METHOD FOR PREPARING FENUGREEK EXTRACT AND PHARMACEUTICAL COMPOSITION COMPRISING FENUGREEK EXTRACT

Abstract

Provided is a method for preparing a fenugreek extract, including the steps of: (1) preparing a fenugreek plant tissue, soaking the fenugreek plant in water from 0.5 hour to 5 hours and between 25 C. and 100 C. for extraction, and (2) filtering the extracted fenugreek plant tissue to obtain the fenugreek extract. Also provided is an active substance that comprises the fenugreek extract obtained by the method. Also provided is a pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease, which comprises the active substance and a pharmaceutically acceptable carrier.

Claims

1. A method for preparing a fenugreek extract, comprising the steps of: (1) preparing a fenugreek plant tissue, soaking the fenugreek plant tissue in water for 0.5 hour to 5 hours and at a temperature between 25 C. and 100 C. for extraction, to obtain an extracted fenugreek plant tissue; and (2) filtering the extracted fenugreek plant tissue to obtain the fenugreek extract.

2. The method according to claim 1, wherein the fenugreek plant tissue is fenugreek seed or whole-plant fenugreek.

3. The method according to claim 1, wherein the water is at a weight ratio of 1 to 20 times of the fenugreek plant tissue.

4. The method according to claim 1, wherein the extraction period is between 0.5 hour and 3 hours, and the temperature of the extraction is between 50 C. and 95 C.

5. The method according to claim 2, wherein the extraction period is between 0.5 hour and 3 hours, and the temperature of the extraction is between 50 C. and 95 C.

6. The method according to claim 3, wherein the extraction period is between 0.5 hour and 3 hours, and the temperature of the extraction is between 50 C. and 95 C.

7. A fenugreek extract, which is obtained by the method according to claim 1.

8. An active substance for preventing or treating nonalcoholic fatty liver disease, wherein the active substance comprises the fenugreek extract according to claim 7.

9. The active substance according to claim 8, wherein the active substance comprises red yeast rice extract, rice bran extract, artichoke extract, taurine, ginseng extract or any combination thereof.

10. The active substance according to claim 8, wherein the active substance comprises 12 to 16 parts by weight fenugreek extract, 3 to 14 parts by weight red yeast rice extract, 0.5 to 5 parts by weight rice bran extract, 0.5 to 3 parts by weight artichoke extract, 0.8 to 25 parts by weight taurine, and 0.1 to 10 parts by weight ginseng extract.

11. A pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease, wherein the pharmaceutical composition comprises the active substance according to claim 8 and a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 is an index histogram of lipid droplets formation in Group A (fenugreek water extract obtained by preparation example 1) of the present invention; each group is duplicate, meanstandard deviation (SD), Student's t test, * for p<0.05, ** for p<0.01, *** for p<0.001, and MTS as an analytical method for cell viability.

[0026] FIG. 2 is an index histogram of lipid droplets formation in Group B (fenugreek alcohol extract obtained by preparation example 2) of the present invention; each group is duplicate, meanSD, Student's t test, * for p<0.05, ** for p<0.01, and *** for p<0.001.

[0027] FIG. 3 is an index histogram of lipid droplets formation in Group C (fenugreek water extract obtained by preparation example 1) of the present invention; each group is duplicate, meanSD, Student's t test, * for p<0.05, ** for p<0.01, and *** for p<0.001.

[0028] FIG. 4 is an index histogram of lipid droplets formation in Group D (fenugreek alcohol extract obtained by preparation example 2) of the present invention; each group is duplicate, meanSD, Student's t test, * for p<0.05, and *** for p<0.001.

[0029] FIG. 5 is a graph of the H&E staining of liver tissue slides of the control group, high fat diet group and YE2 group (fenugreek water extract obtained by preparation example 1) of the present invention.

[0030] FIG. 6 is a histogram of the macrovesicular steatosis score of the control group, the high fat diet group and the YE2 group, meanSD, one-way ANOVA; * represents a comparison with the control group p<0.05; represents a comparison with the high fat diet group p<0.05.

[0031] FIG. 7 is a histogram of the microvesicular steatosis score of the control group, the high fat diet group and the YE2 group, meanSD, one-way ANOVA; * represents a comparison with the control group p<0.05; represents a comparison with the high fat diet group p<0.05.

[0032] FIG. 8 is a histogram of the ballooning degeneration score of the control group, the high fat diet group and the YE2 group, meanSD, one-way ANOVA; * represents a comparison with the control group p<0.05; represents a comparison with the high fat diet group p<0.05.

[0033] FIG. 9 is a histogram of the total cholesterol content of the control group, the high fat diet group and the YE2 group, meanSD, one-way ANOVA; * represents a comparison with the control group p<0.05; represents a comparison with the high fat diet group p<0.05; mg/L.

[0034] FIG. 10 is a histogram of the serum GOT level of the control group, the high fat diet group and the YE2 group, meanSD, one-way ANOVA; * represents a comparison with the control group p<0.05; represents a comparison with the high fat diet group p<0.05; U/L.

[0035] FIG. 11 is a histogram of the serum GPT level of the control group, the high fat diet group and the YE2 group, meanSD, one-way ANOVA; * represents a comparison with the control group p<0.05; represents a comparison with the high fat diet group p<0.05; U/L.

[0036] FIG. 12 is an index histogram of lipid droplets formation of the fenugreek water extract formula of the present invention; each group is four-repeat, meanSD, Student's t test, *** for p<0.001.

[0037] FIG. 13 is a histogram of cell viability of HepG2 cells; each group is four-repeat, meanSD, Student's t test, *** for p<0.001.

[0038] FIG. 14 is a histogram of the serum triglyceride levels; each group is ten-repeat, meanSD; values with different superscripts are significantly different at p<0.05 by one-way ANOVA with Duncan's multiple range test.

[0039] FIG. 15 is a histogram of the serum cholesterol levels; each group is ten-repeat, meanSD; values with different superscripts are significantly different at p<0.05 by one-way ANOVA with Duncan's multiple range test.

[0040] FIG. 16 is a histogram of the serum non-esterified fatty acid (referred to as serum NEFA) levels; each group is ten-repeat, meanSD; values with different superscripts are significantly different at p<0.05 by one-way ANOVA with Duncan's multiple range test.

[0041] FIG. 17 is a histogram of the serum ALT levels; each group is ten-repeat, meanSD; values with different superscripts are significantly different at p<0.05 by one-way ANOVA with Duncan's multiple range test.

[0042] FIG. 18 is a histogram of the serum AST levels; each group is ten-repeat, meanSD; values with different superscripts are significantly different at p<0.05 by one-way ANOVA with Duncan's multiple range test.

[0043] FIG. 19 is a graph of the H&E staining of liver tissue slides of each group.

[0044] FIG. 20 is a histogram of the total liver cholesterol levels of each group; each group is ten-repeat, meanSD; values with different superscripts are significantly different at p<0.05 by one-way ANOVA with Duncan's multiple range test.

[0045] FIG. 21 is a histogram of the total liver triglyceride levels of each group; each group is ten-repeat, meanSD; values with different superscripts are significantly different at p<0.05 by one-way ANOVA with Duncan's multiple range test.

[0046] FIG. 22 is a histogram of the total liver non-esterified fatty acid (referred to as Total liver NEFA) levels of each group; each group is ten-repeat, meanSD; values with different superscripts are significantly different at p<0.05 by one-way ANOVA with Duncan's multiple range test.

[0047] FIG. 23 is a high performance liquid chromatography (referred to as HPLC) profile of the fenugreek water extract of the present invention for total saponin at OD 203 nm.

[0048] FIG. 24 is a HPLC profile of the fenugreek whole plant extract of the present invention for total saponin at OD 203 nm.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Preparation Example 1: Water Extraction of Fenugreek

[0049] The fresh or dried fenugreek seed was extracted with water, wherein the water was at a weight ratio of 1 to 20 times, preferably 5 to 10 times the weight of the fenugreek seed. The fenugreek seed was soaked in the water for a period from half hour to 5 hours and at a temperature between 25 C. to 100 C., preferably for a period from half hour to 3 hours and at a temperature between 50 C. to 95 C., and then filtered (solid-liquid separation, such as by porosity, weight or density, carried out by any known physical filtration method) to obtain the fenugreek water extract (referred to as YE2). The fenugreek water extract could be concentrated or dried by any known food processing method.

Preparation Example 2: Organic Solvent Extraction of Fenugreek

[0050] The fresh or dried fenugreek seed was extracted with 50% alcohol solvent, wherein the alcohol solvent was at a weight ratio of 1 to 20 times, preferably 5 to 10 times the weight of the fenugreek seed. The fenugreek seed was soaked in the alcohol solvent from half hour to 5 hours and between 25 C. to 100 C., preferably half hour to 3 hours and between 50 C. to 95 C., and then filtered to obtain the fenugreek alcohol extract (referred to as YE3). The fenugreek alcohol extract could be concentrated or dried by any known food processing method.

Example 1 Cell Experiment: Simultaneous Administration and Stimulation, for Simulation of Prevention Mechanism

[0051] (1) HepG2 cells (liver hepatocellular carcinoma cells) were seeded at 2.510.sup.4 cells/well and incubated for 24 hours.

[0052] (2) The incubated cells were divided into the following groups:

[0053] Control group (CG) was added with cell culture medium and 1% dimethyl sulfoxide (DMSO) to treat the HepG2 cells for 6 hours.

[0054] Lipid droplets induced group (LDIG) was added with 1% DMSO and 500 mM lipid droplets induced reagent [oleic acid (OA), C18:1 and paltimic acid (PA), C16:0 were dissolved in the cell culture medium at a volume ratio of 2:1 (v/v)] to treat the HepG2 cells for 6 hours.

[0055] Positive control group (PCG) was added with 0.01 g/mL phosphatidylcholine and 500 mM lipid droplets induced reagent to treat the HepG2 cells for 6 hours. Group A was added with the dried fenugreek water extract which was obtained by preparation example 1 and was dissolved in 1% DMSO to give a final concentration of 10.sup.6 m/mL, 10.sup.5 m/mL, 10.sup.4 m/mL, 10.sup.3 m/mL, 10.sup.2 m/mL and 10.sup.1 m/mL, respectively, simultaneously with 500 mM lipid droplets induced reagent to treat the HepG2 cells for 6 hours.

[0056] Group B was added with the dried fenugreek alcohol extract which was obtained by preparation example 2 and was dissolved in 1% DMSO to give a final concentration of 10.sup.6 m/mL, 10.sup.5 m/mL, 10.sup.4 m/mL, 10.sup.3 m/mL, 10.sup.2 m/mL and 10.sup.1 m/mL, respectively, simultaneously with 500 mM lipid droplets induced reagent to treat the HepG2 cells for 6 hours.

[0057] (3) The above groups were examined by cell viability assay (MTS assay) and oil red-O stain, and the oil red OD value/MTS OD value was used as the index of lipid droplets.

[0058] Referring to FIGS. 1 and 2, the fenugreek water extract and the fenugreek alcohol extract both dose-dependently inhibited lipid droplet formation in HepG2 cells. The half maximal inhibitory concentration (IC.sub.50) of group A was 0.1 ng/mL, and the IC.sub.50 of group B was 0.3 ng/mL. The fenugreek water extract at concentration 10.sup.5 g/mL in group A already has a great inhibitory effect.

Example 2 Cell Experiment: Stimulation after Administration, for Simulation of Prevention Mechanism

[0059] (1) HepG2 cells were seeded at 2.510.sup.4 cells/well and incubated for 24 hours.

[0060] (2) The incubated cells were divided into the following groups:

[0061] Control group (CG) was added with 1% DMSO to treat the HepG2 cells for 16 hours, and then treated with cell culture medium for 16 hours.

[0062] Lipid droplets induced group (LDIG) was added with 1% DMSO to treat the HepG2 cells for 16 hours, and then treated with 500 nM lipid droplets induced reagent for 16 hours.

[0063] Positive control group (PCG) was added with 0.01 g/mL phosphatidylcholine to treat the HepG2 cells for 16 hours, and then treated with 500 mM lipid droplets induced reagent for 16 hours.

[0064] Group C was added with the dried fenugreek water extract which was obtained by Preparation Example 1 and was dissolved in 1% DMSO to give a final concentration of 10.sup.6 m/mL, 10.sup.5 m/mL, 10.sup.4 m/mL, 10.sup.3 m/mL, 10.sup.2 m/mL and 10.sup.1 m/mL, respectively, and the HepG2 cells were treated for 16 hours, and then treated with 500 mM lipid droplets induced reagent for 16 hours.

[0065] Group D was added with the dried fenugreek alcohol extract which was obtained by Preparation Example 2 and was dissolved in 1% DMSO to give a final concentration of 10.sup.6 m/mL, 10.sup.5 m/mL, 10.sup.4 m/mL, 10.sup.3 m/mL, 10.sup.2 m/mL and 10.sup.1 m/mL, respectively, and the HepG2 cells were treated for 16 hours, and then treated with 500 mM lipid droplets induced reagent for 16 hours.

[0066] (3) The above groups were examined by cell viability assay (MTS assay) and oil red-O stain, and the oil red OD value/MTS OD value was used as the index of lipid droplets.

[0067] Referring to FIGS. 3 and 4, the IC.sub.50 of group C was 0.3 ng/mL, and the IC.sub.50 of group D was 0.9 ng/mL. The index value of the fenugreek water extract at concentration 10.sup.3 m/mL in group C was 0.38390.0203, having the greatest inhibitory effect on lipid droplets formation.

Example 3: Animal Experiment

[0068] (1) 7 weeks old male C57BL/6J mice were chosen for the experiment.

[0069] (2) Divided into the following groups:

[0070] Control group (CG): 6 mice were fed with chow diet, including 4.8% kcal fat with 0% kcal cholesterol for 12 weeks.

[0071] High fat diet (HFD) group: 6 mice were fed with high fat diet, including 42% kcal fat with 0.2% kcal cholesterol for 12 weeks.

[0072] YE2 group: 6 mice were fed with high fat diet simultaneously with 0.108 g/kg/day the dried fenugreek water extract obtained by Preparation Example 1 for 12 weeks.

[0073] (3) The mice were sacrificed, and the liver tissue slides were stained by hematoxylin and eosin stain (H & E stain), according to the grading criteria for macrovesicular steatosis established by 2005 Kleiner et al.: fraction 0 was <5% (percentage of macrovesicular in the liver tissue), fraction 1 was 5% to 33%, fraction 2 was >33% to 66%, fraction 3 was >66%; for microvesicular steatosis: fraction 0 represented no occurrence of microvesicular steatosis, fraction 1 represented occurrence of microvesicular steatosis; and for ballooning degeneration: fraction 0 represented no ballooning degeneration, fraction 1 represented a small amount of ballooning degeneration, fraction 2 represented majority of ballooning degeneration. In addition, mice blood was extracted and analyzed for total cholesterol, sGOT and sGPT.

[0074] Referring to FIG. 5, the liver of the HFD group was filled with adipose, hollow with loose organization, while the YE2 group was more similar to the control group. Referring to FIG. 6, compared with the HFD group, the macrovesicular steatosis was significantly decreased in the YE2 group. Referring to FIG. 7, the level of microvesicular steatosis in the YE2 group was similar to the HFD group. Referring to FIG. 8, the YE2 group compared with the HFD group, the level of the ballooning degeneration was significantly decreased. Referring to FIG. 9, the total cholesterol content of the YE2 group was similar to that of the HFD group. Referring to FIG. 10, the YE2 group compared with the HFD group, the sGOT content was significantly decreased. Referring to FIG. 11, compared with the HFD group and the control group, the sGPT content was significantly decreased in the YE2 group.

Example 4 Cell Experiment: Formula of Fenugreek Extract

[0075] (1) HepG2 cells were seeded at 2.510.sup.4 cells/well and incubated for 24 hours.

[0076] (2) The incubated cells were divided into the following groups:

[0077] Control group (CG) was added with 1% DMSO to treat the HepG2 cells for 16 hours, and then treated with cell culture medium for 16 hours.

[0078] Lipid droplets induced group (LDIG) was added with 1% DMSO to treat the HepG2 cells for 16 hours, and then treated with 500 nM lipid droplets induced reagent for 16 hours.

[0079] Dried fenugreek water extract which was obtained by Preparation Example 1, red yeast rice extract, rice bran extract, artichoke extract, taurine, and ginseng extract were mixed at different parts by weight into 8 experiment groups (EG) as shown in Table 1 below, wherein the latter five materials are commercially available. In addition, in a preferred embodiment, Vitamin E, SiO.sub.2, MAG and Fibersol-2 may be additionally added as desired. Each experiment group was respectively added to treat the HepG2 cells for 16 hours, and then treated with lipid droplets induced reagent for 16 hours.

TABLE-US-00001 TABLE 1 Content of each group (parts by weight) fenugreek water red yeast rice bran artichoke ginseng extract rice extract extract extract taurine extract 1 2 12~16 3~14 0.5~5 0.5~3 0.8~25 0.1~10 3 12~16 4 3~14 5 0.5~5 6 0.5~3 7 0.8~25 8 0.1~10 Note: means not added.

[0080] (3) The above groups were examined by MTS assay and oil red-O stain, and the oil red OD value/MTS OD value was used as the index of lipid droplets.

[0081] Referring to FIG. 12, the percentage of the lipid droplet formation of the lipid droplets induced group was 100%, the experiment group 1: 97%, the experiment group 2: 38.6%, the experiment group 3: 57%, the experiment group 4: 58.4%, the experiment group 5: 79.3%, the experiment group 6: 78.1%, the experiment group 7: 72.4%, and the experiment group 8: 60.1%. Lipid droplet formation was significantly decreased in the experiment groups 2 to 7, wherein the experiment group 2, which has all of the 6 materials, has the lowest percentage of lipid droplet formation.

[0082] Referring to FIG. 13, there was no significant change in cell viability OD ratio of the control group, the lipid droplets induced group, and the experiment groups 1 to 8, and thus the cell viability could be maintained in groups 2 to 8.

Example 5 Animal ExperimentFormula of Fenugreek Extract

[0083] (1) 8 weeks old male C57BL/6J mice were chosen for the experiment.

[0084] (2) Divided into the following groups:

[0085] Control group (CG): 10 mice were given 0.2 mL ddH.sub.2O daily and fed with a controlled diet after the start of the second week. The controlled diet was fed with general feed and tube-fed with ddH.sub.2O, continuously until the 18th week.

[0086] High fat diet (HFD) group: 10 mice were given 0.2 mL ddH.sub.2O daily and fed with a high fat diet after the start of the second week. The high fat diet was fed with 60% high fat feed and tube-fed with ddH.sub.2O, continuously until the 18th week, wherein the 60% high fat feed was 20% kcal protein, 20% kcal carbohydrate and 60% kcal fat, the formula by E. A. Ulman, Ph. D., deployed in Research Diets, Inc., 8/26/98 With 3/11/99, product number D12492.

[0087] Low dose group (LDG): 10 mice were given 0.2 mL 1(0.0051 g/kg/day) fenugreek water extract formula (same as the proportion of experiment group 2 in Example 4) daily and fed with the high fat diet after the start of the second week, continuously until the 18th week.

[0088] Medium dose group (MDG): 10 mice were given 0.2 mL 5(0.0255 g/kg/day) fenugreek water extract formula (same as the proportion of experiment group 2 in Example 4, the medium dose group given 5 times the dose of the low dose group) daily and fed with the high fat diet after the start of the second week, continuously until the 18th week.

[0089] High dose group (HDG): 10 mice were given 0.2 mL 10(0.0510 g/kg/day) fenugreek water extract formula (same as the proportion of experiment group 2 in Example 4, the high dose group given 10 times the dose of the low dose group) daily and fed with the high fat diet after the start of the second week, continuously until the 18th week.

[0090] (3) Blood samples were extracted for serum triglyceride, serum cholesterol, serum non-esterified fatty acid, serum ALT, and serum AST analysis at the 18th week. The mice were sacrificed at the 18th week to analyze the total cholesterol, total triglyceride and non-esterified fatty acid in the liver tissues, and the liver tissue slides were stained by H & E stain.

[0091] Referring to FIG. 14, serum triglycerides were significantly lower in the low dose group, the medium dose group, and the high dose group compared with the high fat diet group. Referring to FIG. 15, compared with the high fat diet group, the serum cholesterols were dose-dependently decreased in the low dose group, the medium dose group, and the high dose group. Referring to FIG. 16, serum non-esterified fatty acid of the low dose group, the medium dose group, and the high dose group were significantly decreased compared with the high fat diet group. Compared with the high fat diet group, the serum ALT in the low dose group, the medium dose group, and the high dose group were decreased. Referring to FIG. 18, compared with the high fat diet group, the serum AST in the low dose group, the medium dose group, and the high dose group were decreased.

[0092] Referring to FIG. 19, the liver tissue of the high fat diet group was filled with fats and appeared hollow and loose. From the low dose group, the medium dose group and the high dose group, the liver cells became denser with the increase of the dose, and the high dose group was similar to the control group. Referring to FIG. 20, compared with the high fat diet group, the total cholesterol of total liver decreased in the low dose group, the medium dose group, and the high dose group. Referring to FIG. 21, the total triglyceride of total liver significantly decreased in the low dose group, the medium dose group, and the high dose group, as compared with the high fat diet group. Referring to FIG. 22, compared with the high fat diet group, the non-esterified fatty acid of total liver decreased in the low dose group, the medium dose group, and the high dose group.

Example 6: Analysis of Fenugreek Plant Tissue Components

[0093] The fenugreek water extract obtained by Preparation Example 1, and a whole-plant fenugreek extract obtained from the fresh or dried whole-plant fenugreek (including the leaves, stem, and roots of the fenugreek) according to the preparation method of Preparation Example 1 were respectively analyzed by HPLC. The analytical conditions were as follows: column: C18 (5 m250 mm); mobile phase: methanol:water=90:10; detection wavelength: 203 nm; injection volume: 20 L; retention time: 30 minutes; and flow rate: 1 mL/min.

[0094] Referring to FIGS. 23 and 24, the results showed that the similarity of the saponins profile of the fenugreek water extract (FIG. 23) and the whole-plant fenugreek extract (FIG. 24) was high, and thus either fenugreek seed or the whole-plant fenugreek extract by the water extract should have a similar effect.

[0095] Even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and features of the invention, the disclosure is illustrative only. Changes may be made in the details, especially in matters of shape, size, and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed.