CINNAMOYL-C-GLYCOSIDE CHROMONE ISOLATED FROM ALOE BARBADENSIS

Abstract: A composition of matter comprising at least 15 % by weight of the compound: 8-C- beta -D-[2-O-(E)-cinnamoyl]glycopyranosyl-2-[(R)-2-hydroxy]propyl-7-methoxy-5-methylchromone. This compound, found in very small amounts in leaves of the Aloe barbadensis plant, when concentrated to the desired level, exhibits anti-inflammatory activity.

Description: CINNAMOYL-C-GLYCOSIDE CHROMONE ISOLATED FROM ALOE BARBADENSIS BACKGROUND OF THE INVENTION The present invendon describes a compound having biological activity which has been isolated from the Aloe barbadensis plant. This heretofore-unreported compound, systematically named 8-C-j3-D-[2-0-(E)- cinnamoyI]glycopy_anosyl-2-[(-?)-2-hydroxy]propyl-7-methoxy-5-methyl- chromone, has a molecular weight of 541 Da, and the following chemical structure: This compound exhibits potent anti-inflammatory activity as measured in vivo assays. The compound has also been shown to inhibit EGF-induced DNA synthesis in in vitro testing- in epithelial cell lines. Exudates from aloe plants have been employed for centuries as folk remedies for the treatment of bums, sores and other wounds. In recent years, a great deal of research has been concentrated on identifying aloe components which have clinical activity. Two factors which prompt this interest are aloe's propensity to loose at lease some of its therapeutic properties shortly after harvesting, and the desire to eliminate components which may be harmful - e.g., certain anthraquinones. See, e.g., McAnalley, U.S. Patent 4,966,892. The results of these research efforts suggest that the aloe plant contains several components which have wound-healing properties. The literature contains a report of a 5-methylchromone C- glucoside which has been identified in Cape Aloe, viz. , 2-acetonyl-7-O-0-D- glucopyranosyl-8-C- 3-D-[2'-0-(E)-j.-coumaroyl]glucopy_anosyl-5-methyl- chrome. Giovanna Speranza, P. Gramatica, G. Dada, P. Manitto, "Aloeresin C, A Bitter C, 0-Diglucoside from Cape Aloe", Phytochemistry, 1985, 24(7):1571-1573. This prior an compound has a molecular weight of 556.5 Da, the glucosylation group is coumanc acid, not cinnamic acid, and no biological activity is ascribed to the compound. Other reports of chromones in aloe are found in the literature. "Aloe Revisited -- The Structure of Aloeresin A", P. Gramatica, D. Monti, G. Speranza, P. Manitto, Tetrahedron Letters, 1982, 23(23) :2423-2424; "Chromones in Aloe Species Part 1. Aloesin - a C-glucosyl-7-hydroxy- chromone", D.K. Holdswoπh, PM, 19(4):322-325; "Structure Determination of Polysaccharides in Aloe arborescens var. natalensis" , Akira Yagi, H. Nishimura, T. Shida, I. Nishioka, Planta Medica, 1986, 213-218; "Chro¬ mones in Aloe Species Pan II - Aloesone", D.K. Holdswoπh, PM, 22(1):54- 58; and "A C-Glucosylated 5-Methylchromone from Kenya Aloe", Giovanna Speranza. G. Dada. L. Lunazzi, P. Gramatica, P. Maniπo. Phytochemistry. 1986. 25(9):2219-2222. SUMMARY OF THE INVENTION The present invention relates to a composition of matter which contains 8-C-/3-_)-[2-{ (E)-ci__n__noyI]glycopy_anosyl-2-[(Λ)-2-hydroxy]propyl- 7-me_hoxy-5-methylchromone (hereafter, the "540 Compound") at a concentration of at least 15 % by weight. In vivo data indicates that concentra¬ tions below about 15% by weight do not result in statistically significant anti- inflammatory activity. The balance of die composition can contain polysac- charides normally found in Aloe (chiefly manoses), Aloin, conjugated anthaquinones, conjugated chromones and anthraquinone aglycones. The balance of the composition can also include any of the liquid carriers or vehicles commonly used to formulate ointments or lotions, such as emulsified fatty alcohols, ethoxylated stearyl alcohols, fatty ether phosphates (e.g., oleyl ether phosphates), ethoxylated lanolin alcohols, beeswax, glycerol monostea- rate, emulsified polydimethylsiloxanes, glycerin, mineral oil and surfactants. Oϋier suitable vehicles will be apparent to formulators of ointments and lotions. The previously-unidentified 540 Compound is found in the Aloe barbadensis plant at very small levels: in decolorized gel (the most commonly used form of aloe) at < > 0.0001 % by weight; in non-decolorized gel at < 0.0011 % by weight; in decolorized whole leaf extract at <0.0002% by weight: and, in non-decolorized whole leaf extract at < 0.0025% by weight. Thus, in order to prepare compositions having the desired clinical properties, it is necessary to treat liquid solutions containing dissolved or suspended aloe extracts in order to concentrate the 540 Compound to the desired level of 15% or more. Prior art experiences with aloe plant extracts has shown that the majority of the biologically-active components in the plant are located in die clear gel found in the core of aloe leaves. We have found the 540 Compound in such gels; however, as noted above, we have discovered tiiat the 540 Compound is present in greater levels in the leaf rind. Two processes have been developed for concentrating the 540 Compound. In the first process, freshly-harvested leaves from the plant Aloe barbadensis were washed, gel was separated from the rind, and the gel- containing liquid slurry was filtered to remove solid particles. (Because of the greater levels of 540 Compound in the leaf rind, an alternative, preferred, procedure would be to grind the whole leaf to form a slurry). Dialyization of the filtrate (Spectraphor #1 tubing, 24 hours x 3) resulted in two fractions ~ the dialysate and the retained material. Two liters of 80% ethanol was added to a 500 mL portion of the retained material ( - 10 g solids), the sample was agitated and centrifuged at 20,000 rpm for 15 minutes, and die precipate was discarded. Ethanol was stripped from the supernatant in a rotoevaporator, yielding 1.31 grams of a product. A 1.14 gram sample of this product was extracted with a mixture of CHC1 3 and water which partitioned the sample into die aqueous phase and organic phase in the ratio of 3 : 1 , respectivel . The chloroform phase was evaporated and lyophilized, and then taken up in 50% ethanol to a concentration of 20 mg/ml. This sample was analyzed using high performance liquid chromatography (HPLC). A pool eluting from an ODS 1 column at about 14 minutes was found to contain a component which inhibited DNA synthesis induced by EGF in NMuMG epithelial cells. Subsequent analysis revealed tins to be the 540 Compound. Several commercial processes employed in the processing of Aloe barbadensis gel utilize activated charcoal to improve the color of material. This treatment is particularly common wim so-called "whole-leaf processing" wherein the entire leaf is ground and processed. Whole leaf processing produces a darker product than that which is produced using gel which has been separated from the leaf rind. However, when die dialysis separation oudined above was repeated on such charcoal-treated material, the product did not exhibit the same level of biological activity. One hypodiesis was tf at the factor which was responsible for the biological activity had been trapped or entrained on the activated charcoal. Subsequent testing confirmed this hypothesis. Elution of activated charcoal with organic solvents which had been used to treat a slurry of ground Aloe barbadensis leaves produced a solution which contained die 540 Compound. Details of this processing are set out in the following examples. Data relating to in vivo testing of die 540 Compound are set out at FIG. 1. DESCRIPTION OF THE DRAWING FIG. 1 illustrates the topical anti-inflammatory activity of the 540 Compound in comparison with other Aloe barbadensis extracts and hydrocortisone when tested on laboratory mice. Data points represent the mean ± sd for three mice. Irritant alone was utilized for die "croton oil control" group. The "control - no treatment" group data were obtained from untreated ears. Ear thickness was measured by means of an Oditest caliper. Example I Separation of 540 Compound from Activated Charcoal. 150 g of activated charcoal which had been used in d e processing of Aloe barbadensis whole leaf extract was subjected to two cycles of elution with refluxing 25% Acetone in CH 2 C1-. The first elution was conducted with 750 L (because of the need for "wetting" of die dried adsorbed charcoal). After extraction for four hours with stirring, d e liquid was separated by filtration on a cellulose filter and the moist cake was washed with 250 mL of the eluting solvent. The moist cake was then extracted with die same solvent system (500 mL) for 7 hours. The second elute was removed as above and d e moist cake washed. The Acεtone CH 2 CI : eluates were combined with their respective washes and were stripped by rotary evaporation under reduced pressure. The oily residue was transferred to scintillation and evaporation completed under a stream of nitrogen. The residues were dissolved in absolute Ethanol (EtOH) at a concentration of 100 mg/mL. The third and fouπh elutions were performed with refluxing absolute . EtOH. Each elution employed 500 mL. After the third elution, the wash was kept separate from the eluate and there was no wash after die fourth elution. Stripping of the tfiird eluate. wash after third elution and the fourth eluate yielded syrups. These were dried under high vacuum for three days. The residues were dissolved in 50% EtOH in water to 100 mg/mL (wash after third elution and fouπh eluate) or 200 mg/mL (third eluate). Dilutions of 10 mg/mL were made of all eluates as stocks for analysis. Since die first and second eluates (25% Acetone CH 2 Cl-) so closely resembled each other, they were pooled into Pool A. In similar fashion, the third eluate. the wash after the third eluate and fouπh eluate closely resembled each other, and were pooled into Pool B. HPLC analysis was performed on A and B. The composition of the two samples is set out in Table I: TABLE I From HPLC, integration of arbitrary ODU areas under the curve. " Dubois Assay, direct determination on Pool. Samples of d e two products were lyophdized and tested for anti-inflammatory activity on laboratory mice. In addition, die samples were analyzed to confirm the chemical structure of die active component. Example II Confirmation of Chemical Structure. Structural elucidation of the cinnamoyl-C-glycoside chromone found in the sample, Eluate A. prepared in accordance with Example I, was accomplished by use of nuclear magnetic resonance ( l H) spectroscopy and mass spectrometry, evaluation of ultraviolet absorbance, melting point and thin-layer chromatographic behavior. The results were as follows: (1) Analytical data and methodology: Melting point: sublimes >90°C Thin-layer chromatography TLC plate: silica Solvent system: ethyl acetate/ methanol/ water (100:13:3, v/v/v) R f : 0.69 UV spectrum Solvent: CH } OH ,__ (log ε) 246 nm (4.22), 254 nm (4.27), 284 nm (4.36) Proton NMR analysis Instrument: General Electric model QE 300 spectrometer Frequency: 300 mHz Solvent: CD 3 OD Reference: CH,OH, 3.30 ppm Spectral data: 1.30 (3H, d, 6 Hz) (2-y-methyl); 2.74 (3H, s) (5- methyl); 2.82 (2H, dd, 14 Hz, 6.5 Hz) (2-a-methylene); 3.42- 3.50 (1H, m) (5'-H); 3.61 (1H, dd, 10Hz) (4 * -H); 3.70-3.80 (2H, m) (3' 6'a-H); 3.89 (3H, s) (7-methoxy); 4.00 (1H. m) 6 * b-H); 4.37 (1H, m) (2-0-carbinol); 5.21 (1H. d, 10 Hz) (V- H); 5.73 (1H, dd, 10 Hz) (2'-H); 6.13, (1H, s) (3-H); 6.27 ( IH. d. 16 Hz) (cinnamoyl 0-H); 6.81 (1H. s) (6-H): 7.37 (2H, m) (ciππamoyl m-H); 7.45 (1H, d, 16 Hz) (cinnamoyl α-H); 7.50 (3H, m) (cinnamoyl o-, p-H) Mass spectral analysis Instrument: Finnigan MAT 4615 quadrupole mass spectrometer Ionization: electron impact (70 eV) Source temperature: 160°C Sample introduction: direct insertion probe Sample heating: ballistic; peak evaporation at approximately 250°C Spectral data (relative intensity in parendiesis): m/z 540 (12). [M]"; m/z 522 (5), [M-H-0] " ([ -l-D; m z 496 (4), [M-CH 3 CHθr (l) ([M-44D; m/z 410 (16), [M- ([M-130] * ); m/z 392 (10), [M-(130÷18)]"; m/z 376 (4), [M-(18+cinnamate)^ α, ([M-(18^ 146)]'); m/z 366 (8), [M-(130-44)]-; m/z 343 (18), [M-lδ÷ W÷SS)]-" 31 ; m/z 277 (50), ΓM-(130÷ 133)] *(4 '; m/z 259 (100), [M- (130+ 18+ 133)] * ; m/z 233 (92), [M-( 130-44+ 133)] * ; m/z 193 (73), [M-(130+84 (5 > + 133)] + ; m/z 131 (40), [cinnamoyl] * ; m/z 103 (33), [ H 3 CHCHT '" alpha-cleavage of the 2-hydroxypropyl side chain C1 involves hydrogen transfer 01 tentative assignment ' ■ " 133 represents C2 - C6 of die C-glycoside <5) retro-Diels-Alder fragmentation diat includes die hydroxy- propyl side chain Example HI Biological Activity. Samples of Eluate A and Eluate B prepared in accordance with Example I were dissolved in ethanol (100 mg/mL) and tested on mice in accordance with the following procedure. In order to induce ear swelling, a solution of croton oil was applied to the inner side of the ear of laboratory mice. The test materials were applied to die same location 30 minutes after the croton oil treatment. Measurements of ear thickness were made 18 hours after treatment. The results are shown in Table II. TABLE II ' Aloe barbadensis gel supplied by Alo corp. Irving, Texas. : Milligrams of experimental ear punch - milligrams of control ear punch. Mean ± Standard Error of the Mean, groups of 12 animals each. 1 Untreated ear injured with 25 μg of Croton oil. swelling was 7.3 ±0.3 mg. Example IV Measurement of Anti-Inflammatory Activity. A lyophilized sample of a product prepared in accordance with Eluate A of Example I was extracted into methanol, and the solvent evaporated in vacuo. The residue was dissolved in water and partitioned between water and ethyl acetate. The respective solvents were removed in vacuo and the remaining solid tested in parallel with the 540 Compound for topical anti-inflammatory activity. The results of this experiment are reproduced graphically at FIG. 1. From this experiment, it can be seen that the 540 Compound exhibits potent anti-inflammatory activity - comparable to an equal dose of hydrocortisone. Ear thickness after treatment witii the 540 Compound was significantiy less than die croton oil control group; statistical significance was determined by a one-way analysis of variance, with multiple comparisons assessed by Fisher's PLSD. Moreover, a 500-μg portion of the ethyl acetate phase components was similarly active at 6 hours after treatment, but showed diminished potency at 24 hours. Components in the water phase were only slightly active at 24 hours after treatment. Male Balb/c mice weighing 20-25 g were utilized for these experiments. Three animals were used for each group. In order to induce ear swelling, 10 μL of croton oil solution (25 mg croton oil dissolved in a vehicle of pyridine/water/diethyl ether (4: 1:5, v/v/v)) was applied by means of an Eppendori pipette to the inner side of the left ear of each mouse. Each test material was dissolved in the same vehicle and applied to die same location on die left ear. 30 minutes after croton oil treatment. Animals treated with hydrocortisone were evaluated in parallel as a model for potent aπά-inflamma- tory activity. Ear swelling in a control group in which animals were treated with croton oil alone was also assessed. Ear thickness was measured by means of an Oditest caliper. Measurement was made before application of croton oil, and at 6 and 24 hours after treatment with the test solutions. The results of this testing are depicted in FIG. 1.

Claims: WHAT IS CLAIMED IS: 1. A composition of matter comprising at least 15% by weight of the compound: 8-C-)3-D-[2-0-(E)-c_nnamoyI]glycopyranosyl-2-[(-")- " 2 hydroxy]propyI-7-methoxy-5-methylchromone. 2. The composition of claim 1 further including between 15% and 85% by weight of one or more components selected from the group consisting of polysaccharides, aloin, conjugated andiraquinones, conjugated chromones and andiraquinone aglycones. 3. The composition of claim 1 further including between 15% and 85% of a liquid carrier.

Inventors: WALLER TODD A, WALLER, TODD, A.
Applicants: NAM YANG INTERNATIONAL INC DOI, NAM YANG INTERNATIONAL, INC. DOING BUSINESS AS ALO
Application year: 1996
Main classification: A61K31
IPC codes: A61K31, A61K36, A61K45, A61P29, C07D315