COMPOSITION FOR PREVENTING, ALLEVIATING OR TREATING METABOLIC SYNDROME ACCOMPANIED BY OBESITY AND/OR DIABETES, CONTAINING, AS ACTIVE INGREDIENT, COMPLEX (IB COMPLEX) OF INDIAN GOOSEBERRY EXTRACT AND SPROUT BARLEY EXTRACT

Abstract

The present invention relates to a composition for prevention, improvement or treatment of metabolic syndrome comprising a complex of Indian gooseberry (amla) extract and barley sprout extract as an active ingredient. Specifically, the complex of Indian gooseberry extract and barley sprout extract reduces a body weight, blood sugar, insulin in blood and glycated hemoglobin in blood, and thereby it shows a synergistic effect in improvement of obesity or diabetes, and therefore it can be used as a pharmaceutical composition or health functional food for prevention or treatment of metabolic syndrome such as obesity and diabetes.

Claims

1. A method for prevention, improvement or treatment of metabolic syndrome, comprising: administering a complex of Indian gooseberry (Emblica officinalis) extract and barley sprout (Hordeum vulgare) extract as an active ingredient to a subject in need thereof.

2. The method according to claim 1, wherein the weight ratio of Indian gooseberry extract to barley sprout extract (Indian gooseberry extract:barley sprout extract) is 4:1-1:1.

3. The method according to claim 1, wherein the complex of Indian gooseberry extract and barley sprout extract has a synergistic effect compared to each single extract.

4. The method according to claim 1, wherein the complex of Indian gooseberry extract and barley sprout extract retains the efficacy of fat accumulation inhibition and weight loss, by inhibiting fat digestion, absorption and synthesis, by promoting lipolysis, or by inhibiting the enzymatic activity of pancreatic lipase; or retains the efficacy of reduction of blood sugar, insulin and glycated hemoglobin, by regulating absorption of blood sugar and promoting glucose metabolism.

5. The method according to claim 1, wherein the metabolic syndrome is obesity or diabetes.

6. The method according to claim 1, wherein the Indian gooseberry extract comprises 1 to 5 mg/g of ellagic acid under the condition of acid hydrolysis.

7. The method according to claim 1, wherein the Indian gooseberry extract comprises 5 to 25 mg/g of free ellagic acid under the condition of no acid hydrolysis.

8. The method according to claim 1, wherein the barely sprout extract comprises 6 to 11 mg/g of saponarin.

9. The method according to claim 1, wherein the Indian gooseberry extract or barley sprout extract is squeezed one, or extracted one using an extraction solvent selected from the group consisting of water, lower alcohols of 1 to 4 carbon atoms and mixtures thereof.

10. The method according to claim 1, wherein the Indian gooseberry extract or barley sprout extract is in a form of dry powder.

11. The method according to claim 1, wherein the complex of Indian gooseberry (Emblica officinalis) extract and barley sprout (Hordeum vulgare) extract is comprised in a pharmaceutical composition, and is administered to the subject in need thereof by administering the pharmaceutical composition.

12. The method according to claim 1, wherein the complex of Indian gooseberry (Emblica officinalis) extract and barley sprout (Hordeum vulgare) extract is comprised in a food composition, and is administered to the subject in need thereof by administering the food composition.

13. The method according to claim 1, wherein the complex of Indian gooseberry (Emblica officinalis) extract and barley sprout (Hordeum vulgare) extract is prepared by a method, comprising: obtaining Indian gooseberry extract from Indian goose berries comprising squeezing and drying processes; obtaining barley sprout extract from barley sprout comprising squeezing and drying processes; and mixing the Indian gooseberry extract and barley sprout extract in a weight ratio of 4:1 to 1:1 to obtain a complex thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 is a drawing which shows the pancreatic lipase inhibitory activity for 11 kinds of natural substances including Indian gooseberry extract and barley sprout extract.

[0066] FIG. 2 is a drawing which shows the synergistic effect for the pancreatic lipase inhibitory activity of the complex of Indian gooseberry extract and barley sprout extract, compared when treated as a single substance.

[0067] FIG. 3 is a drawing which shows the pancreatic lipase inhibitory activity depending on the mixing ratio of the Indian gooseberry extract and barley sprout extract.

[0068] FIG. 4 is a drawing which shows the intracellular cAMP level depending on the mixing ratio of the Indian gooseberry extract and barley sprout extract.

[0069] FIG. 5 is a drawing which shows the intracellular glycerol release depending on the mixing ratio of the Indian gooseberry extract and barley sprout extract.

[0070] FIG. 6 is a drawing which shows the synergistic effect for the protein expression ratio of intracellular P-IRS1/IRD1 of the complex of Indian gooseberry extract and barley sprout extract, compared when treated as a single substance.

[0071] FIG. 7 is a drawing which shows the synergistic effect for the protein expression ratio of intracellular P-PI3K/PI3K of the complex of Indian gooseberry extract and barley sprout extract, compared when treated as a single substance.

[0072] FIG. 8 is a drawing which shows the synergistic effect for the protein expression ratio of intracellular GLUT4 of the complex of Indian gooseberry extract and barley sprout extract, compared when treated as a single substance.

[0073] FIG. 9 is a drawing which shows the synergistic effect for inhibition of intracellular fat accumulation of the complex of Indian gooseberry extract and barley sprout extract, compared when treated as a single substance.

[0074] FIG. 10 is a drawing which shows the blood sugar (glucose) from experimental animals of the complex of Indian gooseberry extract and barley sprout extract, compared when treated as a single substance.

[0075] FIG. 11 is a drawing which shows the insulin in blood from experimental animals of the complex of Indian gooseberry extract and barley sprout extract, compared when treated as a single substance.

[0076] FIG. 12 is a drawing which shows the glycated hemoglobin (Hemoglobin A1c; HbA1c) in blood from experimental animals of the complex of Indian gooseberry extract and barley sprout extract, compared when treated as a single substance.

DETAILED DESCRIPTION

[0077] Hereinafter, the present invention will be described in more detail by examples. These examples are intended to illustrate the present invention more specifically, and it will be obvious for those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1. Test Substance Preparation Method

1.1 Preparation of Indian Gooseberry (Amla) Extract (Comparative Example 1)

[0078] After washing fruits of Indian gooseberry (Amla, origin Nepal) and squeezing, the Indian gooseberry fruit extract was obtained. Then, after filtering and concentrating, and then drying, the Indian gooseberry extract to be used in the present invention was prepared.

1.2 Preparation of Barley Sprout Extract (Comparative Example 2)

[0079] After harvesting barley sprout (origin Korea) and squeezing, to remove insoluble fibers such as cellulose, it was filtered and then dried. The dried powder was homogenized to prepare the barley sprout extract to be used in the present invention.

1.3 Preparation of Complexes (Examples 1 to 5)

[0080] The Indian gooseberry (Amla) extract and barley sprout extract prepared in the Comparative example 1 to Comparative example 2 were combined in a weight ratio as the following Table 1 to prepare complexes.

TABLE-US-00001 TABLE 1 Combining ratio Indian of Indian gooseberry Barley sprout gooseberry:barley extract extract Classification sprout (% by weight) (% by weight) Example 1 1:1 50 50 Example 2 4:1 80 20 Example 3 2:1 66.67 33.33 Example 4 1:2 33.33 66.67 Example 5 1:4 20 80

1.4 Candidate Material Preparation Method

[0081] Through domestic and foreign document reviews, anti-obesity or anti-diabetic active materials were searched, and natural materials obtained considering the availability of domestic raw materials and availability of intake as food ingredients were selected as candidate materials and used for research. Candidate materials, A, B, C and D represent lotus leaves, balloon-flowers, Cudrania tricuspidata and cinnamon, respectively, and they were extracted by adding hot water and were subjected to spray drying, and then were used for research. E and F represent Mori folium and Nelumbo nucifera, and were extracted by adding hot water and then were concentrated and were subjected to spray drying, and were used for research. G, H and I represent beetroot, pomegranate, and tart cherry, and they were concentrated and were subjected to spray during and then were used for research.

1.5 Marker Component Content Confirmation

[0082] For the marker component content analysis and raw material standardization of the Indian gooseberry (Amla) extract and barley sprout extract, for each raw material of the Indian gooseberry (Amla) extract (Comparative example 1-1, Comparative example 1-2, Comparative example 1-3) and barley sprout extract (Comparative example 2-1, Comparative example 2-2, Comparative example 2-3), three kinds of raw materials were produced.

[0083] The marker component of the Indian gooseberry (Amla) extract was set to ‘ellagic acid’, and the marker component of the barley sprout extract was set to saponarin, and they were analyzed. The content of the marker component for the Indian gooseberry (Amla) extract was arranged in Table 2 and Table 3 depending on the analysis method, and the content of the marker component for the barley sprout extract was arranged in Table 4.

[0084] More specifically, analysis of the content of the marker component of each extract was conducted as follows.

[0085] The analysis method used for quantification of ellagic acid of the Indian gooseberry (Amla) extract was as follows.

[0086] The content of ellagic acid in the Indian gooseberry (Amla) extract was measured using High performance chromatography after acid hydrolysis. It was separated by a gradient method using Capcellpak C18 UG120 (4.6 mm×250 mm, 5 μm) as a column, and using a 6:4 mixed solution of 0.85% phosphate in distilled water and methanol (A) and methanol (B) as a moving bed, and ellagic acid was detected in a wavelength of 370 nm with a UV detector. As a result, the content of ellagic acid in the Indian gooseberry (Amla) extract prepared according to the experimental method 1.1 was confirmed in the range of 1-5 mg/g.

TABLE-US-00002 TABLE 2 Classification Content of ellagic acid(mg/g) Comparative example 1-1 3.17 Comparative example 1-2 1.91 Comparative example 1-3 4.65

[0087] In the analysis method used for quantification of free ellagic acid of the Indian gooseberry (Amla) extract, the analysis method under the condition that did not undergo acid hydrolysis was as follows.

[0088] The content of the free ellagic acid in the Indian gooseberry (Amla) extract was measured using High performance chromatography, after methanol ultrasonic extraction. It was separated by a gradient method using Capcellpak C18 UG120 (4.6 mm×250 mm, 5 μm) as a column, and using a 6:4 mixed solution of 0.85% phosphate in distilled water and methanol (A) and methanol (B) as a moving bed, and ellagic acid was detected in a wavelength of 370 nm with a UV detector. As a result, the content of free ellagic acid in the Indian gooseberry (Amla) extract prepared according to the experimental method 1.1 was confirmed in the range of 5-25 mg/g.

TABLE-US-00003 TABLE 3 Classification Content of free ellagic acid (mg/g) Comparative example 1-1 11.73 Comparative example 1-2 5.36 Comparative example 1-3 24.15

[0089] The analysis method used for quantification of saponarin of the barley sprout extract was as follows.

[0090] The content of the saponarin in the barley sprout extract was measured using High performance chromatography. It was separated by a gradient method using Capcellpak C18 UG120 (4.6 mm×250 mm, 5 μm) as a column, and using 0.1% formic acid (A) and methanol (B) as a moving bed, and saponarin was detected in a wavelength of 340 nm with a UV detector. As a result, the content of saponarin in the barley sprout extract prepared according to the experimental method 1.2 was confirmed in the range of 6-11 mg/g.

TABLE-US-00004 TABLE 4 Classification Content of saponarin (mg/g) Comparative example 2-1 6.56 Comparative example 2-2 7.38 Comparative example 2-3 10.54

2. Efficacy Evaluation

2.1 Pancreatic Lipase Inhibitory Activity Measurement

[0091] Pancreatic lipase is an enzyme of hydrolyzing triglyceride (TG) that is triglyceride into monoglyceride (MG) and fatty acid, and progresses fat digestion, and helps intestinal epithelial cells absorb decomposition products. Accordingly, as fat absorption into intestinal cells and digestive tracts is inhibited with decomposition of triglyceride, when the activity of pancreatic lipase is inhibited, the pancreatic lipase activity inhibitory ability is a very useful test method to predict the anti-obesity activity. In the present example, the pancreatic lipase inhibitory activity was measured as follows. At first, Tris buffer (100 mM Tris-HCl, 5 mM CaCl2. pH 7.0) 169 μL and 20 μL sample were added and mixed to 6 μL enzyme solution in which porcine pancreatic lipase was dissolved at the concentration of 0.5 g/200 mL in enzyme buffer (10 mM MOPS, 1 mM EDTA, pH6.8), and then incubated at 37° C. for 15 minutes. Then, after adding 5 μL substrate solution (10 mM of p-nitrophenylbutyrate in dimethyl formamide) and incubating at 37° C. for 30 minutes, the absorbance was measured at 405 nm using a UV-visible spectrophotometer. The pancreatic lipase inhibitory activity (%) was shown in the following equation 1, and X indicates the absorbance with a sample added, and Y indicates the absorbance without a sample added. The measured value was shown as a mean value of three repeated experiments.

Pancreatic lipase inhibitory activity (%)=(1−(X−Y)/Y)×100  Equation 1

2.2 Confirmation of Synergistic Effect of Complex for Pancreatic Lipase Inhibitory Activity

[0092] To investigate the synergistic effect of the pancreatic lipase inhibitory ability of the complex of the Indian gooseberry extract and barley sprout extract of the present invention compared to the case of treating as a single substance, the activity of the single substance and complex was analyzed at the same concentration, and to confirm the synergistic effect of the complex, Colby equation was utilized. Colby equation was shown in the following Equation 2, and the synergistic effect could be recognized when the measured value exceeded the E value derived by Colby equation. E indicates the predicted effect of the complex (A+B), and A indicates the effect of the single substance A (%), and B indicates the effect of the single substance B (%).

E=A+B−(AB/100)  Equation 2

2.3 Cell Culture and Differentiation Induction

[0093] The effect that the complex of the Indian gooseberry extract and barley sprout extract of the present invention affected fat synthesis and fat decomposition in the adipocyte differentiation process compared to the case of treating as a single substance was confirmed. In the present example, 3T3-L1 cell was distributed from American Type Cultured Collection (ATCC; Rockville, Md., USA) and experimented. The 3T3-L1 preadipocyte was cultured in an incubator at 37° C. and 5% CO.sub.2 (Thermo Fisher Scientific Inc., Pittsburgh, Pa., USA) using high-glucose Dulbeco's modified Eagle's medium (DMEM) containing 10% newborn calf serum (NCS) and 1% penicillin-streptomycin, 1% L-glutamine, 1% sodium pyruvate, 1% hepes, and 1% NEAA mixture, and the culture solution was replaced per 2 days, and when the cell was attached 80% or more on the bottom of the flask as a single layer, the cell surface was washed using PBS solution, and after adding 0.25% trypsin-EDTA, it was left in the incubator for 3 minutes and the cells were separated. Then, the cells were collected by centrifugation at 1600 rpm for 5 minutes using a centrifuge (GYROZEN 416G), and for differentiation of cells, cells of 1×10.sup.5 cells/well were equally aliquoted in a 6-well plate (TPP) using DMEM culture solution comprising, and 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, 1% L-glutamine, 1% sodium pyruvate, 1% hepes, 1% NEAA mixture, gentamycin, and when 100% confluent, induction of differentiation was initiated by mixing 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM), insulin (10 μg/mL), and dexamethasone (DEX, 1 μM), which were the adipogenic cocktail (MDI solution), in DMEM culture solution containing 10% FBS and 1% penicillin-streptomycin, 1% L-glutamine, 1% sodium pyruvate, 1% hepes, 1% NEAA mixture and gentamycin. The differentiation period lasted 9 days in total, and for the first 3 days of differentiation, the same culture solution was replaced, and for the middle 3 days of differentiation, the culture solution was replaced with DMEM comprising 10% FBS containing only Insulin (10 μg/mL) every day, and for the latter 3 days of differentiation, it was replaced with DMEM culture solution containing 10% FBS every day. In the lipogenesis mechanism, the sample was treated at the same time as the start of differentiation every day, and in the lipolysis mechanism, it was treated for 3 days from 3 days before the end of differentiation.

2.4 Confirmation of Synergistic Effect of Complex for Intercellular cAMP Level

[0094] cAMP is involved in promoting triglyceride decomposition and promoting heat generation by activating hormone sensitive lipase (HSL) which is lipase. In the present example, to measure the intercellular cAMP level, cAMP ELISA kit (Cell biolabs Inc., San Diego, Calif. USA) was used, and it was measured as follows. At first, 1×10.sup.6 cell was added to lysis buffer and homogenized, and centrifuged at 13,000 rpm for 5 minutes, and the supernatant was used as a sample. The sample and standard reagent were aliquoted by 50 μL in each well, and Peroxidase cAMP Tracer Conjugate reagent was aliquoted by 25 μL and then Rabbit Anti-cAMP Polyclonal Antibody reagent was aliquoted by 50 μL, and was left at room temperature for 2 hours placing a plate cover. In 2 hours, it was aliquoted by 250 μL with wash buffer, and a total of 5 washing operations were conducted, and 100 μL of the substrate solution warmed at a room temperature was aliquoted in each well, and then it was left at a room temperature for 20 minutes. In 20 minutes, stop solution 100 μL was aliquoted in each well to stop the reaction, and the absorbance was measured at the 450 nm wavelength.

2.5 Confirmation of Synergistic Effect of Complex for Intercellular Glycerol Release

[0095] Fat decomposition is a process that triglyceride in adipocytes is hydrolyzed into free acid and glycerol, and to confirm the effect on fat decomposition in adipocytes, glycerol to be increased during fat decomposition was measured. In the present example, to measure the effect on the amount of glycerol released in the process of differentiation of adipocytes, the glycerol content in the culture solution was measured using a free glycerol reagent applying glycerol phosphate oxidase-TRINDER enzyme reaction method by the method such as McGowan, and the like, and it was measured as follows. At first, each sample was treated by concentration, and each culture solution was collected and used at the latter of differentiation (Day 9), respectively, and the culture solution (1 mL) and free glycerol reagent (800 μL) were mixed and were reacted in a 37° C. hot plate for 10 minutes, and then the optical density was measured at the 540 nm wavelength using ELISA reader (Molecular Devices, USA). The glycerol content was measured by preparing a standard curve using free glycerol as a standard reagent, and the protein content was measured using BSA as a standard reagent by Bradford method.

2.6 Confirmation of Synergistic Effect of Complex for Intercellular IRS, PI3K and GLUT4

[0096] IRS (Insulin receptor substrate) is an insulin receptor substrate distributed in systemic tissue, and is involved in the insulin signaling pathway and regulates glucose and lipid metabolism, and PI3K (Phosphoinositide 3-Kinase) is an enzyme which is involved in the insulin signaling pathway, and functions to regulate blood sugar absorption and inhibit fat synthesis by interacting with IRS. GLUT4 (Glucose transport 4) is a glucose receptor mostly present in adipocytes and muscle cells, and promotes glucose transport from cells to cell membranes by the action of insulin. When insulin resistance occurs by visceral fat accumulation, GLUT4 translocation from cytoplasm into cell membranes does not occur smoothly. In the present example, to measure the protein expression of IRS, PI3K and GLUT4, protein quantification of cell lysate of western blotting was conducted using Bradford assay. Protein (40 μg) was loaded in 10% Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad), and transferring was progressed using Trans-Blot® Turbo™ Transfer system (Bio-Rad). The membrane was blocked in blocking buffer (5% skim milk in Tris buffered saline with 1% Tween® 20) for 1 hour, and after washing, it was reacted with IRS, PI3K and GLUT4 primary antibodies. After washing and reacting the HRP-polymerized secondary antibody (Cell Signaling, 1:3000) for 1 hour, and then it was washed and color was developed using EzWest Lumi plus (ATTO, Tokyo, Japan), and it was analyzed using Ez-Capture II (ATTO) and CS Analyzer 3.0 software (ATTO).

2.7 Confirmation of Synergistic Effect of Complex for Inhibition of Intercellular Fat Accumulation

[0097] To measure the effect on adipocyte differentiation in the process of differentiating from preadipocytes to adipocytes of the complex of the Indian gooseberry extract and barley sprout extract of the present invention, Oil red O staining was performed. In other words, according to the cell culture and differentiation induction protocol, the DMEM culture solution containing 10% FBS comprising the adipogenic cocktail and the extract were replaced every day, and at the latter of differentiation (Day 9), Oil red O staining was conducted, respectively. The culture solution was sucked, and it was washed with PBS solution twice and then PBS solution was completely sucked, and 10% formalin was added and fixed at a room temperature for 5 minutes, and then 10% formalin was sucked, and new 10% formalin was added again, and fixed at a room temperature for 2 hours or more. Then, formalin was sucked, and 60% isopropanol was added and sucked immediately, and then the flask was completely dried, and Oil red O solution was added to stain fat globules for 60 minutes. After staining, they were washed with distilled water 4 times and intercellular accumulation of the fat globules was observed with a microscope and a camera. To qualify the contents of fat globule accumulation, 100% isopropanol was added under the well dried condition, and Oil red O dye was eluted, and then the optical density was measured at the 520 nm wavelength using ELISA reader (Molecular Devices, USA). Then, 100% isopropanol was used as blank.

2.8 Confirmation of Effect on Body Weight Change of Obesity-Induced Mice of Complex

[0098] The effect on the body weight change of obesity-induced mice from high fat diet of the complex of the Indian gooseberry extract and barley sprout extract of the present invention was confirmed. As an experimental animal, 4-week male C57BL/6J mice around 20 g were supplied from Saeron Bio Inc. (Uiwang-si, Korea). They were used for the experiment, after passing through the adaptation period for 1 week under the condition that the light and dark was 12 hours (light/dark cycle) and the temperature was 23±2° C. and the relative humidity was 50±5%. During the adaptation period, AIN-93G diet and drinking water were allowed to be consumed freely, and the body weight was measured to separate 8 mice per each group by the random method. At the end of the adaptation period, the sample intake was progressed as free diet intake for 15 weeks, and the dietary intake and body weight were measured every week, and after the end of the experiment, the weight gain was divided by the dietary intake (total food consumption)) for the same period to calculate the food efficiency ratio (FER). The classification of the experimental group and the composition of the experimental diet were shown in Table 5, and the equation of the diet efficiency was shown in the following equation 3.

FER=weight gain (g)/total food consumption (g)×100  Equation 3

TABLE-US-00005 TABLE 5 Experimental group Experimental diet Normal group (NC) AIN 93G diet Control group (C) 60% high fat diet Positive control 60% high fat diet + Metformin 250 mg/kg b.w. group (Met) Example 3 100 mg/kg 60% high fat diet + IG:BP (2:1) 100 mg/kg b.w. Example 3 200 mg/kg 60% high fat diet + IG:BP (2:1) 200 mg/kg b.w. Example 3 400 mg/kg 60% high fat diet + IG:BP (2:1) 400 mg/kg b.w.

2.9 Confirmation of Effect on Organ and Fat Tissue Weight Change of Obesity-Induced Mice of Complex

[0099] The effect on the organ and fat tissue weight change of obesity-induced mice from high fat diet of the complex of the Indian gooseberry extract and barley sprout extract of the present invention was confirmed. For tissue excision, after blood-gathering, organ (liver, kidney, spleen) and white fat tissue (subcutaneous fat, visceral fat (epididymal fat and intraperitoneal fat)) were excised, and then were washed with physiological saline solution and moisture was removed with filter paper, and then the weight was measured.

2.10 Confirmation of Effect on Change of Glucose, Insulin and HbA1c in Blood of Obesity-Induced Mice of Complex

[0100] The effect on the change of Glucose, Insulin and HbA1c in blood of obesity-induced mice from high fat diet of the complex of the Indian gooseberry extract and barley sprout extract of the present invention was confirmed. For blood analysis, at the end of the experiment, the experimental animals were anesthetized with isoflurane after 12 hours of fasting, and blood was collected through the hepatic vein, and the blood was analyzed by whole blood and serum separated by centrifugation (14,000 rpm, 20 min, 4° C.), and was measured using ELISA kit (Biovision).

3. Experimental Result

3-1. Pancreatic Lipase Inhibitory Activity for 11 Kinds of Natural Substances

[0101] The pancreatic lipase inhibitory activity for 11 kinds of natural substances prepared by the preparation method suggested in the experimental method was measured. As suggested in FIG. 1 and Table 6, it was confirmed that the lipase inhibitory activity of the sample of Comparative example 1 was the highest as 83.77±0.27% at the same concentration, and it was confirmed that the lipase inhibitory activity of the sample of Comparative example 2 was excellent as 42.81±2.09% next. This result confirmed that in the pancreatic lipase inhibitory activity for 11 kinds of natural substances, the Indian gooseberry extract and barley sprout extract were the most excellent, and the Indian gooseberry extract and barley sprout extract reduced the fat absorption in the body by acting as a pancreatic lipase inhibitor, and retained the anti-obesity activity.

TABLE-US-00006 TABLE 6 Pancreatic lipase Classification Sample inhibitory ability (%) A lotus leaves 25.36 B balloon-flowers 13.48 C Cudrania tricuspidata 36.75 Comparative Indian gooseberry 83.77 example 1 D Cinnamon 14.39 E Mori folium 14.75 F Nelumbo nucifera 1.05 G Beetroot 13.62 H Pomegranate 31.97 I Tart cherry 26.91 Comparative Barley sprout 42.81 example 2

3-2. Synergistic Effect of Complex of Indian Gooseberry and Barley Sprout for Pancreatic Lipase Inhibitory Activity

[0102] The pancreatic lipase inhibitory activity for the single substance and complex of the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental method was measured. The pancreatic lipase inhibitory activity of the Indian gooseberry extract was indicated by Comparative example 1, and the pancreatic lipase inhibitory activity of the barley sprout extract was indicated by Comparative example 2, and the pancreatic lipase inhibitory activity of the complex at the same concentration was represented by Example 1-1, and the result measured by utilizing Colby equation to confirm the synergistic effect of the complex was represented by Example 1-2. As suggested in Table 2 and Table 7, the pancreatic lipase inhibitory activity of the Indian gooseberry extract was 94.42±0.66%, and the barley sprout extract showed efficacy of 40.83±3.14%, and the pancreatic lipase inhibitory activity of the complex at the same concentration was 112.64±3.20%, and the pancreatic lipase inhibitory activity of the complex was more excellent than the single substance. The expected value of the synergistic effect of the complex derived using Colby equation was 96.70%, and Example 1-2 shows an excellent effect of 130.06±2.21% about 134% or more then the expected value. This confirmed that the complex of the Indian gooseberry extract and barley sprout extract had a significantly synergistic effect in inhabiting the enzymatic activity of pancreatic lipase than each single substance, and inhibited digestion and absorption of ingested fat and retained the anti-obesity activity.

TABLE-US-00007 TABLE 7 Concen- Pancreatic lipase tration inhibitory ability Classification Sample (mg/mL) (%) Comparative Indian gooseberry 50 94.42 example 1 extract Comparative Barley sprout extract 50 40.83 example 2 Example 1-1 1:1 Mixture of Indian 50 112.64 gooseberry extract and barley sprout extract Example 1-2 1:1 Mixture of Indian 100 130.06 gooseberry extract and barley sprout extract

3-3. Confirmation of Indian Gooseberry and Barley Sprout Complex Combined in Various Weight Ratios on Pancreatic Lipase Inhibitory Activity

[0103] To select the optimal ratio of the complex of the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental method, the pancreatic lipase inhibitory activity was measured. According to the experimental result 3-2, by confirming that the Indian gooseberry and barley sprout showed the synergistic effect in the complex than each single substance, the preparation process and economic feasibility were considered, and then the optimal ratio which the complex of the Indian gooseberry and barley sprout showed the most excellent efficacy was to be selected. As suggested in FIG. 3 and Table 8, the pancreatic lipase inhibitory activity according to the mixing ratio of the Indian gooseberry and barley sprout at the same concentration was 103.45±2.14, 108.37±1.92 and 105.62±0.40, respectively, at 4:1, 2:1 and 1:1 of the ratio of Indian gooseberry and barley sprout, which was similar, and was more excellent than 83.30±1.63 and 60.98±0.33 measured at 1:2 and 1:4. By the result, it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract having a significantly synergistic effect than each single substance could inhibit digestion and absorption of fat by acting as an excellent pancreatic lipase inhibitor in the ratio of Indian gooseberry:barley sprout of 4:1˜1:1 than any other mixing ratio.

TABLE-US-00008 TABLE 8 Indian Pancreatic lipase gooseberry:barley Concentration inhibitory activity Classification sprout mixing ratio (mg/mL) (%) Example 2 4:1 50 103.45 Example 3 2:1 50 108.37 Example 1 1:1 50 105.62 Example 4 1:2 50 83.30 Example 5 1:4 50 60.98

3-4. Confirmation of Effect of Indian Gooseberry and Barley Sprout Combined in Various Weight Ratios for Intercellular cAMP Level

[0104] The intercellular cAMP level was measured by combining the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental method in various weight ratios. As suggested in FIG. 4, the single substance of the Indian gooseberry and barley sprout showed the activity of 1479.3±16.8 and 1300.0±81.9, respectively, at the same concentration, and the cAMP level according to the mixing ratio of Indian gooseberry and barley sprout was 2472.3±163.8 and 2508.3±106.3, at the ratio of Indian gooseberry:barley sprout of 4:1 and 2:1, respectively, and a significantly synergistic effect was shown compared to the single substance. Subsequently, the excellent activity was shown in order of 2132.7±149.4, 1763.7±101.1 and 1469.7±24.5 measured at 1:1, 1:2 and 1:4. By the result, it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract having a significantly synergistic effect than each single substance could promote fat decomposition through cAMP expression regulation.

3-5. Confirmation of Effect of Indian Gooseberry and Barley Sprout Complex Combined in Various Weight Ratios for Intercellular Glycerol Release

[0105] The intercellular glycerol release was measured by combining the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental method in various weight ratios. As suggested in FIG. 5, the single substance of Indian gooseberry and barley sprout showed the activity of 0.55±0.03 and 0.47±0.03, respectively, at the same concentration, and the glycerol release according to the mixing ratio of Indian gooseberry and barley sprout was 1.01±0.17 and 1.01±0.01, respectively, at the ratio of Indian gooseberry:barley sprout of 4:1 and 2:1, which was a significantly synergistic effect, compared to the single substance. Subsequently, the excellent activity was shown in order of 0.77±0.06, 0.69±0.06 and 0.60±0.09 measured at 1:1, 1:4 and 1:2. By the result, it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract having a significantly synergistic effect than each single substance decomposed triglyceride, that is neutral fat, present in fat globules accumulated in adipocytes, thereby increasing the glycerol release.

3-6. Synergistic Effect of Indian Gooseberry and Barley Sprout Complex for Intercellular IRS1

[0106] The expression ratio of the intercellular P-IRS1/IRS1 was confirmed by combining the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental result. As suggested in FIG. 6, the single substance of Indian gooseberry and barley sprout showed the expression ratio of 0.42 and 0.32, respectively, at the same concentration, and the complex of Indian gooseberry and barley sprout, Example 3 showed a significantly synergistic effect compared to the single substance, as the expression ratio of 0.86. In addition, the treatment group of 50, 100 μg/mL showed the expression ratio of 0.51 and 0.63, respectively, and the complex of Indian gooseberry and barley sprout increased the expression ratio of the intercellular P-IRS1/IRS1 in the concentration dependent way, and by the result, it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract could regulate blood sugar absorption and inhibit fat synthesis from glucose metabolism by increasing the expression ratio of P-IRS1/IRS1.

3-7. Synergistic Effect of Indian Gooseberry and Barley Sprout Complex for Intercellular PI3K

[0107] The expression ratio of the intercellular P-PI3K/PI3K was confirmed by combining the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental result. As suggested in FIG. 7, the single substance of Indian gooseberry and barley sprout showed the expression ratio of 1.93 and 1.26, respectively, at the same concentration, and the complex of Indian gooseberry and barley sprout, Example 3 showed a significantly synergistic effect compared to the single substance, as the expression ratio of 4.31. In addition, the treatment group of 50, 100 μg/mL showed the expression ratio of 1.05 and 1.47, respectively, and the complex of Indian gooseberry and barley sprout increased the expression ratio of the intercellular P-PI3K/PI3K in the concentration dependent way, and by the result, it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract could regulate blood sugar absorption and inhibit fat synthesis from glucose metabolism.

3-8. Synergistic Effect of Indian Gooseberry and Barley Sprout Complex for Intercellular GLUT4

[0108] The expression ratio of the intercellular GLUT4 was confirmed by combining the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental result. As suggested in FIG. 8, the single substance of Indian gooseberry and barley sprout showed the expression ratio of 0.18 and 0.11, respectively, at the same concentration, and the complex of Indian gooseberry and barley sprout, Example 3 showed a significantly synergistic effect compared to the single substance, as the expression ratio of 0.38. In addition, the treatment group of 50, 100 μg/mL showed the expression ratio of 0.14 and 0.33, respectively, and the complex of Indian gooseberry and barley sprout increased the expression ratio of the intercellular GLUT4 in the concentration dependent way, and by the result, it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract could regulate blood sugar absorption and inhibit fat synthesis from glucose metabolism by promoting GLUT4 signaling.

3-9. Synergistic Effect of Indian Gooseberry and Barley Sprout Complex for Inhibition of Intercellular Fat Accumulation

[0109] Oil red O staining was performed to measure the effect on adipocyte differentiation by combining the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental method. As suggested in FIG. 9, the single substance of Indian gooseberry and barley sprout showed the content of intercellular triglyceride of 0.60±0.02 and 1.10±0.05, respectively, at the same concentration, and the complex of Indian gooseberry and barley sprout, Example 3 was 0.29±0.03, and a significantly synergistic effect compared to the single substance was shown. In addition, in the treatment group of 50, 100 μg/mL, it was 0.88±0.02 and 0.57±0.03, respectively, and it was confirmed that the complex of Indian gooseberry and barley sprout inhibited fat accumulation in adipocytes in the concentration dependent way.

3-10. Confirmation of Effect on Weight Change of Obesity-Induced Mice of Indian Gooseberry and Barley Sprout Complex

[0110] To confirm the effect on the weight change of obesity-induced mice from high fat diet by combining the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental method, the body weight of the experimental animals was measured. As suggested in Table 9, The control group in which obesity was induced by high fat diet showed the weight increase of 30.92±3.53, and the complex of Indian gooseberry and barley sprout, Example 3 showed the concentration-dependent body weight reduction, as 28.13±3.93, 25.78±3.35 and 24.70±1.85, respectively, according to the administration groups of 100, 200 and 400 mg/kg. Also in the result of comparing the diet efficiency, it was confirmed that it was reduced in the Indian gooseberry and barley sprout complex in the concentration dependent way, compared to the control group, and thereby it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract showed an excellent effect in body weight reduction.

TABLE-US-00009 TABLE 9 HFD supplementation Groups NC C Met Example3 100 Example3 200 Example3 400 Initial body weight (g) 19.11 ± 0.23 19.35 ± 0.84  19.26 ± 0.66  19.40 ± 0.72.sup.  19.57 ± 0.40  18.82 ± 0.53 .sup.  Final body weight (g) 32.24 ± 3.74 50.27 ± 3.67 39.46 ± 2.16   47.53 ± 3.56.sup.ab 45.35 ± 3.30 43.52 ± 2.16   Weight gain (g)* 13.13 ± 3.67 30.92 ± 3.53 20.20 ± 1.89.sup.c  28.13 ± 3.93.sup.ab 25.78 ± 3.35 24.70 ± 1.85.sup.b  Food intake (g/day/mouse)  2.49 ± 0.19  2.91 ± 0.23 2.66 ± 0.18 .sup. 2.89 ± 0.18.sup.a  2.98 ± 0.21 2.71 ± 0.20.sup.b FER**  5.03 ± 1.41 10.12 ± 1.15 7.25 ± 0.68  9.28 ± 1.30.sup.ab  .sup. 8.25 ± 1.07.sup.bc 8.68 ± 0.65.sup.b indicates data missing or illegible when filed

3-11. Confirmation of Effect on Organ and Fat Tissue Weight Change of Obesity-Induced Mice of Indian Gooseberry and Barley Sprout Complex

[0111] To confirm the effect on the organ and fat tissue weight change of obesity-induced mice from high fat diet of the complex of the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental method, organ (liver, kidney, spleen) and white fat tissue (subcutaneous fat, visceral fat (epididymal fat and intraperitoneal fat)) of the experimental animals were measured. As suggested in Table 10, the total amount of the white fat tissue and the weight of the subcutaneous fat tissue and visceral fat tissue of the control group in which obesity was induced by high fat diet were 7.06±0.72, 3.57±0.62 and 3.49±0.32, respectively, and in the complex of Indian gooseberry and barley sprout, Example 3, compared to the control group, according to the administration group of 100, 200 and 400 mg/kg, the total amount of the white fat tissue was reduced to 5.52±0.86, 5.37±0.31 and 3.95±0.41, respectively, and the weight of the subcutaneous fat tissue was reduced to 2.62±0.43, 2.31±0.18 and 1.54±0.31, and the weight of the visceral fat tissue was reduced to 2.90±0.67, 3.06±0.14 and 2.41±0.15, and thereby it was confirmed that the complex of Indian gooseberry and barley sprout reduced the weight of fat tissue in the concentration dependent way, and it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract showed an excellent effect on inhibition of body fat accumulation.

TABLE-US-00010 TABLE 10 HFD supplementation Groups NC C Met Example3 100 Example3 200 Example3 400 Organ weight (g) Liver 1.19 ± 0.14 3.35 ± 0.64 .sup. 1.57 ± 0.21.sup.bc 1.81 ± 0.25.sup.b  1.72 ± 0.17.sup.b  .sup. 1.58 ± 0.20.sup.bc Kidney 0.29 ± 0.03.sup.b  0.40 ± 0.04 0.36 ± 0.02 0.37 ± 0.04 0.37 ± 0.01 0.37 ± 0.04.sup.a  Spleen 0.09 ± 0.02.sup.c  0.14 ± 0.03 0.10 ± 0.01 .sup. 0.13 ± 0.03.sup.ab .sup. 0.13 ± 0.03.sup.ab  0.11 ± 0.03.sup.abc Adipose tissue weight (g) Total WAT 1.87 ± 0.32 7.06 ± 0.72 4.11 ± 1.52 5.52 ± 0.86 5.37 ± 0.31 3.95 ± 0.41 Subcutaneous WAT 0.77 ± 0.14 3.57 ± 0.62 1.74 ± 0.85 2.62 ± 0.43.sup.b  2.31 ± 0.18 1.54 ± 0.31 Visceral WAT 1.10 ± 0.19.sup.c  3.49 ± 0.32 2.37 ± 0.68.sup.b  .sup. 2.90 ± 0.67.sup.ab .sup. 3.06 ± 0.14.sup.ab 2.41 ± 0.15.sup.b  indicates data missing or illegible when filed

3-12. Confirmation of Effect on Blood Sugar Change of Obesity-Induced Mice of Indian Gooseberry and Barley Sprout Complex

[0112] To confirm the effect on the blood sugar change of obesity-induced mice from high fat diet of the complex of the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental method, the blood sugar of the experimental animals was measured. As suggested in FIG. 10, the single substance of Indian gooseberry and barley sprout showed the blood sugar numerical value of 4.74±0.64 and 6.99±0.79, respectively, at the same concentration of 400 mg/kg, and in the complex of Indian gooseberry and barley sprout, Example 3, the blood sugar was 4.63±0.51, and a significant reduction effect was shown compared to the single substance. In addition, in the treatment group of 100, 200 and 400 mg/kg, it was confirmed that the complex of Indian gooseberry and barley sprout reduced blood sugar in the concentration dependent way, and thereby it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract showed an excellent effect on blood sugar reduction.

3-13. Confirmation of Effect on Insulin Change in Blood of Obesity-Induced Mice of Indian Gooseberry and Barley Sprout Complex

[0113] To confirm the insulin change of obesity-induced mice from high fat diet of the complex of the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental method, the insulin in blood of the experimental animals was measured. As suggested in FIG. 11, the single substance of Indian gooseberry and barley sprout showed the insulin numerical value in blood of 0.67±0.01 and 0.76±0.02, respectively, at the same concentration of 400 mg/kg, and in the complex of Indian gooseberry and barley sprout, Example 3, the insulin in blood was 0.56±0.01, and a significant reduction effect was shown compared to the single substance. In addition, in the treatment group of 100, 200 and 400 mg/kg, it was confirmed that the complex of Indian gooseberry and barley sprout reduced insulin in the concentration dependent way, and thereby it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract showed an excellent effect on reduction of insulin in blood.

3-14. Confirmation of Effect on Glycated Hemoglobin (HbA1c) in Blood of Obesity-Induced Mice of Indian Gooseberry and Barley Sprout Complex

[0114] To confirm the change of glycated hemoglobin in blood of obesity-induced mice from high fat diet of the complex of the Indian gooseberry extract and barley sprout extract prepared by the preparation method suggested in the experimental method, the HbA1c in blood of the experimental animals was measured. As suggested in FIG. 12, the single substance of Indian gooseberry and barley sprout showed the HbA1c in blood of 7.50±0.11 and 9.67±0.57, respectively, at the same concentration of 400 mg/kg, and in the complex of Indian gooseberry and barley sprout, Example 3, the insulin in blood was 7.15±0.22, and a significant reduction effect was shown compared to the single substance. In addition, in the treatment group of 100, 200 and 400 mg/kg, it was confirmed that the complex of Indian gooseberry and barley sprout reduced HbA1c in blood in the concentration dependent way, and thereby it was confirmed that the complex of the Indian gooseberry extract and barley sprout extract showed an excellent effect on reduction of HbA1c in blood.