514



874
699.217
1.1651
72.04


37
919
2497.783
16.5514



910
483.491
0.8056
37.18


38
926
2336.725
15.4842



794
1165.059
1.9412
17.70


39
926
2336.725
15.4842



910
484.392
0.8071
31.53
















TABLE 6.2A







2D NMR (HMQC) data of Y1












Assignment
w1
w2
Data Height
















 1C-H
110.904
6.038
24704052



 2C-H
105.023
4.914
15964054



 3C-H
104.615
5.339
11824459



 4C-H
89.823
3.243
7951700



 5C-H
89.712
3.222
7911944



 6C-H
85.528
5.038
10494958



 7C-H
85.417
5.054
8839478



 8C-H
85.338
4.831
10297378



 9C-H
85.248
4.817
12857784



10C-H
83.397
4.948
19288902



11C-H
78.655
4.381
7900396



12C-H
78.602
4.366
8972991



13C-H
77.420
4.784
13438428



14C-H
77.007
4.497
7206874



15C-H
76.570
3.951
12216028



16C-H
74.994
4.092
11486882



17C-H
74.790
4.106
12904558



18C-H
74.220
5.593
10130728



19C-H
74.062
5.574
9532875



20C-H
73.856
6.173
11098625



21C-H
73.802
6.156
10393206



22C-H
73.350
4.468
11007188



23C-H
73.277
4.446
7281630



24C-H
71.577
4.453
8645994



25C-H
71.219
5.701
24595648



26C-H
70.611
3.615
9076031



27C-H
70.067
4.256
9261139



28C-H
69.616
4.567
18994736



29C-H
68.771
3.895
8451744



30C-H
68.543
4.455
12573076



31C-H
63.679
3.604
8239119



32C-H
63.781
3.415
7458621



33C-H
63.862
3.393
7841054



34C-H
62.192
4.312
10795595



35C-H
62.172
4.287
8408334



36C-H
62.266
4.162
7944312



37C-H
62.071
4.131
10031945



38C-H
61.811
4.529
12476046



39C-H
61.793
4.440
11880108



40C-H
61.705
4.463
8055856



41C-H
47.663
3.034
7772454



42C-H
46.740
1.693
8583785



43C-H
39.795
3.034
18038864



44C-H
38.453
1.337
7912356



45C-H
34.666
1.567
7317314



46C-H
32.884
1.264
7887187



47C-H
29.954
1.421
58253544



48C-H
29.632
1.242
16326471



49C-H
29.318
1.117
9248448



50C-H
27.723
1.292
47380240



51C-H
27.251
1.831
59260700



52C-H
21.835
2.317
68135008



53C-H
20.604
1.919
59543524



54C-H
20.377
1.852
66987324



55C-H
19.850
1.369
68067232



56C-H
16.547
1.167
54857956



57C-H
16.223
0.807
25159514



58C-H
15.890
2.028
14256620



59C-H
15.674
1.978
12750140



60C-H
15.693
1.940
10562185



61C-H
15.378
0.758
16911768



62C-H
15.149
0.799
12756197



63C-H
138.670
5.985
8925989



64C-H
137.837
5.881
6871625



65C-H
123.420
5.365
15877691

















TABLE 6.3







2D NMR (HMBC) data of Y1 peaklist












ADDRESS

FREQUENCY













row
[Hz] F1
[PPM] F1



#
col
[Hz] F2
[PPM] F2
INTENSITY














1
148
25871.928
171.4391




362
3702.490
6.1692
11.03


2
148
25871.928
171.4391



756
1390.051
2.3161
93.44


3
169
25232.139
167.1995



796
1153.459
1.9219
28.04


4
169
25232.139
167.1995



803
1112.088
1.8530
29.81


5
293
21555.236
142.8347



806
1097.351
1.8284
25.74


6
314
20935.686
138.7293



786
1215.627
2.0255
33.12


7
314
20935.686
138.7293



796
1153.008
1.9212
46.88


8
318
20813.068
137.9168



786
1213.951
2.0227
9.66


9
318
20813.068
137.9168



792
1175.438
1.9585
29.47


10
318
20813.068
137.9168



803
1110.584
1.8505
46.19


11
369
19321.684
128.0342



785
1218.100
2.0296
13.34


12
369
19321.684
128.0342



792
1175.050
1.9579
28.29


13
369
19321.684
128.0342



796
1153.691
1.9223
26.99


14
369
19321.684
128.0342



803
1111.953
1.8528
65.83


15
486
15874.223
105.1898



477
3026.433
5.0427
16.48


16
486
15874.223
105.1898



545
2628.184
4.3791
9.96


17
486
15874.223
105.1898



557
2559.578
4.2648
10.46


18
565
13533.447
89.6787



491
2942.427
4.9027
10.78


19
565
13533.447
89.6787



861
774.469
1.2904
41.26


20
565
13533.447
89.6787



874
696.834
1.1611
36.06


21
587
12903.256
85.5028



362
3702.844
6.1698
20.78


22
587
12903.256
85.5028



375
3625.743
6.0413
12.97


23
587
12903.256
85.5028



847
852.970
1.4212
35.15


24
587
12903.256
85.5028



853
821.094
1.3681
30.07


25
622
11874.164
78.6836



447
3202.899
5.3367
9.13


26
622
11874.164
78.6836



563
2519.490
4.1980
9.75


27
647
11136.594
73.7961



476
3029.853
5.0484
19.44


28
647
11136.594
73.7961



625
2159.425
3.5981
9.88


29
660
10736.229
71.1431



873
702.447
1.1704
30.15


30
668
10508.083
69.6313



873
702.134
1.1699
11.82


31
673
10374.060
68.7432



873
702.539
1.1706
41.29


32
709
9310.579
61.6961



588
2374.061
3.9557
9.48


33
740
8380.918
55.5357



861
775.074
1.2914
42.71


34
740
8380.918
55.5357



874
696.643
1.1608
30.33


35
740
8380.918
55.5357



913
469.072
0.7816
21.32


36
781
7183.864
47.6035



847
853.277
1.4217
43.72


37
781
7183.864
47.6035



853
821.688
1.3691
35.29


38
786
7044.034
46.6769



848
851.746
1.4192
9.09


39
786
7044.034
46.6769



880
661.983
1.1030
8.36


40
786
7044.034
46.6769



913
469.396
0.7821
27.00


41
813
6236.901
41.3285



806
1097.285
1.8283
38.52


42
813
6236.901
41.3285



910
485.026
0.8082
43.99


43
822
5962.607
39.5109



806
1097.624
1.8289
32.36


44
822
5962.607
39.5109



861
774.567
1.2906
41.54


45
822
5962.607
39.5109



874
696.338
1.1603
63.83


46
822
5962.607
39.5109



910
484.561
0.8074
32.10


47
828
5792.666
38.3848



853
821.024
1.3680
9.58


48
828
5792.666
38.3848



913
469.203
0.7818
39.07


49
832
5676.145
37.6127



847
852.643
1.4207
42.80


50
832
5676.145
37.6127



853
821.900
1.3695
53.76


51
832
5676.145
37.6127



912
471.157
0.7851
10.57


52
837
5531.661
36.6553



853
822.175
1.3699
8.78


53
837
5531.661
36.6553



913
468.520
0.7807
45.56


54
847
5226.124
34.6307



805
1099.040
1.8312
27.35


55
856
4961.126
32.8747



910
484.632
0.8075
32.47


56
871
4515.250
29.9201



477
3025.946
5.0419
7.66


57
871
4515.250
29.9201



837
913.407
1.5219
8.91


58
871
4515.250
29.9201



853
821.483
1.3688
52.28


59
883
4184.067
27.7255



795
1160.819
1.9342
8.46


60
883
4184.067
27.7255



850
836.992
1.3946
11.68


61
883
4184.067
27.7255



874
696.591
1.1607
77.80


62
913
3294.604
21.8315



745
1453.362
2.4216
16.90


63
913
3294.604
21.8315



767
1324.146
2.2063
15.41


64
923
2998.099
19.8668



477
3025.408
5.0410
12.57


65
923
2998.099
19.8668



683
1819.240
3.0313
9.99


66
923
2998.099
19.8668



842
883.190
1.4716
18.02


67
923
2998.099
19.8668



847
853.156
1.4215
51.49


68
923
2998.099
19.8668



864
757.915
1.2629
16.19


69
923
2998.099
19.8668



879
666.328
1.1103
10.30


70
940
2480.249
16.4352



861
775.278
1.2918
42.66


71
940
2480.249
16.4352



884
636.584
1.0607
14.60


72
940
2480.249
16.4352



900
544.736
0.9077
12.91


73
940
2480.249
16.4352



921
420.806
0.7012
12.28


74
946
2313.568
15.3307



902
531.201
0.8851
14.80


75
946
2313.568
15.3307



923
407.732
0.6794
12.79
















TABLE 6.4







2D NMR (COSY) data of Y1.












Assignment
w1
w2
Data Height
















 1H-H
6.704
6.322
9224233



 2H-H
6.678
6.372
9898637



 3H-H
6.419
4.456
20015776



 4H-H
6.368
6.681
11333652



 5H-H
6.320
6.706
11307411



 6H-H
6.171
5.044
134583264



 7H-H
6.043
4.952
12934876



 8H-H
5.980
2.030
80531096



 9H-H
5.980
2.021
105609248



 10H-H
5.881
1.963
81350000



 11H-H
5.881
1.952
75881512



 12H-H
5.701
5.700
215204304



 13H-H
5.700
5.590
18340750



 14H-H
5.699
5.575
15251625



 15H-H
5.582
5.701
24882492



 16H-H
5.583
4.930
32200604



 17H-H
5.583
4.269
54166860



 18H-H
5.582
4.260
100437000



 19H-H
5.582
4.251
50904084



 20H-H
5.335
4.478
54908116



 21H-H
5.335
4.462
48000284



 22H-H
5.334
4.100
20768318



 23H-H
5.048
6.174
89161792



 24H-H
4.949
4.792
11766272



 25H-H
4.950
4.782
9033592



 26H-H
4.930
5.583
17147768



 27H-H
4.929
4.269
30517720



 28H-H
4.927
4.251
28975240



 29H-H
4.901
4.390
27806448



 30H-H
4.900
4.373
34077108



 31H-H
4.831
4.790
99849200



 32H-H
4.820
4.310
7275091



 33H-H
4.819
4.296
7797132



 34H-H
4.822
4.157
13857122



 35H-H
4.821
4.138
18211468



 36H-H
4.794
4.953
30630578



 37H-H
4.787
4.827
86722272



 38H-H
4.568
4.109
17535982



 39H-H
4.567
4.093
15653847



 40H-H
4.542
4.466
32239438



 41H-H
4.522
4.453
145233152



 42H-H
4.517
4.205
7769614



 43H-H
4.523
3.952
73572400



 44H-H
4.507
4.436
11835048



 45H-H
4.468
5.337
79724560



 46H-H
4.468
4.099
83781344



 47H-H
4.461
3.954
44029948



 48H-H
4.454
6.528
9453154



 49H-H
4.454
6.418
79266688



 50H-H
4.450
4.381
13566321



 51H-H
4.449
4.321
8581714



 52H-H
4.444
4.214
29697092



 53H-H
4.445
4.205
23505830



 54H-H
4.443
4.195
23169768



 55H-H
4.382
4.902
75968808



 56H-H
4.380
4.214
34194940



 57H-H
4.379
4.204
34462264



 58H-H
4.380
4.195
34413552



 59H-H
4.302
4.823
16076232



 60H-H
4.302
4.148
221634448



 61H-H
4.262
5.582
80620088



 62H-H
4.261
4.930
59383108



 63H-H
4.203
4.900
12911051



 64H-H
4.204
4.433
20195584



 65H-H
4.150
4.832
17371252



 66H-H
4.153
4.819
18323128



 67H-H
4.149
4.305
216562864



 68H-H
4.099
5.337
25030224



 69H-H
4.100
4.571
36121208



 70H-H
4.101
4.472
68659520



 71H-H
4.095
4.460
36864516



 72H-H
4.028
4.468
7964866



 73H-H
4.028
4.458
8388422



 74H-H
3.961
4.463
41897776



 75H-H
3.953
4.539
34935532



 76H-H
3.952
4.523
50083884



 77H-H
3.953
4.447
24637258



 78H-H
3.893
1.162
59572844



 79H-H
3.887
1.175
95668312



 80H-H
3.766
3.516
11119055



 81H-H
3.744
3.494
6884830



 82H-H
3.598
3.413
146852352



 83H-H
3.598
3.404
128194976



 84H-H
3.517
3.767
10865892



 85H-H
3.508
3.759
9535602



 86H-H
3.480
3.741
8054603



 87H-H
3.475
3.734
6268863



 88H-H
3.409
3.599
107289744



 89H-H
3.406
3.592
110786000



 90H-H
3.229
2.109
12003553



 91H-H
2.110
1.838
8593652



 92H-H
2.107
1.822
11474128



 93H-H
2.107
1.811
10279236



 94H-H
2.105
1.794
8116300



 95H-H
2.029
5.992
47626580



 96H-H
2.027
5.984
47768532



 97H-H
2.026
5.972
51874608



 98H-H
2.030
2.118
7536440



 99H-H
2.027
1.919
89540200



100H-H
2.024
5.964
41580316



101H-H
1.959
5.891
49355596



102H-H
1.955
5.882
46234528



103H-H
1.956
5.870
51174608



104H-H
1.954
1.848
77092128



105H-H
1.923
2.030
54674196



106H-H
1.917
2.020
54160620



107H-H
1.848
1.958
62600468



108H-H
1.834
1.756
16637570



109H-H
1.829
1.732
26336268



110H-H
1.828
1.711
15651960



111H-H
1.835
1.677
25025304



112H-H
1.830
1.553
106561752



113H-H
1.828
1.536
92597192



114H-H
1.820
3.231
15325426



115H-H
1.819
3.220
13169861



116H-H
1.818
2.114
14925469



117H-H
1.818
2.106
16020545



118H-H
1.742
1.848
24286072



119H-H
1.745
1.827
35995120



120H-H
1.669
1.827
14851102



121H-H
1.546
1.834
96039856



122H-H
1.543
1.819
72780528



123H-H
1.545
1.270
7218780



124H-H
1.546
1.255
36723868



125H-H
1.544
1.241
22604678



126H-H
1.476
1.271
6846019



127H-H
1.477
1.255
10952480



128H-H
1.471
1.242
8182750



129H-H
1.474
0.727
7531610



130H-H
1.348
0.812
23199942



131H-H
1.348
0.796
27081584



132H-H
1.344
0.784
22663138



133H-H
1.260
1.540
12084268



134H-H
1.263
1.489
14416738



135H-H
1.263
1.474
17048864



136H-H
1.267
0.736
16594936



137H-H
1.266
0.719
16949332



138H-H
1.252
1.558
13171360



139H-H
1.250
1.525
19080828



140H-H
1.237
0.837
11506187



141H-H
1.167
3.901
86146744



142H-H
1.167
3.877
80709560



143H-H
0.840
1.234
8462904



144H-H
0.798
1.351
39178912



145H-H
0.798
1.342
32789168



146H-H
0.730
1.482
11378742



147H-H
0.731
1.270
11791943



148H-H
0.729
1.255
15935769



149H-H
4.460
4.531
92968608



150H-H
4.203
4.382
45014720



151H-H
4.203
4.373
32816148



152H-H
1.795
1.675
4628933

















TABLE 6.5







Proton NMR chemical shift data of Y1










FREQUENCY












#
ADDRESS
[Hz]
[PPM]
INTENSITY














1
5803.6
5312.007
8.8510
1.05


2
6201.8
5224.701
8.7055
180.00


3
6517.2
5155.558
8.5903
0.09


4
6612.7
5134.626
8.5554
1.05


5
8992.9
4612.799
7.6859
0.41


6
9356.8
4533.029
7.5530
62.60


7
9733.1
4450.514
7.4155
0.40


8
9952.7
4402.383
7.3353
0.13


9
9996.2
4392.832
7.3194
0.90


10
10361.5
4312.746
7.1860
134.51


11
10641.7
4251.327
7.0837
0.05


12
10739.1
4229.980
7.0481
0.87


13
12483.6
3847.521
6.4108
0.10


14
12504.0
3843.034
6.4033
0.10


15
13117.1
3708.630
6.1794
0.31


16
13161.7
3698.843
6.1631
0.31


17
13503.5
3623.905
6.0382
0.38


18
13617.2
3598.988
5.9967
0.08


19
13649.9
3591.811
5.9848
0.19


20
13679.2
3585.404
5.9741
0.18


21
13712.9
3577.998
5.9617
0.08


22
13886.8
3539.884
5.8982
0.09


23
13920.0
3532.594
5.8861
0.19


24
13952.5
3525.479
5.8742
0.17


25
13983.6
3518.658
5.8629
0.06


26
14413.7
3424.364
5.7058
0.36


27
14428.7
3421.080
5.7003
0.36


28
14720.0
3357.206
5.5939
0.19


29
14735.6
3353.801
5.5882
0.18


30
14766.4
3347.042
5.5769
0.20


31
14781.9
3343.652
5.5713
0.17


32
15028.7
3289.533
5.4811
0.05


33
15343.4
3220.543
5.3661
0.33


34
15404.6
3207.131
5.3438
0.25


35
15439.6
3199.461
5.3310
0.25


36
16193.8
3034.111
5.0555
0.14


37
16238.8
3024.240
5.0391
0.15


38
16381.2
2993.014
4.9870
0.17


39
16518.3
2962.967
4.9370
0.12


40
16553.7
2955.209
4.9240
0.12


41
16599.3
2945.198
4.9074
0.08


42
16633.5
2937.700
4.8949
0.08


43
16843.2
2891.736
4.8183
0.18


44
16905.3
2878.117
4.7956
0.17


45
16927.0
2873.370
4.7877
0.19


46
17523.6
2742.566
4.5697
0.33


47
17586.3
2728.818
4.5468
0.18


48
17617.6
2721.965
4.5354
0.23


49
17635.4
2718.066
4.5289
0.31


50
17665.9
2711.371
4.5177
0.32


51
17716.2
2700.349
4.4994
0.21


52
17760.5
2690.632
4.4832
0.20


53
17810.9
2679.588
4.4648
0.55


54
17835.7
2674.141
4.4557
0.72


55
17857.9
2669.267
4.4476
0.54


56
18007.8
2636.409
4.3928
0.17


57
18046.2
2627.986
4.3788
0.26


58
18083.2
2619.886
4.3653
0.19


59
18189.9
2596.493
4.3263
0.11


60
18233.6
2586.912
4.3104
0.27


61
18277.8
2577.209
4.2942
0.31


62
18331.5
2565.443
4.2746
0.21


63
18367.6
2557.542
4.2614
0.23


64
18412.4
2547.721
4.2451
0.19


65
18458.8
2537.540
4.2281
0.10


66
18491.9
2530.283
4.2160
0.21


67
18532.6
2521.359
4.2011
0.28


68
18567.2
2513.770
4.1885
0.14


69
18645.4
2496.625
4.1599
0.25


70
18666.5
2491.997
4.1522
0.25


71
18699.9
2484.686
4.1400
0.24


72
18721.5
2479.943
4.1321
0.19


73
18778.7
2467.413
4.1113
0.20


74
18792.8
2464.305
4.1061
0.20


75
18821.9
2457.939
4.0955
0.19


76
18835.7
2454.909
4.0904
0.18


77
19186.2
2378.063
3.9624
0.17


78
19213.7
2372.029
3.9523
0.29


79
19239.6
2366.350
3.9429
0.16


80
19338.7
2344.635
3.9067
0.08


81
19366.7
2338.490
3.8964
0.22


82
19396.9
2331.872
3.8854
0.21


83
19428.2
2325.007
3.8740
0.06


84
20015.2
2196.324
3.6596
0.05


85
20048.6
2188.994
3.6474
0.08


86
20131.1
2170.900
3.6172
0.13


87
20161.7
2164.211
3.6061
0.28


88
20208.1
2154.040
3.5891
0.31


89
20679.1
2050.765
3.4170
0.24


90
20727.6
2040.135
3.3993
0.23


91
20782.4
2028.119
3.3793
0.08


92
21179.2
1941.123
3.2343
0.14


93
21213.6
1933.579
3.2218
0.13


94
21586.4
1851.862
3.0856
0.07


95
21649.4
1838.049
3.0626
0.09


96
21715.8
1823.490
3.0383
0.67


97
21747.5
1816.527
3.0267
0.23


98
23153.9
1508.199
2.5130
0.06


99
23693.5
1389.916
2.3159
2.02


100
24231.4
1271.971
2.1194
0.22


101
24282.2
1260.831
2.1008
0.22


102
24341.3
1247.878
2.0792
0.16


103
24413.6
1232.024
2.0528
0.15


104
24464.0
1220.989
2.0344
0.83


105
24468.7
1219.963
2.0327
0.83


106
24496.7
1213.827
2.0225
0.82


107
24540.5
1204.212
2.0065
0.24


108
24614.2
1188.066
1.9796
0.24


109
24658.3
1178.391
1.9635
0.88


110
24686.0
1172.311
1.9533
0.86


111
24690.9
1171.249
1.9516
0.85


112
24774.5
1152.908
1.9210
1.65


113
24824.7
1141.905
1.9027
0.22


114
24966.5
1110.815
1.8509
2.07


115
25021.9
1098.679
1.8306
2.09


116
25154.1
1069.689
1.7823
0.22


117
25213.5
1056.676
1.7607
0.20


118
25280.4
1042.010
1.7362
0.33


119
25347.2
1027.363
1.7118
0.24


120
25396.2
1016.604
1.6939
0.38


121
25591.3
973.839
1.6226
0.16


122
25626.4
966.149
1.6098
0.22


123
25673.7
955.785
1.5926
0.16


124
25759.1
937.061
1.5614
0.45


125
25837.0
919.968
1.5329
0.32


126
25932.6
899.023
1.4980
0.22


127
25995.9
885.130
1.4748
0.28


128
26142.6
852.967
1.4212
1.52


129
26284.2
821.936
1.3695
1.99


130
26355.3
806.354
1.3436
0.49


131
26405.2
795.414
1.3253
0.53


132
26437.0
788.423
1.3137
0.61


133
26495.4
775.620
1.2924
1.83


134
26547.6
764.186
1.2733
0.62


135
26578.6
757.400
1.2620
0.75


136
26635.0
745.018
1.2414
0.66


137
26710.2
728.529
1.2139
0.35


138
26742.2
721.515
1.2022
0.34


139
26774.1
714.532
1.1906
0.38


140
26818.0
704.909
1.1745
1.47


141
26852.6
697.329
1.1619
2.39


142
26939.0
678.382
1.1303
0.28


143
26967.1
672.226
1.1201
0.28


144
26987.0
667.851
1.1128
0.31


145
27035.8
657.144
1.0949
0.25


146
27059.9
651.863
1.0861
0.21


147
27091.3
644.992
1.0747
0.18


148
27116.3
639.514
1.0656
0.13


149
27306.1
597.891
0.9962
0.21


150
27331.8
592.271
0.9869
0.20


151
27362.1
585.622
0.9758
0.18


152
27426.7
571.457
0.9522
0.07


153
27465.2
563.025
0.9381
0.12


154
27550.1
544.395
0.9071
0.06


155
27604.4
532.507
0.8873
0.09


156
27723.7
506.347
0.8437
0.23


157
27778.4
494.349
0.8237
0.48


158
27825.7
483.975
0.8064
1.88


159
27892.5
469.335
0.7820
1.79


160
27999.1
445.968
0.7431
0.28


161
28054.1
433.917
0.7230
0.23


162
28134.1
416.375
0.6938
0.08


163
28228.5
395.678
0.6593
0.07


164
28262.5
388.209
0.6468
0.09
















TABLE 8.2







2D NMR (HMQC) of R1 peaklist.












ADDRESS

FREQUENCY













row
[Hz] F1
[PPM] F1



#
col
[Hz] F2
[PPM] F2
INTENSITY














1
151
20624.836
136.6695




397
3494.332
5.8223
4.88


2
157
20461.906
135.5898



220
4534.469
7.5554
67.68


3
234
18646.779
123.5620



258
4313.193
7.1867
100.00


4
234
18646.779
123.5620



445
3213.863
5.3550
9.18


5
343
16069.902
106.4864



496
2913.367
4.8543
7.58


6
343
16069.902
106.4864



498
2904.202
4.8390
7.61


7
350
15895.188
105.3287



471
3059.891
5.0985
7.99


8
350
15895.188
105.3287



473
3050.642
5.0830
9.11


9
350
15895.188
105.3287



481
3004.427
5.0060
8.79


10
363
15605.384
103.4083



510
2835.541
4.7246
7.96


11
380
15192.972
100.6755



326
3912.206
6.5186
16.30


12
454
13447.552
89.1095



656
1974.610
3.2901
4.35


13
454
13447.552
89.1095



659
1958.017
3.2625
4.45


14
515
12025.116
79.6838



561
2533.049
4.2206
4.85


15
518
11934.442
79.0830



389
3540.938
5.9000
4.52


16
524
11802.268
78.2071



562
2525.183
4.2075
12.80


17
524
11802.268
78.2071



594
2340.880
3.9004
11.92


18
534
11563.501
76.6250



571
2474.883
4.1237
4.43


19
534
11563.501
76.6250



588
2372.543
3.9532
3.52


20
534
11563.501
76.6250



610
2246.165
3.7426
5.94


21
534
11563.501
76.6250



612
2234.362
3.7229
6.60


22
540
11437.312
75.7888



574
2457.347
4.0945
20.96


23
545
11309.472
74.9417



553
2580.628
4.2999
5.29


24
545
11309.472
74.9417



555
2569.264
4.2810
6.15


25
545
11309.472
74.9417



561
2533.966
4.2222
3.23


26
545
11309.472
74.9417



582
2411.457
4.0180
9.38


27
551
11175.793
74.0558



550
2597.570
4.3281
3.74


28
551
11175.793
74.0558



552
2585.925
4.3087
7.04


29
551
11175.793
74.0558



554
2574.515
4.2897
3.84


30
556
11047.378
73.2049



586
2386.166
3.9759
5.90


31
562
10907.159
72.2758



511
2826.203
4.7091
15.78


32
562
10907.159
72.2758



520
2777.020
4.6271
5.35


33
567
10784.600
71.4636



562
2525.038
4.2073
12.61


34
567
10784.600
71.4636



567
2497.664
4.1617
4.33


35
577
10543.526
69.8662



497
2908.530
4.8463
4.00


36
577
10543.526
69.8662



500
2892.560
4.8196
3.60


37
577
10543.526
69.8662



514
2810.206
4.6824
5.98


38
577
10543.526
69.8662



516
2796.413
4.6594
5.96


39
577
10543.526
69.8662



548
2609.404
4.3478
6.72


40
624
9434.661
62.5183



532
2705.029
4.5072
11.62


41
624
9434.661
62.5183



534
2689.727
4.4817
8.01


42
624
9434.661
62.5183



547
2616.577
4.3598
7.54


43
669
8381.180
55.5375



919
431.689
0.7193
4.80


44
669
8381.180
55.5375



922
414.881
0.6913
5.21


45
719
7203.831
47.7358



827
972.057
1.6197
3.32


46
728
6986.542
46.2960



777
1265.417
2.1085
3.85


47
728
6986.542
46.2960



859
784.626
1.3074
3.19


48
728
6986.542
46.2960



862
764.970
1.2746
3.45


49
759
6273.788
41.5729



724
1575.188
2.6246
3.64


50
759
6273.788
41.5729



728
1556.033
2.5927
3.54


51
817
4904.791
32.5014



849
844.868
1.4077
9.21


52
831
4556.851
30.1957



868
732.276
1.2201
40.19


53
846
4212.870
27.9164



870
719.170
1.1983
45.73


54
858
3932.408
26.0579



865
749.675
1.2491
45.69


55
893
3102.798
20.5605



808
1085.904
1.8094
37.60


56
900
2935.044
19.4489



864
755.857
1.2594
42.87


57
905
2821.322
18.6953



808
1086.381
1.8102
25.73


58
918
2523.121
16.7193



883
644.770
1.0743
36.59


59
918
2523.121
16.7193



895
571.319
0.9519
43.39


60
924
2368.333
15.6936



794
1163.335
1.9384
21.68


61
924
2368.333
15.6936



895
573.050
0.9548
45.90
















TABLE 8.3







HMBC R1 peaklist


DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan,


EXPNO = 26, PROCNO = 1


F1PLO = 173.650 ppm, F1PHI = 11.502 ppm,


F2PLO = 6.832 ppm, F2PHI = 0.515 ppm


MI = 5.00 cm, MAXI = 10000.00 cm, PC = 3.000












ADDRESS

FREQUENCY













row
[Hz] F1
[PPM] F1



#
col
[Hz] F2
[PPM] F2
INTENSITY














1
166
25330.719
167.8561




389
3542.372
5.9024
11.00


2
166
25330.719
167.8561



808
1085.572
1.8088
37.94


3
293
21569.176
142.9299



865
750.004
1.2497
39.69


4
325
20624.461
136.6697



795
1160.826
1.9342
54.15


5
325
20624.461
136.6697



808
1085.191
1.8082
67.95


6
366
19424.748
128.7197



795
1161.341
1.9351
54.29


7
366
19424.748
128.7197



808
1085.293
1.8083
100.00


8
479
16074.168
106.5168



587
2382.396
3.9696
17.03


9
485
15911.199
105.4369



582
2411.570
4.0182
13.64


10
495
15610.658
103.4453



559
2544.112
4.2391
7.40


11
495
15610.658
103.4453



574
2457.717
4.0951
7.72


12
509
15196.733
100.7024



311
3999.566
6.6642
10.46


13
509
15196.733
100.7024



341
3825.973
6.3749
10.97


14
568
13447.739
89.1126



497
2908.018
4.8454
13.78


15
568
13447.739
89.1126



870
718.459
1.1971
52.71


16
568
13447.739
89.1126



896
570.271
0.9502
33.62


17
616
12037.521
79.7676



560
2540.187
4.2325
10.39


18
616
12037.521
79.7676



567
2496.423
4.1596
12.77


19
620
11934.375
79.0841



557
2555.670
4.2583
16.69


20
620
11934.375
79.0841



586
2384.236
3.9727
18.17


21
624
11794.329
78.1561



561
2532.465
4.2196
29.22


22
624
11794.329
78.1561



581
2415.675
4.0251
13.90


23
632
11561.078
76.6104



554
2575.363
4.2911
11.27


24
632
11561.078
76.6104



861
774.214
1.2900
8.95


25
632
11561.078
76.6104



864
757.631
1.2624
31.05


26
632
11561.078
76.6104



868
733.017
1.2214
52.22


27
640
11319.147
75.0073



562
2529.498
4.2147
14.09


28
640
11319.147
75.0073



574
2457.979
4.0955
12.03


29
640
11319.147
75.0073



611
2238.440
3.7297
7.35


30
645
11173.489
74.0421



511
2826.331
4.7093
5.08


31
645
11173.489
74.0421



807
1087.742
1.8124
27.89


32
650
11046.945
73.2035



389
3541.020
5.9001
14.57


33
655
10904.058
72.2566



326
3913.536
6.5208
23.27


34
655
10904.058
72.2566



552
2586.768
4.3101
9.96


35
655
10904.058
72.2566



563
2521.903
4.2021
6.06


36
659
10780.204
71.4359



532
2702.900
4.5036
6.93


37
659
10780.204
71.4359



534
2692.127
4.4857
8.19


38
659
10780.204
71.4359



563
2520.533
4.1998
48.21


39
667
10532.287
69.7931



472
3054.495
5.0895
11.46


40
667
10532.287
69.7931



480
3007.639
5.0114
18.84


41
671
10425.301
69.0841



326
3913.495
6.5208
32.61


42
671
10425.301
69.0841



552
2586.054
4.3089
7.93


43
671
10425.301
69.0841



807
1088.313
1.8134
58.15


44
705
9430.492
62.4919



560
2539.835
4.2319
5.37


45
740
8372.175
55.4789



870
718.660
1.1974
38.08


46
740
8372.175
55.4789



895
571.407
0.9521
44.50


47
780
7199.307
47.7068



883
644.628
1.0741
27.47


48
780
7199.307
47.7068



895
572.447
0.9538
31.41


49
788
6981.523
46.2636



864
757.767
1.2626
51.63


50
788
6981.523
46.2636



868
732.960
1.2213
78.63


51
804
6495.941
43.0459



574
2458.027
4.0956
8.82


52
804
6495.941
43.0459



861
774.879
1.2911
8.89


53
804
6495.941
43.0459



867
737.966
1.2296
6.55


54
810
6313.906
41.8396



445
3213.454
5.3543
8.33


55
810
6313.906
41.8396



755
1397.464
2.3285
5.40


56
810
6313.906
41.8396



865
749.894
1.2495
42.24


57
810
6313.906
41.8396



883
644.620
1.0741
52.86


58
814
6189.256
41.0136



865
749.287
1.2485
6.31


59
814
6189.256
41.0136



883
643.362
1.0720
8.62


60
817
6120.849
40.5603



865
749.159
1.2483
15.69


61
817
6120.849
40.5603



883
644.286
1.0735
17.94


62
817
6120.849
40.5603



896
570.189
0.9501
6.27


63
819
6053.888
40.1166



865
749.605
1.2490
43.77


64
819
6053.888
40.1166



883
644.397
1.0737
47.98


65
819
6053.888
40.1166



896
570.067
0.9499
8.91


66
824
5921.430
39.2388



864
754.133
1.2566
5.80


67
824
5921.430
39.2388



870
718.264
1.1968
70.97


68
824
5921.430
39.2388



896
569.449
0.9488
65.30


69
824
5921.430
39.2388



920
427.451
0.7122
6.70


70
824
5921.430
39.2388



922
414.558
0.6907
7.41


71
827
5818.316
38.5556



870
718.161
1.1966
8.71


72
827
5818.316
38.5556



895
572.960
0.9547
35.78


73
829
5749.308
38.0983



896
570.060
0.9498
8.61


74
837
5529.916
36.6444



829
959.437
1.5986
5.22


75
837
5529.916
36.6444



864
755.929
1.2595
12.15


76
837
5529.916
36.6444



868
732.618
1.2207
8.77


77
837
5529.916
36.6444



895
572.369
0.9537
51.81


78
837
5529.916
36.6444



920
427.331
0.7120
5.36


79
837
5529.916
36.6444



922
414.571
0.6908
6.76


80
840
5443.605
36.0725



777
1264.891
2.1076
5.25


81
840
5443.605
36.0725



864
757.333
1.2619
68.88


82
840
5443.605
36.0725



868
732.776
1.2210
83.66


83
840
5443.605
36.0725



895
571.988
0.9531
11.13


84
858
4903.620
32.4942



883
644.423
1.0738
38.16


85
870
4556.862
30.1964



611
2240.976
3.7340
8.07


86
870
4556.862
30.1964



857
794.987
1.3246
18.78


87
870
4556.862
30.1964



864
756.793
1.2610
79.89


88
870
4556.862
30.1964



878
670.310
1.1169
18.43


89
872
4487.079
29.7340



864
756.125
1.2599
16.38


90
874
4422.445
29.3057



863
758.384
1.2636
8.16


91
877
4356.193
28.8667



864
755.084
1.2581
8.79


92
877
4356.193
28.8667



896
569.898
0.9496
6.22


93
882
4211.267
27.9063



860
779.212
1.2983
19.55


94
882
4211.267
27.9063



864
754.574
1.2573
5.59


95
882
4211.267
27.9063



881
654.994
1.0914
19.01


96
882
4211.267
27.9063



896
570.162
0.9500
87.84


97
891
3938.165
26.0966



854
812.119
1.3532
19.50


98
891
3938.165
26.0966



865
751.828
1.2527
10.44


99
891
3938.165
26.0966



876
686.003
1.1430
17.89


100
891
3938.165
26.0966



896
569.852
0.9495
5.20


101
893
3876.630
25.6888



865
749.874
1.2495
40.70


102
893
3876.630
25.6888



876
686.400
1.1437
5.01


103
919
3095.315
20.5114



398
3488.142
5.8120
7.58


104
919
3095.315
20.5114



797
1150.511
1.9170
9.43


105
919
3095.315
20.5114



818
1022.904
1.7044
9.18


106
919
3095.315
20.5114



868
732.855
1.2211
5.53


107
925
2937.158
19.4633



611
2240.910
3.7339
13.27


108
925
2937.158
19.4633



777
1265.381
2.1084
11.35


109
925
2937.158
19.4633



780
1248.250
2.0799
5.80


110
925
2937.158
19.4633



853
818.856
1.3644
23.23


111
925
2937.158
19.4633



868
733.355
1.2219
89.32


112
925
2937.158
19.4633



875
693.473
1.1555
20.36


113
932
2710.756
17.9630



554
2574.880
4.2903
6.54


114
932
2710.756
17.9630



849
843.916
1.4062
5.24


115
932
2710.756
17.9630



868
732.674
1.2208
6.51


116
939
2527.765
16.7504



829
960.874
1.6010
6.70


117
939
2527.765
16.7504



870
718.686
1.1975
83.68


118
939
2527.765
16.7504



885
631.287
1.0519
21.97


119
939
2527.765
16.7504



893
583.038
0.9715
14.30


120
939
2527.765
16.7504



906
506.673
0.8442
22.28


121
939
2527.765
16.7504



920
426.044
0.7099
7.71


122
939
2527.765
16.7504



922
414.883
0.6913
10.74


123
944
2376.340
15.7470



784
1224.570
2.0404
9.28


124
944
2376.340
15.7470



806
1096.659
1.8273
8.54


125
944
2376.340
15.7470



870
718.378
1.1970
6.60


126
944
2376.340
15.7470



885
633.381
1.0554
19.99


127
944
2376.340
15.7470



906
509.452
0.8489
20.05


128
944
2376.340
15.7470



920
426.988
0.7115
8.96


129
944
2376.340
15.7470



922
415.107
0.6917
10.34
















TABLE 8.4







2D NMR (cosy) of R1 peak list












Assignment
w1
w2
Data Height
















1
6.521
6.522
257252096



2
6.522
4.711
7840252



3
6.129
0.959
3905950



4
5.898
5.901
32367934



5
5.902
4.847
10836326



6
5.902
4.258
10138593



7
5.899
4.127
9360363



8
5.906
3.975
16282743



9
5.821
1.810
7215536



10
5.817
5.822
31028110



11
5.817
1.944
43526536



12
5.816
1.929
44815556



13
5.355
5.355
178856592



14
5.094
5.102
109124320



15
5.094
5.085
116649128



16
5.092
4.205
15712139



17
5.093
4.048
38195456



18
5.011
5.021
112612352



19
5.010
5.004
121053544



20
5.008
4.210
14216868



21
5.011
4.021
38155584



22
4.847
5.913
5875469



23
4.845
5.900
4787344



24
4.845
5.889
7478266



25
4.841
4.855
118994368



26
4.836
4.823
47239140



27
4.836
4.369
92117104



28
4.831
4.134
9652155



29
4.834
4.116
7044446



30
4.844
3.987
44245188



31
4.845
3.962
44209660



32
4.798
4.795
12493983



33
4.795
4.621
15346245



34
4.796
1.821
57265952



35
4.796
1.809
75309312



36
4.792
4.312
21750568



37
4.753
4.360
24472454



38
4.725
4.733
72665064



39
4.721
4.719
99191696



40
4.727
4.226
50380324



41
4.709
6.523
22903380



42
4.702
4.629
20594276



43
4.707
4.612
19038358



44
4.706
4.326
11671381



45
4.670
4.688
34985344



46
4.669
4.660
40038520



47
4.669
4.355
94428136



48
4.670
3.958
8587019



49
4.672
3.941
7812038



50
4.620
4.800
8615562



51
4.619
4.711
21326744



52
4.618
4.633
29866052



53
4.619
4.607
44064012



54
4.621
4.312
23669784



55
4.584
4.355
27276566



56
4.566
4.365
23402122



57
4.492
4.513
55339136



58
4.491
4.482
71439344



59
4.496
4.370
164934224



60
4.495
4.360
174062528



61
4.499
3.912
17609992



62
4.498
3.896
17924064



63
4.377
4.349
24720128



64
4.365
4.843
97382152



65
4.364
4.829
127752656



66
4.356
4.680
67867680



67
4.355
4.667
81378376



68
4.361
4.491
211941920



69
4.374
4.393
38442384



70
4.364
4.372
27706436



71
4.358
4.335
31327086



72
4.364
4.137
14876345



73
4.363
4.113
13081764



74
4.355
3.960
10728486



75
4.357
3.940
9552408



76
4.371
3.913
20295026



77
4.372
3.895
17021364



78
4.309
4.799
25905518



79
4.309
4.619
43016120



80
4.306
4.310
56206892



81
4.294
3.742
80581928



82
4.295
3.731
101483432



83
4.280
4.354
9482896



84
4.251
5.915
17374606



85
4.250
5.901
10701927



86
4.251
5.887
17140758



87
4.253
4.259
32444480



88
4.258
4.125
31047850



89
4.257
3.976
5638105



90
4.229
4.730
34642484



91
4.207
5.092
13039638



92
4.209
5.012
11226875



93
4.217
4.179
33544654



94
4.209
4.058
28007252



95
4.207
4.047
46281960



96
4.211
4.020
33876260



97
4.207
4.005
21146824



98
4.210
3.904
80923600



99
4.204
4.365
9066176



100
4.202
4.032
40055832



101
4.167
3.951
18178542



102
4.150
4.209
11484229



103
4.126
5.912
6719130



104
4.122
5.890
7671593



105
4.126
4.378
9045784



106
4.127
4.273
34269312



107
4.119
4.258
32734018



108
4.133
4.246
34046856



109
4.131
4.140
18941800



110
4.116
4.355
11770206



111
4.092
4.096
279326848



112
4.043
5.095
84940104



113
4.035
4.208
50268828



114
4.042
4.183
15655260



115
4.043
4.064
26875002



116
4.019
5.015
77663464



117
4.015
4.241
9036684



118
4.027
4.226
11811450



119
4.016
4.037
19835598



120
3.973
5.915
24059896



121
3.974
5.901
20609464



122
3.973
5.887
22835978



123
3.975
4.853
37923332



124
3.977
3.995
18827896



125
3.949
4.682
5809583



126
3.950
4.371
10668304



127
3.953
4.348
15916606



128
3.949
4.175
18189762



129
3.948
4.161
30027716



130
3.953
4.149
19945408



131
3.940
3.966
14571032



132
3.903
4.505
10048077



133
3.903
4.488
14760281



134
3.903
4.387
19898852



135
3.912
4.342
16459215



136
3.904
4.239
12064081



137
3.907
4.214
108106008



138
3.898
4.364
32756190



139
3.900
4.199
27604272



140
3.901
3.917
43085776



141
3.897
3.888
38415896



142
3.735
4.300
93317560



143
3.736
4.290
98467152



144
3.736
3.748
65327612



145
3.735
3.725
50388764



146
3.270
3.283
17369858



147
3.269
3.267
22139430



148
3.271
2.354
5853844



149
3.271
2.342
7970904



150
3.271
1.912
5107326



151
3.271
1.896
5093256



152
2.602
2.631
5486520



153
2.600
2.598
5094291



154
2.601
2.590
7143968



155
2.602
2.132
14357642



156
2.602
2.109
26185532



157
2.601
2.086
14739034



158
2.603
1.289
14662965



159
2.341
3.287
5980422



160
2.342
3.264
5054491



161
2.334
2.360
5303651



162
2.336
1.932
6500135



163
2.334
1.872
7760766



164
2.325
2.341
13008529



165
2.325
2.315
11101831



166
2.328
1.923
6345431



167
2.326
1.895
9356538



168
2.289
2.294
14262412



169
2.186
2.193
4223454



170
2.103
2.620
30164346



171
2.103
2.598
27444406



172
2.101
2.109
7249900



173
2.105
1.307
22762886



174
2.102
1.300
18897670



175
2.103
1.279
18250726



176
2.070
2.078
4104108



177
1.967
1.969
13308390



178
1.977
1.609
7894784



179
1.956
1.909
11551598



180
1.961
1.874
10608534



181
1.965
1.625
5946444



182
1.931
5.834
39974876



183
1.931
5.824
42157648



184
1.932
5.812
40474008



185
1.931
5.798
30554264



186
1.931
2.320
12384753



187
1.931
1.942
228192416



188
1.931
1.810
59524928



189
1.913
2.339
13306613



190
1.902
2.001
6772636



191
1.895
3.287
9744711



192
1.896
3.264
9357915



193
1.897
2.362
8772959



194
1.886
1.973
16266749



195
1.885
1.885
5269322



196
1.882
1.616
15011650



197
1.883
1.242
8077304



198
1.878
1.219
8767326



199
1.818
5.840
3029832



200
1.805
5.829
3553817



201
1.804
5.808
3418502



202
1.805
4.822
22320414



203
1.805
4.813
19315348



204
1.806
4.797
27156710



205
1.806
4.785
23402710



206
1.806
4.774
22620674



207
1.806
4.322
15274810



208
1.806
4.312
11189881



209
1.806
4.302
17586586



210
1.802
1.943
42426312



211
1.804
1.932
30620986



212
1.804
1.810
1349507072



213
1.776
1.778
8674281



214
1.703
1.710
5954890



215
1.696
1.702
9409535



216
1.613
1.911
8734371



217
1.611
1.635
17314310



218
1.609
1.610
29144662



219
1.609
1.599
16356248



220
1.613
1.032
9463035



221
1.614
1.017
7358422



222
1.613
0.991
9572058



223
1.406
1.303
9782705



224
1.395
1.406
70684776



225
1.393
1.385
22170084



226
1.390
1.293
11202919



227
1.307
1.318
7420988



228
1.285
2.609
28951250



229
1.284
2.133
16431712



230
1.285
2.110
23106420



231
1.283
2.083
14481833



232
1.279
1.407
11364364



233
1.288
1.384
6974150



234
1.286
1.299
18845462



235
1.288
0.712
21876642



236
1.288
0.696
27076318



237
1.251
1.260
768046912



238
1.221
1.927
5903658



239
1.217
1.223
1002405376



240
1.188
1.197
664805376



241
1.168
1.171
16019256



242
1.137
1.145
30540644



243
1.113
1.120
37708240



244
1.070
1.077
775147520



245
1.058
1.058
17850510



246
1.004
2.349
5175155



247
1.003
1.916
6850918



248
1.003
1.892
5963474



249
1.005
1.630
21215516



250
1.005
1.610
23547650



251
1.000
1.008
25525576



252
1.016
0.959
33680632



253
0.985
0.999
11598959



254
0.963
0.985
13491636



255
0.944
0.953
1674528384



256
0.912
0.912
21859108



257
0.872
0.878
12589871



258
0.697
0.716
28731108



259
0.699
0.693
23421302



260
0.694
1.297
7870457



261
0.692
1.289
8126754

















TABLE 8.5







R1-proton-NMR-peaklist










FREQUENCY












#
ADDRESS
[Hz]
[PPM]
INTENSITY














1
13957.6
3913.934
6.5215
20.81


2
15614.0
3550.810
5.9164
7.03


3
15656.8
3541.408
5.9008
14.16


4
15699.7
3532.016
5.8851
7.43


5
15839.6
3501.350
5.8340
2.56


6
15872.2
3494.204
5.8221
6.47


7
15905.1
3486.992
5.8101
6.47


8
15932.8
3480.916
5.8000
2.49


9
17152.3
3213.554
5.3545
12.90


10
17853.4
3059.848
5.0984
17.52


11
17888.8
3052.093
5.0855
18.03


12
18074.9
3011.286
5.0175
16.80


13
18110.2
3003.544
5.0046
17.26


14
18524.3
2912.751
4.8533
15.71


15
18558.4
2905.282
4.8408
20.27


16
18597.3
2896.749
4.8266
12.17


17
18638.6
2887.709
4.8116
5.88


18
18667.2
2881.421
4.8011
6.29


19
18680.9
2878.427
4.7961
6.65


20
18709.4
2872.187
4.7857
5.58


21
18857.7
2839.672
4.7315
13.15


22
18891.0
2832.356
4.7193
15.61


23
18902.5
2829.844
4.7151
13.51


24
18918.1
2826.431
4.7095
13.25


25
18996.6
2809.208
4.6808
8.60


26
19038.5
2800.032
4.6655
9.32


27
19136.8
2778.471
4.6296
7.89


28
19151.7
2775.208
4.6241
6.99


29
19179.0
2769.220
4.6141
8.39


30
19194.1
2765.907
4.6086
7.85


31
19474.8
2704.369
4.5061
16.52


32
19515.8
2695.385
4.4911
14.33


33
19529.0
2692.503
4.4863
20.98


34
19797.2
2633.699
4.3883
10.45


35
19819.7
2628.754
4.3801
17.64


36
19850.7
2621.968
4.3688
26.32


37
19872.3
2617.228
4.3609
27.93


38
19897.2
2611.771
4.3518
17.82


39
19930.6
2604.438
4.3396
11.93


40
19963.7
2597.191
4.3275
8.30


41
20006.6
2587.791
4.3118
14.07


42
20031.7
2582.289
4.3027
11.75


43
20048.8
2578.545
4.2964
8.57


44
20076.5
2572.467
4.2863
11.71


45
20107.9
2565.584
4.2748
7.33


46
20151.6
2556.001
4.2589
14.11


47
20174.3
2551.024
4.2506
6.12


48
20196.3
2546.196
4.2425
13.56


49
20214.9
2542.128
4.2358
16.39


50
20247.5
2534.968
4.2238
26.96


51
20285.9
2526.555
4.2098
39.00


52
20305.4
2522.271
4.2027
24.70


53
20319.5
2519.190
4.1975
23.22


54
20346.4
2513.298
4.1877
7.91


55
20358.9
2510.546
4.1831
7.69


56
20379.8
2505.959
4.1755
8.17


57
20422.0
2496.719
4.1601
10.22


58
20461.6
2488.043
4.1456
5.87


59
20487.5
2482.352
4.1362
7.27


60
20514.7
2476.402
4.1262
7.42


61
20558.8
2466.727
4.1101
7.60


62
20596.2
2458.528
4.0965
31.95


63
20697.7
2436.266
4.0594
8.35


64
20735.2
2428.056
4.0457
13.12


65
20773.1
2419.740
4.0318
13.77


66
20811.2
2411.385
4.0179
12.76


67
20848.0
2403.322
4.0045
7.29


68
20890.7
2393.971
3.9889
7.87


69
20932.7
2384.749
3.9735
10.17


70
20970.3
2376.499
3.9598
12.68


71
21017.7
2366.108
3.9425
6.44


72
21123.5
2342.921
3.9038
16.37


73
21559.5
2247.327
3.7445
11.72


74
21604.4
2237.496
3.7282
11.07


75
22810.2
1973.141
3.2877
6.60


76
22829.6
1968.890
3.2806
6.56


77
22863.1
1961.542
3.2684
6.70


78
22883.0
1957.180
3.2611
6.59


79
24645.4
1570.792
2.6173
4.82


80
24691.2
1560.756
2.6006
5.30


81
25359.9
1414.147
2.3563
5.53


82
25407.3
1403.761
2.3390
10.56


83
25464.1
1391.301
2.3182
6.50


84
25529.5
1376.964
2.2943
2.87


85
25976.1
1279.056
2.1312
4.43


86
26038.8
1265.317
2.1083
9.07


87
26101.1
1251.649
2.0855
4.68


88
26348.4
1197.433
1.9952
2.18


89
26423.1
1181.067
1.9679
6.86


90
26495.7
1165.147
1.9414
40.57


91
26523.1
1159.138
1.9314
35.58


92
26528.4
1157.984
1.9295
36.42


93
26565.0
1149.953
1.9161
14.14


94
26612.0
1139.641
1.8989
12.71


95
26673.0
1126.283
1.8766
9.66


96
26829.5
1091.957
1.8194
48.60


97
26857.0
1085.929
1.8094
113.02


98
27349.4
977.978
1.6295
11.86


99
27403.6
966.099
1.6097
12.07


100
27963.7
843.313
1.4051
22.40


101
28018.6
831.275
1.3851
8.46


102
28254.5
779.546
1.2989
11.85


103
28298.3
769.948
1.2829
12.55


104
28317.6
765.717
1.2759
13.04


105
28356.2
757.269
1.2618
86.45


106
28387.8
750.328
1.2502
81.24


107
28464.0
733.633
1.2224
88.26


108
28533.4
718.414
1.1970
73.80


109
28677.9
686.739
1.1443
3.89


110
28745.9
671.820
1.1194
3.59


111
28867.8
645.098
1.0749
74.04


112
28987.7
618.804
1.0311
4.13


113
29051.5
604.826
1.0078
8.69


114
29106.8
592.703
0.9876
4.79


115
29207.1
570.711
0.9509
114.09


116
29320.8
545.779
0.9094
3.45


117
29410.0
526.225
0.8768
1.79


118
29492.8
508.068
0.8466
1.41


119
29857.9
428.030
0.7132
9.46


120
29911.7
416.233
0.6935
9.30
















TABLE 8.6







13C NMR of R1 peak list


DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan,


EXPNO = 30, PROCNO = 1


176.659 ppm, F2 = 6.843 ppm,


MI = 0.06 cm, MAXI = 10000.00 cm, PC = 1.400










FREQUENCY












#
ADDRESS
[Hz]
[PPM]
INTENSITY














1
5310.9
25331.195
167.8559
0.12


2
8227.3
22634.219
149.9845
1.57


3
8251.8
22611.535
149.8342
11.79


4
8281.1
22584.428
149.6546
12.50


5
8310.4
22557.355
149.4752
12.30


6
9381.7
21566.594
142.9100
0.11


7
10403.1
20622.062
136.6511
0.14


8
10577.3
20460.975
135.5836
1.08


9
10596.8
20442.969
135.4643
5.78


10
10623.6
20418.133
135.2998
6.16


11
10650.4
20393.346
135.1355
6.01


12
11696.9
19425.617
128.7229
0.14


13
12537.9
18647.846
123.5690
2.11


14
12557.4
18629.844
123.4497
10.62


15
12584.3
18604.896
123.2844
11.25


16
12611.3
18580.010
123.1195
10.98


17
13444.2
17809.705
118.0151
0.11


18
14249.2
17065.322
113.0825
0.15


19
15319.0
16075.968
106.5266
0.21


20
15492.3
15915.713
105.4647
0.24


21
15509.0
15900.229
105.3621
0.25


22
15823.2
15609.646
103.4365
0.18


23
16269.0
15197.378
100.7047
0.16


24
18158.1
13450.413
89.1285
0.12


25
19683.7
12039.604
79.7798
0.13


26
19797.3
11934.502
79.0834
0.14


27
19929.2
11812.533
78.2752
0.23


28
19945.5
11797.527
78.1757
0.42


29
20197.3
11564.589
76.6322
0.15


30
20211.9
11551.142
76.5431
0.16


31
20226.2
11537.928
76.4555
0.14


32
20335.9
11436.434
75.7830
0.10


33
20410.2
11367.753
75.3279
0.13


34
20473.6
11309.069
74.9390
0.38


35
20617.2
11176.295
74.0592
0.13


36
20755.1
11048.805
73.2144
0.15


37
20910.0
10905.522
72.2649
0.21


38
21019.3
10804.420
71.5950
0.15


39
21047.5
10778.391
71.4225
0.37


40
21283.2
10560.453
69.9783
0.12


41
21320.7
10525.739
69.7483
0.12


42
21375.2
10475.339
69.4143
0.14


43
21430.8
10423.882
69.0733
0.13


44
22496.2
9438.663
62.5448
0.19


45
22510.2
9425.676
62.4588
0.20


46
23646.3
8375.039
55.4968
0.13


47
24915.8
7201.028
47.7173
0.13


48
25152.5
6982.204
46.2672
0.11


49
25671.3
6502.366
43.0876
0.12


50
25868.3
6320.202
41.8805
0.10


51
25921.2
6271.273
41.5563
0.11


52
26160.1
6050.418
40.0928
0.10


53
26300.3
5920.724
39.2334
0.13


54
26396.6
5831.667
38.6433
0.10


55
26722.2
5530.549
36.6479
0.11


56
26820.5
5439.679
36.0458
0.13


57
27398.1
4905.475
32.5059
0.10


58
27774.8
4557.162
30.1978
0.23


59
28148.7
4211.391
27.9066
0.23


60
28389.5
3988.645
26.4306
0.10


61
28448.2
3934.385
26.0710
0.17


62
28512.9
3874.571
25.6747
0.10


63
28828.2
3583.030
23.7428
0.11


64
29348.8
3101.522
20.5521
0.23


65
29529.3
2934.591
19.4459
0.22


66
29650.2
2822.798
18.7051
0.26


67
29719.5
2758.712
18.2805
0.11


68
29766.2
2715.508
17.9942
0.10


69
29970.4
2526.664
16.7428
0.24


70
29985.5
2512.765
16.6507
0.17


71
30140.7
2369.192
15.6993
0.22
















TABLE 9.2







2D NMR (HMQC) chemical shift list of O54.












Assignment
w1
w2
Data Height
















1
123.540
5.337
10801059



2
106.679
4.875
7546160



3
106.701
4.860
8527622



4
105.189
5.129
11004760



5
105.382
4.997
11249292



6
105.173
5.114
7915383



7
103.375
4.725
9431190



8
100.663
6.513
18941426



9
88.873
3.329
5975702



10
88.850
3.307
5246604



11
79.767
4.210
7199634



12
79.708
4.184
2885672



13
78.203
4.213
23826162



14
78.196
3.907
13786228



15
76.818
4.148
6859174



16
76.490
3.944
5773632



17
76.642
3.749
7515808



18
76.628
3.730
8437212



19
75.842
4.089
22251034



20
75.318
4.241
2654032



21
75.330
4.213
9853961



22
75.103
4.299
7580958



23
75.169
4.279
6376272



24
74.986
4.025
11823145



25
75.168
4.003
10800605



26
74.084
4.330
4901724



27
74.098
4.309
7830222



28
74.228
4.291
3975150



29
72.281
4.711
17310028



30
72.230
4.625
8709239



31
72.206
4.606
7361591



32
71.479
4.220
14407576



33
71.481
4.208
15081282



34
71.581
4.169
4582404



35
71.613
4.150
8419769



36
71.628
4.121
5804582



37
71.637
4.099
7950110



38
71.667
4.080
3119152



39
70.305
4.890
7622650



40
70.298
4.868
7628303



41
70.265
4.314
4762902



42
69.983
4.676
6227999



43
69.918
4.649
6037791



44
70.105
4.341
9963877



45
69.129
4.783
4704838



46
62.554
4.502
14998714



47
62.532
4.477
14154374



48
62.548
4.362
11342701



49
62.537
4.344
8091356



50
56.159
3.697
1838600



51
56.081
3.758
2144782



52
55.778
3.686
1783080



53
55.590
0.717
5572202



54
55.588
0.694
6671325



55
47.732
1.608
4217994



56
47.785
1.595
3965686



57
46.238
2.131
1734693



58
46.271
2.105
4919338



59
46.299
2.072
2490009



60
46.279
1.304
4826865



61
46.276
1.272
4573323



62
41.612
2.605
4436446



63
41.597
2.572
4591152



64
38.742
1.643
4256302



65
38.730
1.612
3985649



66
38.641
1.031
3538408



67
32.508
1.413
10863677



68
30.189
1.220
58134804



69
27.984
1.242
55524444



70
26.495
2.420
3113032



71
26.468
2.392
3183732



72
25.878
1.917
3435953



73
25.880
1.880
1979946



74
26.066
1.242
63345236



75
25.715
1.212
5054462



76
23.814
1.988
2104036



77
23.705
1.959
3237644



78
23.659
1.900
2754050



79
23.695
1.865
3108091



80
19.416
1.257
40949520



81
18.692
1.802
30867330



82
18.344
1.389
3540974



83
18.308
1.413
6204910



84
17.911
2.330
3260134



85
17.942
2.308
4212457



86
17.914
1.936
2468661



87
17.943
1.904
3914254



88
16.825
0.980
48392540



89
16.640
1.072
52924908



90
15.701
0.947
60375744

















TABLE 9.3







2D NMR (HMBC) chemical shift list of O54.


DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan,


EXPNO = 37, PROCNO = 1


F1PLO = 149.426 ppm, F1PHI = 11.307 ppm,


F2PLO = 6.861 ppm, F2PHI = 0.388 ppm


MI = 2.00 cm, MAXI = 10000.00 cm, PC = 3.000











ADDRESS
FREQUENCY













row
[Hz]F1
[PPM]F1



#
col
[Hz]F2
[PPM]F2
INTENSITY














1
294
21551.752
142.8145




728
1553.288
2.5881
3.15


2
294
21551.752
142.8145



791
1185.345
1.9750
3.03


3
294
21551.752
142.8145



801
1127.853
1.8793
3.00


4
294
21551.752
142.8145



866
744.836
1.2411
41.49


5
392
18639.387
123.5154



728
1554.313
2.5898
2.70


6
392
18639.387
123.5154



730
1540.225
2.5664
2.11


7
392
18639.387
123.5154



794
1164.901
1.9410
2.94


8
392
18639.387
123.5154



801
1127.069
1.8779
3.61


9
478
16106.854
106.7334



585
2391.723
3.9851
20.41


10
478
16106.854
106.7334



653
1992.507
3.3200
3.17


11
485
15890.850
105.3020



494
2924.288
4.8725
3.34


12
485
15890.850
105.3020



516
2797.792
4.6617
3.27


13
485
15890.850
105.3020



547
2614.903
4.3570
5.99


14
485
15890.850
105.3020



583
2405.277
4.0077
11.83


15
495
15600.868
103.3804



560
2539.046
4.2306
4.55


16
495
15600.868
103.3804



574
2456.564
4.0932
3.63


17
509
15198.148
100.7118



312
3996.510
6.6591
11.15


18
509
15198.148
100.7118



341
3824.267
6.3721
12.11


19
509
15198.148
100.7118



560
2539.421
4.2312
2.82


20
570
13411.609
88.8731



495
2921.818
4.8684
18.81


21
570
13411.609
88.8731



797
1147.142
1.9114
2.73


22
570
13411.609
88.8731



827
975.260
1.6250
6.25


23
570
13411.609
88.8731



866
746.125
1.2432
69.46


24
570
13411.609
88.8731



892
589.846
0.9828
48.36


25
616
12031.100
79.7251



561
2533.072
4.2207
10.23


26
616
12031.100
79.7251



568
2492.076
4.1524
12.78


27
624
11792.174
78.1418



469
3075.630
5.1247
3.71


28
624
11792.174
78.1418



481
3002.844
5.0034
2.57


29
624
11792.174
78.1418



534
2690.732
4.4834
2.54


30
624
11792.174
78.1418



547
2617.207
4.3608
2.56


31
624
11792.174
78.1418



561
2532.765
4.2201
29.07


32
624
11792.174
78.1418



573
2460.286
4.0994
15.44


33
624
11792.174
78.1418



580
2419.846
4.0320
13.41


34
624
11792.174
78.1418



585
2390.343
3.9828
8.30


35
624
11792.174
78.1418



868
732.442
1.2204
3.23


36
632
11558.919
76.5961



553
2577.697
4.2950
11.58


37
632
11558.919
76.5961



563
2521.889
4.2020
3.02


38
632
11558.919
76.5961



573
2462.246
4.1026
13.81


39
632
11558.919
76.5961



861
772.953
1.2879
9.11


40
632
11558.919
76.5961



864
755.650
1.2591
33.06


41
632
11558.919
76.5961



868
732.170
1.2200
53.66


42
639
11373.424
75.3669



326
3911.898
6.5181
5.00


43
639
11373.424
75.3669



563
2523.630
4.2049
25.40


44
639
11373.424
75.3669



574
2456.045
4.0923
5.16


45
639
11373.424
75.3669



611
2240.865
3.7338
5.57


46
639
11373.424
75.3669



808
1082.299
1.8034
2.40


47
639
11373.424
75.3669



864
756.608
1.2607
2.78


48
639
11373.424
75.3669



868
732.370
1.2203
5.47


49
646
11168.377
74.0082



511
2828.344
4.7127
8.44


50
646
11168.377
74.0082



520
2772.974
4.6204
6.66


51
646
11168.377
74.0082



563
2524.284
4.2060
5.35


52
646
11168.377
74.0082



808
1082.395
1.8035
49.69


53
655
10899.106
72.2238



326
3911.679
6.5177
25.27


54
655
10899.106
72.2238



498
2902.662
4.8365
2.42


55
655
10899.106
72.2238



511
2829.414
4.7144
5.76


56
655
10899.106
72.2238



523
2756.376
4.5927
2.33


57
655
10899.106
72.2238



532
2703.575
4.5048
2.40


58
655
10899.106
72.2238



552
2588.313
4.3127
9.99


59
655
10899.106
72.2238



563
2523.240
4.2043
7.35


60
655
10899.106
72.2238



808
1083.933
1.8061
2.26


61
659
10785.637
71.4719



326
3912.133
6.5185
2.28


62
659
10785.637
71.4719



494
2925.999
4.8754
2.36


63
659
10785.637
71.4719



516
2800.177
4.6657
3.39


64
659
10785.637
71.4719



532
2702.493
4.5030
7.49


65
659
10785.637
71.4719



534
2691.698
4.4850
7.86


66
659
10785.637
71.4719



547
2614.980
4.3571
2.35


67
659
10785.637
71.4719



562
2525.201
4.2075
46.85


68
659
10785.637
71.4719



584
2398.095
3.9958
2.02


69
665
10604.599
70.2723



326
3911.956
6.5182
2.12


70
665
10604.599
70.2723



469
3075.252
5.1241
18.68


71
665
10604.599
70.2723



481
3002.048
5.0021
3.29


72
665
10604.599
70.2723



562
2525.138
4.2074
3.03


73
665
10604.599
70.2723



573
2460.794
4.1002
6.46


74
665
10604.599
70.2723



808
1081.934
1.8027
3.10


75
666
10552.990
69.9303



326
3912.459
6.5190
2.10


76
666
10552.990
69.9303



468
3077.187
5.1273
13.23


77
666
10552.990
69.9303



481
3002.402
5.0027
10.72


78
666
10552.990
69.9303



563
2522.129
4.2024
3.33


79
666
10552.990
69.9303



573
2461.269
4.1010
4.47


80
666
10552.990
69.9303



808
1081.723
1.8024
2.72


81
671
10429.136
69.1095



326
3911.639
6.5177
38.49


82
671
10429.136
69.1095



552
2589.015
4.3139
8.97


83
671
10429.136
69.1095



808
1081.987
1.8028
63.77


84
705
9425.537
62.4591



560
2542.206
4.2359
3.27


85
705
9425.537
62.4591



562
2526.304
4.2094
2.08


86
740
8376.884
55.5101



827
971.239
1.6183
3.45


87
740
8376.884
55.5101



849
843.561
1.4056
3.43


88
740
8376.884
55.5101



866
746.292
1.2435
48.00


89
740
8376.884
55.5101



892
590.131
0.9833
31.95


90
740
8376.884
55.5101



896
567.050
0.9448
25.60


91
780
7196.908
47.6909



447
3202.461
5.3360
4.85


92
780
7196.908
47.6909



864
756.127
1.2599
2.83


93
780
7196.908
47.6909



868
731.658
1.2191
3.78


94
780
7196.908
47.6909



883
642.229
1.0701
28.25


95
780
7196.908
47.6909



896
567.166
0.9450
32.38


96
780
7196.908
47.6909



922
414.895
0.6913
2.37


97
788
6979.996
46.2535



730
1540.055
2.5661
2.37


98
788
6979.996
46.2535



864
755.679
1.2591
58.65


99
788
6979.996
46.2535



868
731.892
1.2195
85.12


100
804
6505.572
43.1097



553
2577.654
4.2949
4.16


101
804
6505.572
43.1097



574
2455.343
4.0911
4.70


102
804
6505.572
43.1097



755
1393.963
2.3227
3.13


103
804
6505.572
43.1097



858
789.603
1.3157
3.35


104
804
6505.572
43.1097



861
773.435
1.2887
9.90


105
804
6505.572
43.1097



867
737.064
1.2281
9.41


106
804
6505.572
43.1097



883
642.755
1.0710
2.74


107
804
6505.572
43.1097



892
589.037
0.9815
2.09


108
810
6311.923
41.8265



447
3202.295
5.3357
8.24


109
810
6311.923
41.8265



755
1393.660
2.3221
6.43


110
810
6311.923
41.8265



830
955.929
1.5928
2.54


111
810
6311.923
41.8265



866
744.369
1.2403
45.72


112
810
6311.923
41.8265



869
724.741
1.2076
3.80


113
810
6311.923
41.8265



883
642.271
1.0702
53.84


114
810
6311.923
41.8265



892
589.734
0.9826
2.36


115
814
6190.622
41.0227



447
3204.169
5.3389
2.27


116
814
6190.622
41.0227



866
744.967
1.2413
4.20


117
814
6190.622
41.0227



883
641.291
1.0685
13.51


118
814
6190.622
41.0227



892
590.266
0.9835
3.54


119
817
6122.212
40.5694



849
842.541
1.4039
2.02


120
817
6122.212
40.5694



866
744.098
1.2398
10.92


121
817
6122.212
40.5694



883
641.844
1.0695
19.65


122
817
6122.212
40.5694



892
590.010
0.9831
4.84


123
819
6054.448
40.1203



800
1128.653
1.8806
2.01


124
819
6054.448
40.1203



830
955.874
1.5927
5.24


125
819
6054.448
40.1203



849
842.438
1.4037
7.20


126
819
6054.448
40.1203



866
744.338
1.2402
39.00


127
819
6054.448
40.1203



883
642.047
1.0698
49.93


128
819
6054.448
40.1203



892
589.724
0.9826
6.64


129
819
6054.448
40.1203



896
566.654
0.9442
2.44


130
823
5927.002
39.2758



866
746.066
1.2431
48.54


131
823
5927.002
39.2758



883
642.112
1.0699
7.22


132
823
5927.002
39.2758



892
589.816
0.9828
60.87


133
823
5927.002
39.2758



896
566.631
0.9441
5.86


134
823
5927.002
39.2758



922
415.473
0.6923
7.24


135
827
5833.933
38.6590



827
970.335
1.6168
3.11


136
827
5833.933
38.6590



865
746.825
1.2444
10.44


137
827
5833.933
38.6590



883
642.035
1.0698
3.42


138
827
5833.933
38.6590



892
588.957
0.9813
11.20


139
827
5833.933
38.6590



896
567.124
0.9450
55.77


140
830
5737.570
38.0205



864
753.968
1.2563
3.79


141
830
5737.570
38.0205



868
731.483
1.2188
4.47


142
830
5737.570
38.0205



883
641.875
1.0695
2.21


143
830
5737.570
38.0205



892
589.272
0.9819
4.70


144
830
5737.570
38.0205



896
567.206
0.9451
11.09


145
833
5652.819
37.4589



865
751.942
1.2529
5.17


146
833
5652.819
37.4589



868
732.193
1.2200
5.78


147
833
5652.819
37.4589



883
642.313
1.0702
2.64


148
833
5652.819
37.4589



892
590.189
0.9834
2.92


149
833
5652.819
37.4589



896
567.196
0.9451
11.22


150
837
5532.757
36.6633



747
1441.047
2.4011
2.33


151
837
5532.757
36.6633



830
956.266
1.5934
6.70


152
837
5532.757
36.6633



849
840.791
1.4009
7.18


153
837
5532.757
36.6633



864
754.417
1.2570
10.60


154
837
5532.757
36.6633



868
733.768
1.2226
10.09


155
837
5532.757
36.6633



883
642.220
1.0701
2.62


156
837
5532.757
36.6633



896
567.041
0.9448
59.49


157
837
5532.757
36.6633



922
415.510
0.6923
7.27


158
840
5447.502
36.0983



611
2241.469
3.7348
3.44


159
840
5447.502
36.0983



775
1277.736
2.1290
2.18


160
840
5447.502
36.0983



778
1261.898
2.1026
5.77


161
840
5447.502
36.0983



780
1246.496
2.0769
2.77


162
840
5447.502
36.0983



861
771.393
1.2853
7.30


163
840
5447.502
36.0983



864
755.513
1.2589
85.50


164
840
5447.502
36.0983



868
731.684
1.2191
94.68


165
840
5447.502
36.0983



883
642.211
1.0701
2.09


166
840
5447.502
36.0983



896
566.770
0.9444
11.25


167
858
4901.029
32.4771



859
783.807
1.3060
2.31


168
858
4901.029
32.4771



883
642.014
1.0697
38.68


169
858
4901.029
32.4771



922
416.874
0.6946
3.35


170
870
4554.428
30.1803



611
2241.599
3.7350
9.79


171
870
4554.428
30.1803



777
1263.228
2.1048
3.21


172
870
4554.428
30.1803



857
793.884
1.3228
18.60


173
870
4554.428
30.1803



864
755.200
1.2583
93.90


174
870
4554.428
30.1803



879
668.641
1.1141
19.16


175
870
4554.428
30.1803



892
590.200
0.9834
3.11


176
881
4218.921
27.9570



855
807.262
1.3451
16.41


177
881
4218.921
27.9570



864
755.803
1.2593
4.75


178
881
4218.921
27.9570



876
683.031
1.1381
15.67


179
881
4218.921
27.9570



892
589.918
0.9829
73.26


180
881
4218.921
27.9570



922
416.446
0.6939
2.87


181
891
3935.921
26.0817



802
1120.176
1.8665
2.34


182
891
3935.921
26.0817



855
806.480
1.3438
19.11


183
891
3935.921
26.0817



866
745.923
1.2429
8.92


184
891
3935.921
26.0817



868
731.989
1.2197
5.17


185
891
3935.921
26.0817



877
680.851
1.1344
18.56


186
891
3935.921
26.0817



892
589.687
0.9825
4.65


187
893
3878.913
25.7039



791
1185.608
1.9755
2.05


188
893
3878.913
25.7039



810
1072.605
1.7872
2.02


189
893
3878.913
25.7039



855
805.970
1.3429
4.93


190
893
3878.913
25.7039



866
744.248
1.2401
44.75


191
893
3878.913
25.7039



877
680.894
1.1345
4.45


192
893
3878.913
25.7039



892
590.170
0.9834
3.96


193
925
2932.829
19.4346



611
2240.954
3.7339
15.68


194
925
2932.829
19.4346



775
1278.753
2.1307
4.57


195
925
2932.829
19.4346



778
1262.372
2.1034
11.84


196
925
2932.829
19.4346



780
1246.392
2.0768
6.85


197
925
2932.829
19.4346



854
816.734
1.3609
22.66


198
925
2932.829
19.4346



868
732.145
1.2199
93.22


199
925
2932.829
19.4346



875
691.416
1.1521
21.54


200
933
2709.260
17.9531



553
2577.918
4.2954
10.65


201
933
2709.260
17.9531



574
2456.969
4.0939
2.40


202
933
2709.260
17.9531



728
1553.812
2.5890
2.47


203
933
2709.260
17.9531



786
1214.060
2.0229
2.44


204
933
2709.260
17.9531



865
746.759
1.2443
4.64


205
933
2709.260
17.9531



868
731.918
1.2195
5.50


206
938
2536.520
16.8085



653
1992.054
3.3192
2.61


207
938
2536.520
16.8085



829
958.476
1.5970
2.70


208
938
2536.520
16.8085



866
745.904
1.2428
52.11


209
938
2536.520
16.8085



882
650.673
1.0842
17.18


210
938
2536.520
16.8085



895
574.736
0.9576
3.28


211
938
2536.520
16.8085



903
526.041
0.8765
16.84


212
938
2536.520
16.8085



922
416.280
0.6936
8.32


213
944
2375.487
15.7414



830
955.713
1.5924
5.34


214
944
2375.487
15.7414



866
745.684
1.2425
6.94


215
944
2375.487
15.7414



886
627.576
1.0457
21.31


216
944
2375.487
15.7414



892
588.774
0.9810
3.00


217
944
2375.487
15.7414



907
503.819
0.8395
21.33


218
944
2375.487
15.7414



922
415.374
0.6921
10.43
















TABLE 9.5







Proton NMR peak list of O54.


DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan,


EXPNO = 35, PROCNO = 1


F1 = 10.000 ppm, F2 = 0.000 ppm, MI = 2.00 cm,


MAXI = 10000.00 cm, PC = 1.000










FREQUENCY












#
ADDRESS
[Hz]
[PPM]
INTENSITY














1
7886.3
5225.509
8.7069
180.00


2
11038.4
4534.438
7.5554
72.73


3
12044.3
4313.911
7.1879
160.90


4
13878.4
3911.819
6.5180
14.53


5
17112.8
3202.719
5.3364
9.02


6
17674.0
3079.680
5.1314
10.52


7
17709.4
3071.938
5.1185
10.66


8
18004.6
3007.215
5.0107
10.57


9
18039.9
2999.480
4.9978
10.78


10
18330.8
2935.701
4.8915
6.88


11
18377.3
2925.509
4.8745
16.06


12
18411.1
2918.099
4.8622
10.01


13
18591.4
2878.554
4.7963
3.55


14
18621.6
2871.941
4.7853
4.53


15
18660.9
2863.325
4.7709
3.56


16
18768.9
2839.657
4.7315
8.48


17
18801.9
2832.416
4.7194
15.95


18
18929.2
2804.496
4.6729
5.72


19
18974.3
2794.608
4.6564
6.90


20
19049.2
2778.191
4.6291
5.41


21
19063.7
2775.007
4.6238
4.83


22
19091.3
2768.955
4.6137
5.79


23
19106.0
2765.737
4.6083
5.24


24
19395.7
2702.228
4.5025
11.91


25
19447.9
2690.790
4.4835
16.03


26
19708.9
2633.561
4.3881
6.86


27
19731.8
2628.552
4.3798
8.37


28
19761.7
2621.989
4.3688
16.78


29
19784.1
2617.071
4.3606
18.07


30
19814.8
2610.350
4.3494
12.73


31
19834.7
2605.976
4.3421
13.55


32
19863.8
2599.612
4.3315
9.52


33
19909.8
2589.523
4.3147
13.96


34
19947.5
2581.249
4.3009
10.27


35
19990.6
2571.809
4.2852
7.86


36
20116.2
2544.275
4.2393
6.06


37
20155.1
2535.750
4.2251
29.53


38
20188.0
2528.527
4.2131
36.53


39
20218.4
2521.859
4.2020
22.79


40
20258.5
2513.072
4.1873
10.10


41
20308.5
2502.111
4.1691
7.17


42
20349.0
2493.228
4.1543
10.94


43
20384.7
2485.412
4.1412
7.98


44
20454.4
2470.136
4.1158
8.00


45
20496.1
2460.979
4.1005
11.54


46
20522.9
2455.108
4.0908
20.70


47
20619.4
2433.953
4.0555
5.10


48
20655.0
2426.151
4.0425
7.46


49
20704.6
2415.271
4.0244
6.32


50
20741.8
2407.119
4.0108
8.93


51
20763.7
2402.312
4.0028
6.78


52
20800.3
2394.290
3.9894
7.59


53
20840.0
2385.587
3.9749
4.23


54
20899.5
2372.534
3.9532
5.04


55
20961.5
2358.953
3.9305
5.93


56
21011.8
2347.917
3.9122
8.88


57
21462.4
2249.135
3.7476
8.28


58
21507.4
2239.268
3.7311
7.60


59
22606.3
1998.348
3.3297
4.57


60
22625.1
1994.242
3.3228
4.57


61
22658.9
1986.819
3.3105
4.74


62
22678.2
1982.598
3.3034
4.55


63
24608.6
1559.375
2.5983
3.63


64
24657.0
1548.768
2.5806
3.98


65
25113.6
1448.658
2.4138
3.70


66
25160.1
1438.461
2.3968
4.03


67
25341.7
1398.662
2.3305
3.88


68
25394.7
1387.036
2.3111
4.43


69
25900.2
1276.207
2.1264
3.15


70
25962.8
1262.484
2.1036
6.25


71
26025.4
1248.772
2.0807
3.23


72
26432.4
1159.530
1.9320
8.83


73
26518.7
1140.610
1.9005
11.87


74
26686.8
1103.771
1.8391
2.00


75
26772.7
1084.927
1.8077
28.83


76
26800.2
1078.904
1.7977
28.10


77
27239.8
982.535
1.6371
4.71


78
27287.4
972.099
1.6197
8.43


79
27366.6
954.736
1.5908
4.35


80
27864.2
845.631
1.4090
14.94


81
28172.8
777.969
1.2963
9.42


82
28216.7
768.363
1.2803
9.50


83
28235.2
764.288
1.2735
9.84


84
28276.7
755.206
1.2583
58.74


85
28321.2
745.447
1.2421
94.67


86
28380.5
732.444
1.2204
58.53


87
28793.1
641.978
1.0697
50.29


88
28906.6
617.114
1.0282
5.31


89
29031.6
589.696
0.9826
56.49


90
29134.1
567.228
0.9451
50.09


91
29765.3
428.852
0.7146
6.67


92
29818.8
417.128
0.6950
6.36









References

    (1) Chen, Q. 1995. Methods of study on pharmacology of Chinese medicines. Press of People's Public Health, Beijing. p 892.
    (2) Huang, Zh. Sh., Liu, M. P., Chen, Ch. Zh. 1997. Study on effects of Yangshou Dan on improving learning and retention. Chinese Journal of combination of Chinese and west medicine, 9(17): 553.
    (3) Zhang, Y., Zhang, H. Y., Li, W. P. 1995. Study on effects of Anjifu on improving intelligence, Chinese Bulletin of Pharmacology, 11(3): 233.
    (4) Yang, J., Wang, J., Feng, P. A. 2000. Study on effects of Naokkangtai capsule on improving learning and retention in mice, New Chinese Medicine and Clinical Pharmacology, 1(11): 29.
    (5) Yang, J., Wang, J., Zhang, J. Ch. 2000. Study on effects of Crude saponins of peonies on improving learning and retention in mice, Chinese journal of Pharmacology, 2(16): 46.
    (6) Xia, W. J., Jin, M. W., Zhang, L. 2000. Study on treatment of senile dementia caused by angio-aging with Didang tang, Pharmacology and Clinical of Chinese Medicines, 16 (4).
    (7) Bian, H. M., Yu, J. Z., Gong, J. N. 2000. Study on effects of Tongmai Yizhi capsule on improving learning and retention in mice, Pharmacology and Clinical of Chinese Medicines, 16 (5): 40.
    (8) Wei, X. L., Zhang, Y. X. 2000. Study of animal model for studying senile dementia, Chinese journal of Pharmacology, 8(16): 372.
    (9) Bureau of Medicinal Police, Department of Public Health. Guide line for study of effect of medicines for treatment of nervous system diseases, in Guidebook of study of new medicine. p 45.
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## Claims

1. A method for inhibiting cancer cell growth in a subject, comprising administering to the subject an effective amount of saponins from Wenguanguo or Xanthoceras Sorbifolia, wherein the cancer is selected from the group consisting of breast cancer, leukocyte cancer, liver cancer, ovary cancer, bladder cancer, prostate cancer, bone cancer, colon, cervix, lung and brain cancer.

2. The method of claim 1, wherein the cancer is breast cancer.

3. The method of claim 1, wherein the cancer is leukocyte cancer.

4. The method of claim 1, wherein the cancer is liver cancer.

5. The method of claim 1, wherein the cancer is ovary cancer.

6. The method of claim 1, wherein the cancer is prostate cancer.

7. The method of claim 1, wherein the cancer is bone cancer.

8. The method of claim 1, wherein the cancer is brain cancer.

9. The method of claim 1, wherein the cancer is colon cancer.

10. The method of claim 1, wherein the cancer is cervix cancer.

11. The method of claim 1, wherein the cancer is lung cancer.

12. The method of claim 1, wherein the saponins are in a pharmaceutically suitable carrier.

13. A method for inhibiting cancer cells growth in a subject, comprising administering to the subject an effective amount of health food or medicament, wherein the health food or medicament includes an essential amount of extract from Wenguanguo or Xanthoceras Sorbifolia, wherein the cancer is selected from the group consisting of breast cancer, leukocyte cancer, liver cancer, ovary cancer, bladder cancer, prostate cancer, bone cancer, colon, cervix, lung and brain cancer.

14. The method of claim 13, wherein the extract includes an essential amount of saponins from Wenguanguo or Xanthoceras Sorbifolia.

15. The method of claim 13, wherein the cancer is leukocyte cancer.

16. The method of claim 13, wherein the cancer is liver cancer.

17. The method of claim 13, wherein the cancer is ovary cancer.

18. The method of claim 13, wherein the cancer is prostate cancer.

19. The method of claim 13, wherein the cancer is bone or breast cancer.

20. The method of claim 13, wherein the cancer is brain or colon cancer.