Method for preparing anti-obesity composition by using astringent persimmons and mandarin peels

Abstract

Disclosed is a method for preparing an anti-obesity composition by using astringent persimmons and mandarin peels, the method comprising: adding a solvent to astringent persimmons and mandarin peels and then heating the same; degrading the extracted solution by using an enzyme; and filtering the solution degraded by the enzyme and then concentrating the filtered solution.

Claims

1. A method for preparing an anti-obesity composition by using astringent persimmons and mandarin peels, the method comprising: step (a) for adding a solvent to astringent persimmons and mandarin peels and then heating the same; step (b) for degrading the extracted solution by using an enzyme; and step (c) for filtering the solution degraded by the enzyme and then concentrating the filtered solution.

2. The method according to claim 1, wherein the step (a) comprises performing extraction for 1 to 3 hours at a temperature of 80 to 120 C.

3. The method according to claim 1, wherein the solvent is at least one selected from the croup consisting of water, alcohol, benzene, toluene, carbon tetrachloride and isopropyl alcohol (IPA).

4. The method according to claim 1, wherein the step (a) is repeated 2 times.

5. The method according to claim 1, wherein the enzyme of the step (b) includes at least one selected from the group consisting of arabanase, cellulase, beta-glucanase, hemicellulase, and xylanase.

6. The method according to claim 1, wherein the step (b) is performed for 5-25 hours.

7. The method according to claim 1, wherein the filtration of the step (c) is performed using diatomite, activated carbon, zeolite or a hollow fiber membrane filter.

8. The method according to claim 1, wherein the filtering in step (c) is performed until the concentration of the extract reaches 0-10 BX.

9. The method according to claim 1, further comprising, after the step (c), a step for adding a sterilized dextrin.

10. The method according to claim 1, further comprising a step for freeze-drying and pulverizing the extract.

11. An anti-obesity composition prepared according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows DPPH and ABTS inhibition test results using PCM and ascorbic acid according to the present invention, A and B are graphs of DPPH test results, and C and D are graphs of ABTS test results.

(2) FIG. 2 shows a graph of pancreatic lipase inhibition according to the present invention.

(3) FIG. 3 shows a graph respectively showing TG, TC, HDL-cholesterol, and LDL-cholesterol levels of mice of each experimental group after 6 weeks experiment according to the present invention.

DETAILED DESCRIPTION

(4) Hereinafter, the present invention will be described in more detail through the preferred embodiments of the present invention. In explanation of the present invention, a detailed explanation thereof will be omitted when it is determined that a specific explanation of the related known art may blur the main idea of present invention. Throughout the description, when any part includes/comprises a certain component, which means that any part may further include/comprise other components, not exclude other components, unless otherwise specially stated.

(5) The present inventors have noted that the reduction in the activity of pancreatic lipase by conventional chemical synthesis and the method of inhibiting fat absorption by the same cause serious gastrointestinal diseases and excessive cost. As a result of efforts to solve this problem, it was found that extracts of pancreatic lipase inactivation components can be extracted from the natural products, astringent persimmons and mandarin peels which are easily obtainable from the surroundings, thereby leading to the present invention.

(6) Accordingly, the present invention relates to a method for preparing anti-obesity composition by using astringent persimmons and mandarin peels, the method including: step (a) for adding a solvent to astringent persimmons and mandarin peels and then heating the same; step (b) for degrading the extracted solution by using an enzyme; and step (c) for filtering the solution degraded by the enzyme and then concentrating the filtered solution.

(7) The step (a) is a step of softening astringent persimmons and mandarin peels by heat to extract an active ingredient of the astringent persimmons and mandarin peels, a solvent to extract the active ingredient can be used without limitation, but preferably at least one selected from among water, alcohol, benzene, toluene, carbon tetrachloride and IPA can be used. In addition, since the composition of the present invention is administered to a human or animal, the active ingredient is expected to be water-soluble, and more preferably water can be used.

(8) In addition, the step (a) is preferable to heat at a temperature of 80 to 120 C. for 1 to 3 hours such that the active ingredient elute as much as possible, and softens the astringent persimmons and mandarin peels, and more preferably at a temperature of 100 C. for 2 h. In addition, when the step (a) is performed only once, the enzyme degradation of the next step is not completely performed due to insufficient softening, therefore, it is preferable to repeat the step (a) several times, and more preferable to repeat 2 times in order to save time and cost consumption by the fuel used.

(9) In the present invention, an astringent persimmon is, for example, an astringent persimmon of 3 to 10 cm in diameter with immature astringent taste persimmon and green appearance, and which is harvested 6 to 9 months after cultivation. The present invention uses an active ingredient such as polyphenols having an anti-obesity component extracted from persimmon and mandarin peels, and thus, it is preferable to use an astringent persimmon which is expected to have a high content of polyphenol. In addition, when an astringent persimmon is completely matured, the sugar content increases, thereby being difficult to lower the sugar content in concentration process, and sugar-induced weight gain may occur, therefore it is preferable to use an immature astringent persimmon.

(10) The step (b) is a step of degrading by adding an enzyme to softened astringent persimmons and mandarin peels solution, and the enzyme to be used preferably includes at least one selected from the group consisting of arabanase, cellulase, beta-glucanase, hemicellulase, and xylanase, and more preferably Viscozyme from Novozymes (Denmark) is used to effectively degrade the astringent persimmon and mandarin peel composed of various components. In addition, it is preferable to carry out the degradation by enzyme for 5 to 25 hours so that the astringent persimmon and mandarin peel can be sufficiently degraded, more preferably 10 to 20 hours, and most preferably 15 hours. In addition, after completion of the degradation by the enzyme, it is preferable to inactivate the enzyme by heating to 80 to 100 C. in order to prevent side reactions such as saccharification by the enzyme and degradation of the active ingredient.

(11) The step (c) is a step of removing dietary fiber, cell wall and the like excluding the active ingredient after the effective ingredient is eluted, and filtration is preferably performed using diatomite, activated carbon, zeolite or a hollow fiber membrane filter, and more preferably using diatomite which is a filter usable for foods.

(12) In addition, it is preferable to concentrate the filtered solution reaches 0 to 10 BX. Since the composition of the present invention is used for alleviating obesity, it is preferable to concentrate to reach 0 to 10 BX to prevent sugar-induced weight gaining, and more preferably 0 BX.

(13) The unit BX used herein is a unit for measuring sugar content and is often expressed as Brix. This is expressed as sugar content based on the concentration of sugarcane sugar dissolved in 100 g of water, as the value increases, it means that the more sugar is contained.

(14) The present invention may further include, after the step (c), a step for adding sterilized dextrin. Dextrin is added to increase the viscosity of the produced composition and may be added for convenience of transportation and storage of the product. Also, the dextrin may be sterilized at 95 C. for 30 minutes to prevent a denaturing of the extract.

(15) The present invention may further include a step for freeze-drying and pulverizing the extract. Since the produced composition is in a liquid phase, it may easily denatured and may not be easy to transport and store due to heavy weight. Therefore, it is preferable to freeze-dry and then pulverize into a powder form.

(16) In addition, the present invention provides an anti-obesity composition prepared by the above-mentioned method.

(17) Hereinafter, the present invention will be described through the particular examples.

EXAMPLES

(18) Used Raw Materials

(19) Porcine pancreatic lipase (Type 2), Orlistat, morpholinepropanesulphonicacid (MOPS), Tris-HCl, and p-nitrophenyl butyrate (p-NPB), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis-diammonium salt (ABTS) were purchased from Sigma-Aldrich Co. (St Louis, Mo., USA).

(20) Ethylenediaminetetraacetic acid (EDTA) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

(21) Viscozyme was purchased from Novozymes (Denmark).

(22) All other reagents were used as biochemical grade.

(23) Treatment of Experimental Animals

(24) Male healthy 4-week-old ICR mice (about 30-32 g) were purchased from Orient (Gyeonggi-do, Korea). Each mouse was kept at room temperature (223 C.) and humidity (555%) with a 12-h light/dark cycle. The experiments were approved by the Ethics Committee of Animal Experimentation of the University of Daegu Haany. The mice were allowed free access to laboratory pellet chow and water ad libitum.

(25) After adaptation (1 week), all experimental mice except normal mice (n=8) were fed with 60% high-fat diet (HFD; Diet 12492, Research Diets, Inc., 105 New Brunswick, N.J., USA) for 5 days to adapt to a feed. Thereafter, ICR mice (n=32) fed 60% HFD were randomly divided into four groups (HFD control group, orlistat group, and two PCM treatment groups (50 and 200 mg/kg/day))(in each group n=8). The normal group is supplied with a normal feed and the rest of the groups are supplied with 60% HFD until the end of experiment.

(26) The normal and HFD control groups were given water using a stomach tube, while the drug treatment groups were orally given Orlistat or PCM daily using a stomach tube for 6 weeks. After administration for 6 weeks, each mouse was sacrificed after fasting for 12 hours. The blood was immediately centrifuged for 10 minutes at 4 C.

(27) Serum triglyceride and total cholesterol were conducted spectrophotometrically using commercially available kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan). HDL-cholesterol is measured using a commercial kit from Asan Pharm Co., Ltd. (Hwaseong-si, Korea, Cat AM203). LDL-cholesterol levels are calculated though TG, TC and HDL levels, and the following formula.
LDL-cholesterol level (mg/dL)=[TC(HDL-cholesterol)TG]/5

(28) Statistical Processing

(29) All results are expressed as meanstandard error (meanS.E). One-way analysis of variance (ANOVA) in accordance with Dunnett's multiple comparison test (SPSS 18.0 for Windows, SPSS Inc., U.S.A.) for a significance test was performed. When P<0.05, it was determined to be statistically significant.

Example 1

(30) Astringent persimmon (Diospyros kaki Thunb.) was harvested (an astringent persimmon harvested of 3 to 10 cm in diameter with green color harvested at 6-8 months after cultivation) in Gyeongsangbuk-do Agricultural Research & Extension Services (Sangju, Korea) and a dried mandarin peel (Citrus unshiu. S. Marcov.) was purchased from MSC Co., Ltd. (Yangsan, Korea). Each 500 kg was selected and extracted with water and boiled in 100 C. for 2 hours for 2 times. Thereafter enzyme (Viscozyme) degradation was performed for 15 hours. Next, enzyme was inactivated in 90 C. for 30 minutes. After filtration using the diatomite, the extracts were concentrated to reach 0 Bx. The concentrated extracts were added dextrin and were sterilized in 95 C. for 30 minutes. The sterilized extracts were freeze-dried and pulverized by a grinder (the resultant was named as PCM).

Comparative Example 1

(31) The extract was prepared in the same manner as in Example 1 except that an enzymatic degradation process was not performed.

Comparative Example 2

(32) The extract was prepared in the same manner as in Example 1 except that enzymatic degradation process was performed for 5 hours.

Comparative Example 3

(33) The extract was prepared in the same manner as in Example 1 except that enzymatic degradation process was performed for 25 hours.

Experimental Example 1

(34) Antioxidant activity determination of PCM was performed by inhibition of by DPPH radical according to the method of Park et al. 100 L of an ethanolic solution of PCM (blank: 100 L of ethanol) was added to 100 L of an ethanolic solution of DPPH (60 M) using 96-well plate. The ascorbic acid (standard sample) was prepared for eight concentrations (0.5, 1, 2, 5, 10, 20, 50, and 200 g/mL). The reaction mixture was stored in the dark at 25 C. for 30 minutes. The optical density was determined using a microplate reader (M200 PRO, Tecan, Austria). The mixture was measured spectrophotometrically using 540 nm. The antioxidant activity of each sample was expressed in terms of IC.sub.50 (micromolar concentration required to inhibit DPPH radical formation by 50%, calculated from the log-dose inhibition curve). Antioxidant activity was expressed in terms of IC.sub.50, and a lower IC.sub.50 value corresponds to a large inhibition. The radical scavenging activity was calculated using the following formula.
DPPH radical scavenging activity (%)=[1(A.sub.sample/A.sub.blank)]100

(35) As shown in FIG. 1 (A, B), IC.sub.50 of DPPH inhibition of the PCM was 117.464.89 g/mL, and IC.sub.50 value of ascorbic acid (positive control) was 1.260.02 g/mL.

Experimental Example 2

(36) ABTS inhibition of the different extracts was measured according to the modified method of Park et al. ABTS stock solution was dissolved in water to 7.4 mM concentration. The ABTS radical cation (ABTS) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 14 hours at room temperature in the dark. The ABTS solution was diluted with ethanol to obtain an absorbance of 0.700.02 at 750 nm. After adding diluted ABTS solution to PCM solution, the mixture was left standing at room temperature for 15 minutes in the dark. The ascorbic acid (standard sample) was prepared for eight concentrations (0.5, 1, 2, 5, 10, 20, 50, and 200 g/mL). The absorbance at 750 nm was measured using a microplate reader (M200 PRO, Tecan, Austria). The blank was prepared in the same manner, except that distilled water was used instead of the sample. The radical scavenging activity was calculated using the following formula.
ABTS radical scavenging activity (%)=[1(A.sub.sample/A.sub.blank)]100

(37) IC.sub.50 value of PCM against the ABTS was 120.041.67 g/mL and IC.sub.50 value of ascorbic acid as a positive control was 2.270.19 g/mL (FIG. 1 (C, D)).

Experimental Example 3

(38) Pancreatic lipase activity was modified from the method previously reported by Kim et al. Briefly, an enzyme buffer was prepared by the addition of 6 L of a solution of porcine pancreatic lipase in buffer containing 10 mM of MOPS (morpholinepropanesulfonic acid) and 1 mM of EDTA, pH 6.8, to 169 L Tris buffer (100 mM Tris-HCl and 5 mM CaCl.sub.2, pH 7.0). Then, either 20 L of PCM of Example 1 at the test concentration (100, 250, 500, and 1000 g/mL) or orlistat (0.1, 0.25, 0.5, and 1 g/mL) was mixed with 175 L of enzyme buffer and left standing for 15 minutes at 37 C. with 5 L of the substrate solution (10 mM p-NPB (p-nitrophenyl butyrate) in dimethylformamide). The enzymatic reactions were allowed to proceed for 35 minutes at 37 C. Lipase activity was determined by measuring the hydrolysis of p-NPB into p-nitrophenol. Increase in light absorption at 405 nm was measured using a microplate reader (M200 PRO, Tecan, Austria). Inhibition of lipase activity was expressed as the percentage decrease in OD when porcine pancreatic lipase was incubated with the test compounds. Lipase inhibition was calculated according the following formula.
Inhibition (%)=100[(Bb)/(Aa)*100]

(39) Where A is the activity without inhibitor, a is the negative control without inhibitor, B is the activity with inhibitor, and b is the negative control with inhibitor, and the results were expressed as an average (n=4).

(40) As shown in FIG. 2, the lipase inhibition of Orlistat did not rise at least a certain concentration, but the higher the dose of PCM, the higher the lipase inhibition.

Experimental Example 4

(41) The total phenolic content of PCM was quantified by mild modification from the method of Folin-Ciocalteu. 10 L PCM of Example 1 and 790 L of distilled water were mixed and added to well and then mixed with 50 L of Folin-Ciocalteu's reagent for 1 minute. After that 150 L of 20% sodium carbonate solution (Na.sub.2CO.sub.3) was added and the mixture was mixed for 2 hours at 20 C. Lastly, the absorbance of the resulting color was measured at 765 nm. The total phenolic content was expressed as mg gallic acid equivalents per gram extract. Values presented are the average of three measurements. Flavonoid was extracted and quantified by adaptation of the method of Lister et al. 50 L of PCM and 500 L of diethylene glycol were mixed in well. And then 1 N NaOH 5 L was added and the mixture was left standing for 1 hour at 37 C. Finally, the absorbance of the resultant was measured at 420 nm. The flavonoid content was expressed as mg naringin equivalents per gram extract. Values presented are the average of three measurements.

(42) Total phenolic content was measured as gallic acid equivalents (GAE) with reference to standard curve (y=0.023+0.30 and R2=0.997) and was 29.900.14 mg GAE/g of PCM extract. The flavonoid content was 18.330.08 mg naringin equivalent/g of PCM extract, with reference to standard curve (y=0.0195+196 0.04 and R2=0.9999).

Experimental Example 5

(43) To analyze the effects of PCM on HFD-induced obesity, the development of HFD-induced obesity in mice with and without PCM prepared in Example 1 supplementation for 6 weeks was investigated, and measured the effects of PCM on serum lipid profiles such as TG, TC, HDL-cholesterol, and LDL-cholesterol, after the experiment.

(44) As shown in FIG. 3, TG, TC, and LDL-cholesterol levels in mice (Con) after 6 weeks of HDF-supplied were significantly higher than normal food supplied mice (Nor). Orlistat-supplied mice (0) or 200 mg of PCM-supplied mice (PCM200) were at the same level as normal food-supplied mice, and this shows that both Orlistat and PCM may help to lower the TG, TC and LDL-cholesterol levels. In addition, mice (PCM50) supplied with 50 mg of PCM shows a reduction in certain amount of level not as much as Orlistat or PCM200. In addition, HDL-cholesterol which is known to be beneficial to vascular health, was not significantly changed in all mice.

Experimental Example 6

(45) To analyze the effects of PCM on HFD-induced obesity, the development of HFD-induced obesity in mice with and without PCM prepared in Example 1 supplementation for 6 weeks was investigated, and measured the change in body weight and visceral fat, after the experiment.

(46) TABLE-US-00001 TABLE 1 Weight Amount of change Amount of (g/6 Visceral Group Initial (g) Final (g) weeks) fat (g) General 36.06 47.92 1.46 11.86 1.00 24.10 1.0 food 0.96 HDF CON 37.67 55.15 1.78 17.48 1.95 57.5 1.5 0.51 food O 36.71 44.92 1.62 8.21 1.82 42.5 2.8 0.96 PCM50 37.18 48.06 0.17 10.88 0.52 50.6 3.2 0.44 PCM200 36.93 45.98 0.94 9.05 0.90 50.3 2.3 0.38

(47) As shown in Table 1, HFD control mice (CON) increased significantly final body weight compared with normal mice (Normal food) (55.151.78 g, 47.921.46 g). In addition, the visceral fat weight in HFD control mice was significantly increased compared to normal mice (238% of normal mice), but Orlistat and PCM200-treated mice (0, PCM200) were significantly decreased compared with those of HFD control mice. PCM50 treatment showed a tendency to decrease without significance. Above all, body weight change reduced significantly in all drug-treated experimental groups. Overall, PCM may help to alleviate the disorders of HFD-induced obesity.

Experimental Example 7

(48) In order to check the effect according to the method for preparing PCM, experiments were performed in the same manner as in Experimental Example 6 using PCM respectively prepared by the methods of Example 1, Comparative Example 1, Comparative Example and Comparative Example 3. 200 mg of PCM prepared by each method was supplied to experimental mice, and changes of body weight and changes visceral fat according to the preparing method were measured.

(49) TABLE-US-00002 TABLE 2 Weight Amount of Amount Viscozyme change of degradation Initial Final (g/6 Visceral Group time (g) (g) weeks) fat (g) General food 36.06 0.96 47.92 1.46 11.86 1.00 24.1 1.0 HDF CON 37.67 0.51 55.15 1.78 17.48 1.95 57.5 1.5 food Example 1 15 hours 36.93 0.38 45.98 0.94 9.05 0.90 50.3 2.3 Comparative .sup.0 hour 36.51 0.36 53.36 1.42 16.85 1.65 54.8 1.8 Example 1 Comparative 5 hours 38.38 0.54 49.16 0.52 10.78 1.02 52.1 3.2 Example 2 Comparative 25 hours 36.03 0.48 51.02 1.02 14.99 1.32 55.2 2.1 Example 3

(50) As shown in Table 2, it was showed that the Comparative Example 1 exhibited the least effect due to no degradation by Viscozyme, and it was understood that the Comparative Example 2 could not be eluted an active ingredient sufficiently due to short degradation time by Viscozyme. In addition, Comparative Example 3 has sufficient elution time by the Viscozyme, but the eluted active ingredient was degraded by the enzyme or was inactivated by binding with the enzyme, thereby being judged that the effects wears off.

(51) Hereinbefore, preferred embodiments of the present invention have been explained in detail. The explanation of the present invention is only for illustration, and it could be understood that particular embodiment could be easily changed without changing the technical spirit or essential features of the present invention by one of ordinary skilled in the art.

(52) Accordingly, it should be interpreted that the scope of the present invention is represented by claims hereinafter rather than the detailed explanation, and all changes or modifications derived from the meaning, range and equivalent concept of claims are included in the scope of the present invention.