# Patent

## Title
Composition containing a Spiranthes sinensis extract and pharmaceutical applications thereof

## Abstract

The present invention is related to a pharmaceutical composition comprising an effective amount of a Spiranthes sinensis extract and a pharmaceutically acceptable carrier. The present invention improves the economic value of Spiranthes sinensis by verifying the efficacies of said pharmaceutical composition in anti-inflammation, anti-liver fibrosis and/or anti-oxidation.

## Background
BACKGROUND
Technical Field
The present disclosure relates to a method and a pharmaceutical composition for inflammation and/or liver fibrosis treatment; more specifically, to a method and a pharmaceutical composition for inflammation and/or liver fibrosis treatment by using a Spiranthes sinensis extract.
Description of Related Art
Spiranthes sinensis, commonly known as the Chinese Spiranthes, is a species of orchid occurring in much of eastern Asia, west to the Himalayas, south and east to New Zealand, and north to Siberia. Previous phytochemical investigations of Spiranthes sinensis showed the presence of dihydrophenanthrenes, sterols, homocyclotirucallane (Lin, Huang et al. 2000, Lin, Wang et al. 2001), two new prenylated coumarins (Peng, Han et al. 2008) and one novel dimeric phenanthrene and flavone (Li, Liu et al. 2013). However, the medicinal properties of Spiranthes sinensis crude extract and its phytochemicals is still unexplored yet.
Inflammation is recognized as an onset of immune response for infections or disorders of a subject. However, it is also noted that inflammation may also be harmful to a subject if it is out of control, for example, in an allergy response. Moreover, overly (exuberant) wound healing usually happens from the recovery of severe and out-of-control inflammation, which may result in fibrosis in tissues and then lead to carcinogenesis. Case in point, liver fibrosis occurs during inflammation of liver tissue and is recognized to be involved in cirrhosis and liver cancer. Although inflammation is parts of the defense mechanism of immune system, it is important to appropriately reduce the degree of inflammation in necessary.
Liver fibrosis is scarring process, on behalf of the liver damage response. Liver fibrosis is overly (exuberant) wound healing in which excessive connective tissue builds up in the liver. The extracellular matrix (ECM) is overproduced, degraded deficiently, or both. Over time this process can lead to the cirrhosis of liver, once the development of cirrhosis and severe complications might occur including portal hypertension, liver failure, and liver cancer.
In light of the foregoing, as Spiranthes sinensis is expected to have potentials in pharmaceutical applications, further researches of Spiranthes sinensis in applications of treating inflammation and/or liver fibrosis are required for improving the economic value of Spiranthes sinensis. 
SUMMARY
One of the objectives of the present invention is to provide a pharmaceutical composition having efficacies in anti-inflammation, anti-liver fibrosis and/or anti-oxidation; therefore provide novel options for medication.
Another objective of the present invention is to improve the economic value of Spiranthes sinensis by verifying novel pharmaceutical applications thereof.
In order to achieve the above-mentioned objects, the present invention provides a pharmaceutical composition having efficacies in anti-inflammation, anti-liver fibrosis and/or anti-oxidation, comprising an effective amount of a Spiranthes sinensis extract and a pharmaceutically acceptable carrier; wherein said effective amount is 0.6 to 5 mg/60 kg body weight/day.
Preferably, said pharmaceutical composition comprises 5 to 100 μg/mL of said Spiranthes sinensis extract based on the total volume of said pharmaceutical composition.
Preferably, said anti-inflammation comprises down-regulation of inflammatory cytokines, iNOS, Cox-2, NFκB and/or the phosphorylation of ERK.
Preferably, said anti-liver fibrosis comprises accumulating lipid in hepatic stellate cells, down-regulating the proliferation of hepatic stellate cells, inhibiting the mobility of hepatic stellate cells, preventing the activation of hepatic stellate cells, decreasing the synthesis of ECM proteins, or a combination thereof.
Preferably, an administration route of said pharmaceutical composition is via oral administration, intravenous injection, or a combination thereof.
Preferably, said pharmaceutically acceptable carrier is water, alcohol, glycerol, chitosan, alginate, chondroitin, Vitamin E, mineral oil, dimethyl sulfoxide (DMSO), or a combination thereof.
The present invention also provides a method for anti-inflammation, anti-liver fibrosis and/or anti-oxidation, comprising: administrating an effective amount of a Spiranthes sinensis extract to a subject; wherein said effective amount is 0.6 to 5 mg/60 kg body weight/day.
Preferably, said administrating is via oral administration, intravenous injection, or a combination thereof.
Preferably, said Spiranthes sinensis extract is administrated with a pharmaceutically acceptable carrier; wherein said pharmaceutically acceptable carrier is water, alcohol, glycerol, chitosan, alginate, chondroitin, Vitamin E, mineral oil, dimethyl sulfoxide (DMSO), or a combination thereof.
Preferably, said Spiranthes sinensis extract is prepared by the following steps: obtaining a plant of Spiranthes sinensis; mixing said plant with a solvent to obtain a mixture; and drying said mixture to obtain said extract.
Preferably, said plant is dried and/or ground before mixing with said solvent.
Preferably, said solvent is ethyl acetate, acetone, n-hexane, or a combination thereof.
Preferably, said ethyl acetate is at a concentration of 5% to 100% (v/v); wherein said concentration is based on the total volume of said ethyl acetate; more preferably, is 10% to 100% (v/v).
Preferably, said mixing is conducted at 20 to 28° C. Preferably, said mixing is conducted for 1 to 2 days. Preferably, said drying is achieved by vacuum drying, freeze-dried, lyphilization, or a combination thereof.
In light of the foregoing, the present invention discloses the novel pharmaceutical applications of Spiranthes sinensis in anti-inflammation, anti-liver fibrosis and anti-oxidation. Therefore the economic value of Spiranthes sinensis can be improved.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the cell viability experiments for RAW264.7 in Example 2 by using MTT assay. The cells were treated with the present Spiranthes sinensis extract at various concentrations of 5, 20, 50, and 100 μg/mL for 24 h. The concentration is based on the volume of the culture medium.
FIG. 2 shows the effect of the present Spiranthes sinensis extract on NO release. ** P<0.01 as compared with 10 μg/mL of the present Spiranthes sinensis extract group.
FIG. 3 is the result of Real time PCR showing the expression of TNF-alpha, iNOs, IL-1, and IL-6 mRNA. Data represent the mean±SEM from three separate experiments. LPS04: cells treated with 0.4 μg/mL LPS only; negative control. LPS10: cells treated with 1 μg/mL LPS only; negative control. Control: blank control. SS100: cells treated with 1 μg/mL LPS plus 100 μg/mL of Spiranthes sinensis extract. SS50: cells treated with 1 μg/mL LPS plus 50 μg/mL of Spiranthes sinensis extract. SS20: cells treated with 1 μg/mL LPS plus 20 μg/mL of Spiranthes sinensis extract. SS5: cells treated with 1 μg/mL LPS plus 5 μg/mL of Spiranthes sinensis extract. The concentration is based on the volume of the culture medium.
FIG. 4 shows the expression of Cox-2, NFκB, p-ERK, and ERK measured by Western blot. (A) Results of SDS-PAGE. (B), (C), (D) Quantitation of the expression of Cox-2, NFκB, and p-ERK respectively from the results of the Western blot. Control: blank control. LPS04: cells treated with 0.4 μg/mL LPS only; negative control. LPS10: cells treated with 1 μg/mL LPS only; negative control. SS100: cells treated with 1 μg/mL LPS and 100 μg/mL of Spiranthes sinensis extract. SS50: cells treated with 1 μg/mL LPS plus 50 μg/mL of Spiranthes sinensis extract. SS20: cells treated with 1 μg/mL LPS plus 20 μg/mL of Spiranthes sinensis extract. SS5: cells treated with 1 μg/mL of LPS plus 5 μg/mL of Spiranthes sinensis extract. The concentration is based on the volume of the culture medium.
FIG. 5 shows the result of DPPH assay indicating the anti-oxidant activity of the present Spiranthes sinensis extract.
FIG. 6 shows the change in the swelling index of foot. CFA: negative control (injected with Complete Freund's Adjuvant but without treatment). SS: the present Spiranthes sinensis extract. TE: triamcinolone. SE: sevatrim. n=5.
FIG. 7 shows the area under curve calculated from the results shown in FIG. 6. CFA: negative control (induced by CFA but without treatment). SS: the present Spiranthes sinensis extract. TE: triamcinolone. SE: sevatrim.
FIG. 8 shows the results of the gelatin zymography for determination of the gelatinolytic activity of MMP 9 in the Example 7. CFA: negative control (induced by CFA but without treatment). SS: the present Spiranthes sinensis extract. TE: triamcinolone. SE: sevatrim.
FIG. 9 shows the cell viability experiments for NHSCs and THSCs by using MTT assay. The cells were treated the present Spiranthes sinensis extract at various concentrations of 5, 20, 50, and 100 μg/mL for 24 h. The concentration is based on the volume of the culture medium.
FIG. 10 shows the results of the oil red o staining assay in Example 9 under 240× magnification; wherein (A) NHSCs, untreated, or treated with 5, 20, and 50 μg/mL of Spiranthes sinensis extract; (B) THSCs, untreated, or treated with 5, 20, and 50 μg/mL of Spiranthes sinensis extract.
FIG. 11 is a bar diagram showing the quantitation from the results of the experiments shown in FIG. 10.
FIG. 12 shows the effect of the present Spiranthes sinensis extract (5, 20 and 50 μg/mL) and curcumin (25 μM) on fatty acid storage in (A) NHSCs and (B) THSCs at 48 h. * P<0.05; *** P<0.001 as compared with the control group.
FIG. 13 shows the results of the Sirius Red stain in Example 10 under 240× magnification; wherein (A) NHSCs, untreated, or treated with 5, 20, and 50 μg/mL of Spiranthes sinensis extract; (B) THSCs, untreated, or treated with 5, 20, and 50 μg/mL of Spiranthes sinensis extract. The concentration is based on the volume of the culture medium.
FIG. 14 shows the activity of MMP 2 examined by gelatin zymography. (A) NHSCs; (B) THSCs; (C) Quantitation from the results of (A); (D) Quantitation from the results of (B).
FIG. 15 shows the expression of COL1 I, COL1 III, TGF-β R1, and TGF-β R2 in both (A) NHSCs and (B) THSCs; wherein 18S is a reference gene in this examination. N-CU25: NHSCs treated with 25 μM of curcumin; N-CON: untreated NHSCs; N-SS5: NHSCs treated with 5 μg/mL of Spiranthes sinensis extract; N-SS20: NHSCs treated with 20 μg/mL of Spiranthes sinensis extract; N-SS50: NHSCs treated with 50 μg/mL of Spiranthes sinensis extract. T-CU25: THSCs treated with 25 μM of curcumin; T-CON: untreated THSCs; T-SS5: THSCs treated with 5 μg/mL of Spiranthes sinensis extract; T-SS20: THSCs treated with 20 μg/mL of Spiranthes sinensis extract; T-SS50: THSCs treated with 50 μg/mL of Spiranthes sinensis extract. The concentration is based on the volume of the culture medium. Data represent the mean±SEM from three separate experiments.
FIG. 16 shows the expression of PPARγ in both NHSCs and THSCs. N-Control: untreated NHSCs; N-SS5: NHSCs treated with 5 μg/mL of Spiranthes sinensis extract; N-SS20: NHSCs treated with 20 μg/mL of Spiranthes sinensis extract; N-SS50: NHSCs treated with 50 μg/mL of Spiranthes sinensis extract. N-CU25: NHSCs treated with 25 μM of curcumin. T-Control: untreated THSCs; T-SS5: THSCs treated with 5 μg/mL of Spiranthes sinensis extract; T-SS20: THSCs treated with 20 μg/mL of Spiranthes sinensis extract; T-SS50: THSCs treated with 50 μg/mL of Spiranthes sinensis extract. THSCs treated with 25 μM of curcumin. The gene expression was normalized by the expression of 18S. The concentration is based on the volume of the culture medium. Data represent the mean±SEM from three separate experiments.
FIG. 17 shows the expression of RXRα, NrF2 in both NHSCs and THSCs. N-Control: untreated NHSCs; N-SS5: NHSCs treated with 5 μg/mL of Spiranthes sinensis extract; N-SS20: NHSCs treated with 20 μg/mL of Spiranthes sinensis extract; N-SS50: NHSCs treated with 50 μg/mL of Spiranthes sinensis extract. N-CU25: NHSCs treated with 25 μM of curcumin. T-Control: untreated THSCs; T-SS5: THSCs treated with 5 μg/mL of Spiranthes sinensis extract; T-SS20: THSCs treated with 20 μg/mL of Spiranthes sinensis extract; T-SS50: THSCs treated with 50 μg/mL of Spiranthes sinensis extract. THSCs treated with 25 μM of curcumin. The gene expression was normalized by the expression of 18S. The concentration is based on the volume of the culture medium. Data represent the mean±SEM from three separate experiments.



DETAILED DESCRIPTION
The present invention provides the pharmaceutical applications of Spiranthes sinensis by showing its efficacies in anti-inflammation, anti-liver fibrosis, and/or anti-oxidation.
The term “anti-” used herein is referred to as the prevention and/or treatment of a disorder, an illness and/or a disease. The term of “prevention” or “preventing” herein is referred to avoid a subject from the occurrence of a disorder, an illness and/or a disease. The term of “treatment” or “treating” herein is referred to eliminate, stop, or reduce the progress/degree of a disorder, an illness and/or a disease.
The term “anti-inflammation” used herein is referred to as the prevention and/or treatment of an inflammation reaction in a subject. Said anti-inflammation comprises down-regulation of inflammatory cytokines, iNOS, COX-2, NFκB and/or the phosphorylation of ERK. The term of “anti-liver fibrosis” herein is referred to as the prevention and/or treatment of a liver fibrosis in a subject. Said anti-liver fibrosis comprises accumulating lipid in hepatic stellate cells, down-regulating the proliferation of hepatic stellate cells, inhibiting the mobility of hepatic stellate cells, preventing the activation of hepatic stellate cells, decreasing the synthesis of ECM proteins, or a combination thereof. The term of “anti-oxidation” or “anti-oxidant activity” herein is referred to the ability of scavenging, capturing, and neutralizing free radicals.
The term “effective amount” used is herein referred to an amount that is sufficient to perform the aforesaid efficacies (anti-inflammation, anti-liver fibrosis, and/or anti-oxidation). The effective amount obtained in the following animal model (mice) is 0.06 to 0.5 mg/kg body weight/day and the dosage can be calculated as 0.6 to 5 mg/60 kg body weight/day for human.
In one aspect of the present invention, an extract of Spiranthes sinensis is provided; more specifically said extract is an ester extract. Said ester extract is prepared at least by the following steps: obtaining a plant of Spiranthes sinensis; mixing said plant with a solvent to obtain a liquid; and drying said liquid to obtain said ester extract of Spiranthes sinensis. 
Said plant of Spiranthes sinensis may be any parts of Spiranthes sinensis including but not limit to: a root, a stem, a leaf or a whole plant thereof. Preferably, said plant of Spiranthes sinensis is dried before being proceeded to the following steps. More preferably, said plant of Spiranthes sinensis is dried and ground into proper size so that the interaction between said plant and said solvent can be more effective.
Said solvent is ethyl acetate, acetone, n-hexane, or a combination thereof. Preferably, said solvent is ethyl acetate. In a preferable embodiment of the present invention, an ethyl acetate of a concentration of 5 to 100% (v/v) is used; wherein said concentration is based on the total volume of said ethyl acetate; more preferably, is 10% to 100% (v/v).
Said mixing is conducted at 20 to 28° C. Preferably, said mixing is conducted for 1 to 2 days. Alternatively, said mixing can be performed by immersing said plant into said solvent.
Said drying can be achieved by vacuum drying, freeze-dried, lyophilization or a combination thereof. In an alternative embodiment, said extract is in the form of powder after said drying.
In another aspect of the present invention, a pharmaceutical composition is provided, which comprises 5 to 100 μg/mL of said Spiranthes sinensis extract based on the total volume of said pharmaceutical composition. Said pharmaceutical composition has an effective amount of 0.6 to 5 mg/60 kg body weight/day for providing efficacies in anti-inflammation, the anti-liver fibrosis, and/or anti-oxidation.
In yet another aspect of the present invention, a method for anti-inflammation, anti-liver fibrosis and/or anti-oxidation, is provided. The method comprises administrating an effective amount of the ester extract to a subject. Preferably, said ester extract is administrated to said subject in the form of said pharmaceutical composition while the effective amount of the ester extract is 0.6 to 5 mg/60 kg body weight/day.
In a preferable embodiment of the present invention, the route of said administrating is via oral administration, intravenous injection, or a combination thereof.
In a preferable embodiment of the present invention, said pharmaceutically acceptable carrier is water, alcohol, glycerol, chitosan, alginate, chondroitin, Vitamin E, mineral oil, dimethyl sulfoxide (DMSO), or a combination thereof.
The following embodiments recite the research of the present invention. In order to clearly describe the present invention, the researches of the present invention are divided into three parts. The first part was made by an in vitro RAW 264.7 macrophage cell culture system, including the examination in NO content, pro-inflammatory cytokines, nitric oxide synthesis, and anti-oxidation. The second part was performed in a mice model including examining the anti-inflammation activity and the effect on MMP 9 activity in vivo. The last part of the research was made by in vitro THSCs and NHSCs culture system, including examination in fatty acid storage, collagen synthesis, MMP activity, expression of fibrosis-related genes.
The following embodiments are recited for further explaining the advantages of the present invention but not for limiting the claim scope of the present invention.
Example 1: Preparation of Spiranthes sinensis Extract of the Present Invention
The dried Spiranthes sinensis was obtained from a local market, Hualien, Taiwan, in August, 2013. The dried Spiranthes sinensis was ground into powder and extracted with 100% ethyl acetate (preferably, 10 fold dry weight volumes) for one week at room temperature (RT). The liquid extract was in vacuo evaporated to produce its dried powder with a yield of 20.9% on a dry weight basis. For the following experiments, a stock composition was made by dissolving said extract in DMSO at a concentration of 1 mg/mL based on the total volume of said DMSO.
1st Part Example 2-Example 5
RAW 264.7 Macrophage Cell Culture System
Data were expressed as means±SEM. Statistical comparisons of the results were made using one-way analysis of variance (ANOVA). Means within each column followed by the different letters are significantly different at p<0.05 by Tukey's test.
Example 2: Cell Culture and Viability
[RAW264.7]
The RAW264.7 murine macrophage cell line, which is commonly used for study of inflammation, was purchased from the Bioresource Collection and Research Center (BCRC, Taiwan). The cells were incubated at 37° C. in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Gibco Inc., NY, USA) supplemented with 100 U/mL penicillin, 100 μg/mL sodium pyruvate and 10% fetal bovine serum (FBS; Gibco Inc.).
The cells were also treated with 0.01% DMSO as a vehicle control. The cells were seeded into 12-well plates at a density of 6×105 cells/well in 24 h then incubated with 0.1% BSA with serum-free medium 3 h, treated with various concentrations (5, 10, 20, 50, and 100 μg/mL) of the present Spiranthes sinensis extract and then incubated in the presence of LPS (0.4 or 1 μg/mL) for additional 4 h.
[Cell Viability]
In order to examine the bio-compatibility of the extract of the present invention, a MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Invitrogen), a colorimetric based assay) was performed in this example.
Briefly, 8×103 of cells per well was seeded in 96-well plates and were incubated in 5% CO2 at 37° C. for overnight. Cells were treated different concentrations (5, 20, 50, and 100 μg/mL) of Spiranthes sinensis crude extract for 24 h, after incubation 20 μL (5 mg/mL) of MTT solution was added per well and further incubated for 4 h. The media was removed, and formazan was solubilized by adding 100 μL/well of DMSO (Sigma-Aldrich) and OD was measured at 570 nm using a microplate reader (ELISA reader, Thermo Labsystems). Percentage of viable cells was estimated by comparing with untreated control cells. The average absorbance value of the control cells was taken as 100% viability.
The results showed the present extract of Spiranthes sinensis substantially has no harm to the cells at given dosages (FIG. 1).
Example 3: The Effect of the Present Spiranthes sinensis Extract on NO2-Production
NO2-production was determined in the cell culture supernatants by the Griess reaction. RAW 264.7 cells were cultured and treated as recited in the above Example 2 and the supernatant was collected. 200 μL/well of the supernatant were placed in a 96 well plate and 20 μL of 6.5 M hydrochloric acid and 20 μL of 37.5 mM sulfanilic acid were added. After incubation for 10 min at room temperature, 20 μL of 12.5 mM N (1-napthyl) ethylendiamine was added. Optical density was read at 550 nm 30 min later and compared with a standard curve. The results (FIG. 2) showed the inhibition effect of the present Spiranthes sinensis extract on NO release in a dose-dependent manner.
Example 4: The Effect of the Present Spiranthes sinensis Extract on the Expression of iNOS, Pro-Inflammatory Cytokines, COX-2, NFκB and the Phosphorylation and ERK
RAW 264.7 cells were cultured and treated as recited in the above example 2. The expressions of iNOs, TNF-Alpha, IL 1, and IL 6 of the cells were examined by Real-time PCR while the expression of Cox-2, NFκB, p-ERK and ERK were examined by Western blot.
[Real-Time PCR]
Total RNA was prepared from freshly harvested RAW 264.7 cells and was isolated using Trizol extraction. RNA samples were frozen at −80° C. until analyzed. cDNA was then made using an M-MLV RT kit (Invitrogen, Carlsbad, Calif.) according to the manufacturer's protocol.
The expression of selected cytokine and related genes in mouse tissue and in RAW264.7 cells exposed to the present Spiranthes sinensis extract was determined by the SensiFAST SYBR No-ROX Kit (BIOLINE, London, UK), as described previously. PCR cycling conditions for TNF-Alpha included a 95° C. heating step for 2 min at the beginning of every run. The tubes were then cycled at 95° C. for 5 sec, annealed at 60° C. for 10 sec, and extended at 72° C. for 20 sec for forty cycles. PCR conditions for iNOs, IL6, and IL1 included a 95° C. heating step for 2 min at the beginning of every run. The tubes were then cycled at 95° C. for 5 sec, annealed at 64° C. for 10 sec, and extended at 72° C. for 20 sec for thirty-eight cycles. In all cases, optical data were collected during the annealing phase. In order to quantify expression represented by each of the PCR products, an internal reference by β-actin. The primers used in the real-time PCR of this example are listed in the following table 1.







TABLE 1







Primers for Real-time PCR









Name
Sequences 5′->3′
SEQ ID NO





β-actin Forward
AGT GGT ACG ACC AGA GGC ATA C
SEQ ID NO 01


 


β-actin Reverse
ATG GGT CAG AAG GAC TCC TAC G
SEQ ID NO 02


 


iNOs Forward
TCC TAC ACC ACA CCA AAC
SEQ ID NO 03


 


iNOs Reverse
CTC CAA TCT CTG CCT ATC C
SEQ ID NO 04


 


TNF Alpha Forward
AAC CCT CTG GCC CAA GGA
SEQ ID NO 05


 


TNF Alpha Reverse
GGC GAC GGG CTT ATC TGA
SEQ ID NO 06


 


Interleukins (IL)-6 Forward
ATG AAC TCC CTC TCC ACA AGC
SEQ ID NO 07


 


Interleukins (IL)-6 Reverse
TGG CTT TGT CTG GAT TCT TTC
SEQ ID NO 08


 


Interleukins (IL)-1 Forward
AAA GGG GAC TTG AAG AGA G
SEQ ID NO 09


 


Interleukins (IL)-1 Reverse
CTG CTT GAG AGG TGC TGA TGT
SEQ ID NO 10









[Western Blot]

The concentrations of proteins from whole-cell lysates were determined by Bradford assay. Equal amounts (30 μg) of protein were separated by SDS-PAGE and transferred onto PVDF membranes. The blots were then blocked overnight with 5% (wt/vol) nonfat dry milk, and probed with anti-Cox-2 antibodies, anti-NFκB antibodies, phospho-specific antibodies to ERK and anti-ERK antibodies in 5% (wt/vol) BSA dissolved in TBST [20 mM Tris-HCl buffer, pH 7.6, containing 137 mM NaCl and 0.05% (vol/vol) Tween-20]. With the use of horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody, bound antibodies were detected by enhanced chemiluminescence.
According to the results shown in FIGS. 3 and 4, the present Spiranthes sinensis extract diminished LPS-induced inflammatory cytokine and iNOS transcription, expression of Cox-2 and NFκB as well as the phosphorylation of ERK in a dose-dependent manner.
Example 5: The Anti-Oxidant Activity of the Present Spiranthes sinensis Extract
The DPPH radical scavenging activity of Spiranthes sinensis was determined as previously described (Sharma and Bhat 2009). In brief, the reaction mixtures containing various concentrations of the present Spiranthes sinensis extract and 0.04 mM DPPH in a 96-well microtiter plate were incubated at 37° C. for 30 min, and the absorbance was measured at 490 nm. Vitamin C was used as a positive control. The anti-oxidant activity of the present Spiranthes sinensis extract was shown based on the anti-oxidant activity of 0.1 mg of Vitamin C as 100%. The results shown in FIG. 5 indicate that the present Spiranthes sinensis extract has comparable ability with Vitamin C in anti-oxidation.
2nd Part Example 6-Example 7
Mice Model
Data were expressed as means±SEM. Statistical comparisons of the results were made using one-way analysis of variance (ANOVA). Means within each column followed by the different letters are significantly different at p<0.05 by Tukey's test.
Example 6: The Anti-Inflammatory Activity of the Present Spiranthes sinensis Extract
[Animal]
Male C57BL/6 mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan) and kept at controlled environmental conditions with room temperature (22±2° C.), humidity (50±10%). The 12 h light (0600 am-1800 pm) and dark cycle was maintained throughout the study. Animal experiments were approved by the National Dong-Hwa University Animal Ethics Committee and were used according to the “Guide for the Care and Use of Laboratory Animals” of National Dong-Hwa University.
Animals were separated into 6 groups, which are respectively injected with various dosage of the present Spiranthes sinensis extract (0.06, 0.3, 0.5 mg/kg body weight), 0.3 mg/kg body weight of trimcinolone (positive control), 3 mg/kg body weight of sevatrim (positive control) and DMSO (negative control). To produce a chronic inflammatory response, mice were then injected with 20 μL of Complete Freund's Adjuvant (Mycobacterium tuberculosis; Sigma, St. Louis, Mo., USA) subcutaneously in the plantar surface of the right hind paw (i.pl.). The left hind paw was taken as a control.
The anti-inflammatory effect was examined for 4 days and the swelling index was recorded every day after the injection. The swelling index was calculated by the following formula below and presented as a ratio based on the swelling index before injection (Day 0).




  
    Length
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    ⁢
    
      
    
    ⁢
    right
    ⁢
    
      
    
    ⁢
    hindpaw
    ⁢
    
      
    
    ⁢
    
      (
      
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        ⁢
        
          
        
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        paw
      
      )
    
  
  
    Length
    ×
    Width
    ⁢
    
      
    
    ⁢
    of
    ⁢
    
      
    
    ⁢
    left
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    ⁢
    hindpaw
    ⁢
    
      
    
    ⁢
    
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        uninjected
        ⁢
        
          
        
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        paw
      
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The results were shown in FIG. 6 and the area under curve (AUC) was calculated and presented in the bar diagram of FIG. 7. The results show that by injecting with the present Spiranthes sinensis extract, the inflammation (represented by the swelling index) was reduced significantly in a dosage-dependent manner.
Example 7: The Effect of the Present Spiranthes sinensis Extract on the Activity of MMP 9
Mice tested in the experiment of example 6 were subjected to further examination for the activity of MMP 9 (matrix metalloproteinase-9). Blood samples were collected from the tail vein at Day 4. The blood were pre-heated at 55° C. with 2× loaded dye (0.125 M Tris-HCl, pH 6.8, 4% SDS, 0.04% Bromophenol blue, 20% Glycerol). 8% SDS-PAGE gels were prepared containing 10% gelatin. 5 μL of blood sample was loaded into gel and electrophoresis separation was performed at 80V for 2-3 h. After electrophoresis, gel was washed 2 times in 50 mL of 2.5% Triton X-100 per gel, and then incubated in developing buffer (0.05 M Tris-HCl, pH 8.8, 5 mM CaCl2, 0.02% NaN3) at 37° C. for 16 h. Finally, gel was stained in 0.1% Coomassie blue R-250 (Bio-Rad) for 4 h and then destained by fixing buffer (45% methanol, 10% acetic acid). Gels were scanned using Epson scanner and quantified using multi-gauge software (Fujifilm). This method has capability to detect the active form and latent form of MMP 9 at their correct molecular weight size.
The results in FIG. 8 indicated an inhibitory effect on the activity of MMP 9 by treating with the present Spiranthes sinensis extract. MMP 9 is a kind of gelatinase and is recognized to be up-regulated during liver fibrosis. Therefore, the results hinted the present Spiranthes sinensis extract's efficacy in treating inflammation.
3rd Part Example 8-Example 12
THSCs and NHSCs Cell Culture System
Data were expressed as means±SEM. Statistical comparisons of the results were made using one-way analysis of variance (ANOVA). Means within each column followed by the different letters are significantly different at p<0.05 by Tukey's test.
Example 8: Cell Culture and Viability
[NHSCs and THSCs]
Hepatic Stellate Cells (HSCs), one of non-parenchymal cell, is the major cell type involved in liver fibrosis, which is the formation of scar tissue in response to liver damage. In normal liver, HSCs keep in quiescent form and is principal storage site of vitamin A and lipids. The stored amount of vitamin A decreases progressively in liver injury. When the liver is damaged, stellate cells are changed into an activated state (Hjelkrem, Morales et al. 2012). The activated stellate cell is characterized by proliferation, contractility, and chemotaxis. Therefore, the quiescent form of HSC presents the indication of liver condition.
Non chemical-induced hepatic stellate cells (NHSCs >95% purity) were isolated from male Sprague-Dawley rat liver, and thioacetamide (TAA)-induced hepatic stellate cells (THSCs >95% pure) were isolated from TAA (Fluka) induced fibrotic liver of male Sprague-Dawley rat as our previous study described. Both the cell lines were maintained in DMEM (pH 7.4) supplemented with 10% FBS, and incubated at 37° C. with 5% CO2 in water saturated incubator. Media was changed in every 2 days and the cells were passage during 80%-90% confluence using trypsin/EDTA (Sigma-Aldrich).
[Cell Viability]
In order to examine the bio-compatibility of the extract of the present invention, a MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Invitrogen), a colorimetric based assay) was performed in this example.
Briefly, 1×104 of cells per well (for NHSCs and THSCs) was seeded in 96-well plates and were incubated in 5% CO2 at 37° C. for overnight. Cells were treated different concentrations (5, 20, 50, and 100 μg/mL) of Spiranthes sinensis crude extract for 24 h, after incubation 20 μL (5 mg/mL) of MTT solution was added per well and further incubated for 4 h. The media was removed, and formazan was solubilized by adding 100 μL/well of DMSO (Sigma-Aldrich) and OD was measured at 570 nm using a microplate reader (ELISA reader, Thermo Labsystems). Percentage of viable cells was estimated by comparing with untreated control cells. The average absorbance value of the control cells was taken as 100% viability.
The results showed that the present extract of Spiranthes sinensis substantially has no harm to the cells at given dosages (FIG. 9).
Example 9: The Effect of the Present Spiranthes sinensis Extract in Lipid Accumulation
In this example, an Oil red O staining assay and an AdipoRed assay were used for examining the effect of the present Spiranthes sinensis extract in Lipid accumulation.
[Oil Red O Staining Assay]
Briefly, 1×105 of cells per well (NHSCs or THSCs) were seeded in 6-well plates and were incubated in 5% CO2 at 37° C. overnight for confluence. Afterward, cells were treated with various concentrations (5, 20, and 50 μg/mL) of the present Spiranthes sinensis extract for 24 h, respectively. Cells were washed twice with PBS, and then fixed by 3.7% paraformaldehyde (pre-warmed) for 10 min. Oil red O stock solution 0.5% (w/v) in isopropanol (Fluka) was diluted with 60% volume of water, filtered, and stained at RT under the dark for 1 h. Cells were washed with 50% isopropanol for 5 sec and counterstained with Hematoxylin (Sigma-Aldrich) for 1 min. Cells were washed with distilled water for three times and stored using glycerol. Images were acquired using ZEISS inverted microscope connected with Canon 700 D camera.
The results are shown in FIG. 10. It is noted that, the present Spiranthes sinensis extract increases the abundant perinuclear lipid droplets both in NHSCs and THSCs when compared with the control group. The results of FIG. 10 are also quantified as the bar chart shown in FIG. 11 from which it is more obvious that the treatment of the present Spiranthes sinensis extract respectively increases the fatty acid storage of NHSCs and THSCs in about 3 fold and about 1.5 fold compare with the untreated control groups.
[AdipoRed Assay]
1×104 of cells per well were seeded in 96-well plates and incubated in 5% CO2 at 37° C. overnight. Cells were treated with various concentrations (5, 20, 50, and 100 μg/mL) of Spiranthes sinensis extract for 48 h. After incubation, the culture supernatant was removed and each well carefully rinsed with 200 μL of PBS. And then, each well was filled with 200 μL of PBS and 5 μL of AdipoRed Reagent (Lonza, Walkersville, Md., USA). After incubation at room temperature for 10 min, placed the plate in the fluorimeter and fluorescence with excitation at 485 nm and emission at 572 nm (Multimode Plate Reader, PerkinElmer Inc Waltham, Mass., USA) was acquired.
The result (FIG. 12) showed that the Spiranthes sinensis extract increased the lipid accumulation in NHSCs (1.8 fold) and THSCs (1.4 fold) when compared with the control group. The observation was consistent with the results of the aforesaid Oil red O staining assay and further verified the capability of the present Spiranthes sinensis extract in lipid accumulation.
Example 10: The Effect of the Present Spiranthes sinensis Extract in Collagen Accumulation
In this example, a Sirius Red stain was used for examining the effect of the present Spiranthes sinensis extract in Collagen accumulation. Briefly, 3×104 of cells per well were seeded in 12-well plates and were incubated in 5% CO2 at 37° C. overnight for confluence. Afterward, cells were treated with various concentrations (5, 20, and 50 μg/mL) of the present Spiranthes sinensis extract for 24 h, respectively. Cells were washed twice with PBS, and then fixed by 3.7% paraformaldehyde (pre-warmed) for 10 min. Picro-Sirius Red solution (ScyTek, Logan, Utah, USA) was stained at RT under the dark for 1 h. Cells were washed twice with ddH2O and counterstained with Hematoxylin (Sigma-Aldrich) for 1 min. Cells were washed with distilled water for three times and stored using glycerol. Images were acquired using ZEISS inverted microscope connected with Canon 700 D camera.
The results are shown in FIG. 13. Both NHSCs and THSCs treated with the present Spiranthes sinensis extract (5, 20, and 50 μg/mL) of for 24 h show reduced perinuclear collagen comparing with the control groups.
Example 11: The Effect of the Present Spiranthes sinensis Extract on the Activity of MMP 2
MMPs, a serials protein enzyme of biodegradable ECM, play an important role in normal or diseased cells. Liver fibrosis is a wound healing response, in which the damaged regions are refilled by ECM. Fibrosis reflects a balance between matrix production and degradation, though the degradation of ECM is a key determining factor event in hepatic fibrosis. MMPs contribute to the regression of liver fibrosis through the cleavage of fibrillar ECM and promotion of apoptosis among the activated HSCs. Hence, MMPs play dual roles of both bad and good in liver fibrosis depending on the timing.
The present Spiranthes sinensis extract has shown an inhibitory effect on the activity of MMP 9 (See Example 7). In this example, the activity of MMP 2 was also determined by gelatin zymography. 2×105 of HSCs were seeded into 6-well plates, after 70 to 80% confluence; cells were starved in DMEM containing 0.1% BSA for 6 h, and then treated with different concentrations (5, 20, 50, and 100 μg/mL) of the present Spiranthes sinensis extract for 24 h, respectively. The conditioned medium was collected, centrifuged at 12,000×g at 4° C. for 30 min to remove the cell debris. The supernatant was collected and quantified using Bradford dye (Bio-Rad). 8% SDS-PAGE gels were prepared containing 10% gelatin. Proteins were pre-heated at 55° C. with 2× loaded dye (0.125 M Tris-HCl, pH 6.8, 4% SDS, 0.04% Bromophenol blue, 20% Glycerol). 7.5 μg of protein sample was loaded into gel and electrophoresis separation was performed at 80V for 2-3 h. After electrophoresis, gel was washed 2 times in 50 mL of 2.5% Triton X-100 per gel, and then incubated in developing buffer (0.05 M Tris-HCl, pH 8.8, 5 mM CaCl2, 0.02% NaN3) at 37° C. for 16 h. Finally, gel was stained in 0.1% Coomassie blue R-250 (Bio-Rad) for 4 h and then destained by fixing buffer (45% methanol, 10% acetic acid). Gels were scanned using Epson scanner and quantified using multi-gauge software (Fujifilm).
The results were shown in FIG. 14; wherein the activity of MMP-2 was quantified by densitometer. The MMP-2 activity was reduced in both NHSCs and THSCs treated with 5, 20, 50, and 100 μg/mL of the present Spiranthes sinensis extract, respectively.
Example 12: The Effect of the Present Spiranthes sinensis Extract on the Expression of COL1 I, COL1 III, TGF-β R1, TGF-β R2, RXRα, NrF2 and PPARγ
The expressions of COL1 III (collagen alpha 1 type III), COL1 I (collagen alpha 1 type I), TGF-β R1 (TGF-β Receptor 1), TGF-β R2 (TGF-β Receptor 2), RXRα (retinoid X receptor), NrF2 (Nuclear factor (erythroid-derived 2)-like 2) and PPARγ (peroxisome proliferator-activated receptor γ) were examined by Real-time PCR; wherein 18S was used as a control.
For RNA isolation and cDNA synthesis, 4×105 of HSCs were seeded into 6-well plates, after 70 to 80% confluence, and then treated with different concentrations (5, 20, 50, and 100 μg/mL) of the present Spiranthes sinensis extract in DMEM containing 5% FBS for 8 and 12 h, respectively. The positive control group was treated with 25 μM of curcumin. Total RNA was prepared from freshly harvested HSCs and was isolated using Trizol extraction. RNA samples were frozen at −80° C. until analyzed. cDNA was then made using an M-MLV RT kit (Invitrogen, Carlsbad, Calif.) according to the manufacturer's protocol.
For Real-time PCR, the SensiFAST SYBR No-ROX Kit (BIOLINE, London, UK) was used. PCR cycling conditions for RXRα, NrF2 and PPARγ included a 95° C. heating step for 2 min at the beginning of every run. The tubes were then cycled at 95° C. for 5 sec, annealed at 64° C. for 10 sec, and extended at 72° C. for 20 sec for forty cycles. PCR conditions for collagen alpha 1 type III, collagen alpha 1 type I, TGF-β Receptor 1, and TGF-β Receptor 2 included a 95° C. heating step for 2 min at the beginning of every run. The tubes were then cycled at 95° C. for 5 sec, annealed at 62° C. for 10 sec, and extended at 72° C. for 20 sec for 45 cycles. In all cases, optical data were collected during the annealing phase. In order to quantify expression represented by each of the PCR products, an internal reference by 18S. The primer sets used in this example were listed in the following table 2.







TABLE 2







primer sets for real-time PCR









Name of the




primer
Sequences 5′->3′
SEQ ID NO





18S Forward
ACG GAC CAG AGC GAA AGC AT
SEQ ID NO 11


 


18S Reverse
TGT CAA TCC TGT CCG TGT CC
SEQ ID NO 12


 


PPARγ Forward
AGC ATG GTG CCT TCG CTG ATG C
SEQ ID NO 13


 


PPARγ Reverse
AAG TTG GTG GGC CAG AAT GGC A
SEQ ID NO 14


 


Collagen alpha 1 
GAA AAA ACC CTG CTC GGA ATT
SEQ ID NO 15


type III Forward




 


Collagen alpha 1 
GGA TCA ACC CAG TAT TCT CCA CTC T
SEQ ID NO 16


type III Reverse




 


TGF-β Receptor 1 
CAT CGG CAA AGG TCG GTT T
SEQ ID NO 17


Forward




 


TGF-β Receptor 1 
AAT ATC TTC ACG GCA ACT TCT TCT C
SEQ ID NO 18


Reverse




 


TGF-β Receptor 2 
TCA CCT ACC ACG GCT TCA CTC T
SEQ ID NO 19


Forward




 


TGF-β Receptor 2 
CGC CCT TTT CTT TTC CTT CA
SEQ ID NO 20


Reverse




 


RXRα Forward
GCC GGC CTC TGA CTG TGA
SEQ ID NO 21


 


RXRα Reverse
GCA CCA CAA TGT CCC AGT GA
SEQ ID NO 22


 


NrF2 Forward
GAC AAA CAT TCA AGC CGA TTA GAG G
SEQ ID NO 23


 


NrF2 Reverse
ACT TTA TTC TTC CCT CTC CTG CGT
SEQ ID NO 24









FIG. 15 showed that NHSCs and THSCs treated with the present Spiranthes sinensis extract for 8 h had reduced expression of COL1 I, COL1 III, TGFBR1, and TGFBR2.
FIGS. 16 and 17 showed that RXRα, NrF2 and PPARγ gene expression increased in both NHSCs and THSCs treated with the present Spiranthes sinensis extract for 12 h treatment.
REFERENCES


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    2. Sun, M. Effects of population size, mating system, and evolutionary origin on genetic diversity in Spiranthes sinensis and S-hongkongensis. Conserv Biol 10, 785-795 (1996).
    3. Lin, Y. L., Huang, R. L., Don, M. J. & Kuo, Y. H. Dihydrophenanthrenes from Spiranthes sinensis. J Nat Prod 63, 1608-1610 (2000).
    4. Lin, Y. L., Wang, W. Y., Kuo, Y. H. & Liu, Y. H. Homocyclotirucallane and two dihydrophenanthrenes from Spiranthes sinensis. Chem Pharm Bull 49, 1098-1101 (2001).
    5. Li, C. Y., et al. New dimeric phenanthrene and flavone from Spiranthes sinensis. J Asian Nat Prod Res 15, 417-421 (2013).
    6. Peng, J. Y., et al. Two new prenylated coumarins from Spiranthes sinensis (Pers.) Ames. J Asian Nat Prod Res 10, 279-283 (2008).
    7. Nakerakanti, S. & Trojanowska, M. The Role of TGF-beta Receptors in Fibrosis. The open rheumatology journal 6, 156-162 (2012).
    8. Fu, Y., Zheng, S., Lin, J., Ryerse, J. & Chen, A. Curcumin protects the rat liver from CC14-caused injury and fibrogenesis by attenuating oxidative stress and suppressing inflammation. Molecular pharmacology 73, 399-409 (2008).
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## Claims

1. A method for treating inflammation in a subject in need thereof comprising administering an effective amount of an ethyl acetate extract of Spiranthes sinensis to said subject, wherein said effective amount is 0.6 to 5 mg/60 kg body weight/day, and wherein said ethyl acetate extract of Spiranthes sinensis is obtained by:
i.) obtaining a Spiranthes sinensis plant;
ii.) mixing said Spiranthes sinensis plant with ethyl acetate to obtain a mixture, wherein ethyl acetate is the only solvent used; and
iii.) drying said mixture to obtain the ethyl extract of Spiranthes sinensis.

2. The method of claim 1, wherein said administrating is via oral administration, intravenous injection, or a combination thereof.

3. The method of claim 1, wherein said Spiranthes sinensis extract is administrated with a pharmaceutically acceptable carrier; selected from the group consisting of water, alcohol, glycerol, chitosan, alginate, chondroitin, Vitamin E, mineral oil, dimethyl sulfoxide (DMSO), or a combination thereof.

4. The method of claim 1, wherein said plant is dried and/or ground before mixing with said solvent.

5. The method of claim 1, wherein said mixing is conducted at 20 to 28° C.

6. The method of claim 1, wherein said mixing is conducted for 1 to 2 days.

7. The method of claim 1, wherein said drying is achieved by vacuum drying.

8. A method for treating inflammation in a subject in need thereof comprising:
a.) preparing an ethyl acetate extract of Spiranthes sinensis by:
i.) obtaining a Spiranthes sinensis plant;
ii.) mixing said Spiranthes sinensis plant with ethyl acetate to obtain a mixture, wherein said ethyl acetate is the only solvent used; and
iii.) drying said mixture to obtain said extract; and

b.) administering 0.6 to 5 mg/60 kg body weight/day of the ethyl acetate extract of Spiranthes sinensis to the subject.