CN106109513B

CN106109513B - Chinese chestnut pulp extract and preparation method and application thereof

Info

Publication number: CN106109513B
Application number: CN201610496604.9A
Authority: CN
Inventors: 郭传明; 崔龙
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-06-29
Filing date: 2016-06-29
Publication date: 2020-04-07
Anticipated expiration: 2036-06-29
Also published as: CN106109513A

Abstract

The invention relates to a chestnut pulp extract and a preparation method and application thereof, belonging to the technical field of natural product extraction and application. The preparation method comprises the following steps: extraction: the Chinese chestnut pulp is used as a raw material, ethanol water solution with the volume percentage concentration of 40-80% is used as a solvent, solvent extraction is carried out, and the solvent is recovered, so that the Chinese chestnut pulp extract is obtained. The semen Castaneae pulp extract has effect in inhibiting diacylglycerol acyltransferase (DGAT), and can be used as DGAT inhibitor for treating diabetes, obesity and other complications caused by diabetes and obesity.

Description

Chinese chestnut pulp extract and preparation method and application thereof

Technical Field

The invention relates to the technical field of natural product extraction and application, in particular to a chestnut pulp extract and a preparation method and application thereof.

Background

With the rapid development of global economy, the incidence of obesity is increasing day by day. It is estimated that by 2050, the prevalence of obesity will increase to 60% in adult males, 50% in adult females, and 25% in children under 16 years of age. Obesity epidemic will lead to a series of complications, such as diabetes; cardiovascular diseases: myocardial infarction, hypertension, hyperlipidemia, etc. Among them, obesity is the most dangerous factor among many factors causing the onset of diabetes.

Obesity is mainly treated by three aspects: improving lifestyle, medication and surgical treatment. Lifestyle changes often require the patient's own volition and supervision to ensure implementation, while paying attention to physical changes and maintaining nutritional balance, but often have a high recurrence rate. Surgical methods are mostly used for the treatment of complications of severely obese patients, but in view of the high surgical costs and the lifelong nature of medical care, the prophylactic treatment of obesity using drugs is a crucial tool.

Triglyceride (TG) is a main form of energy storage of eukaryotes, when energy taken in by a human body exceeds energy consumed by the human body, the surplus energy is stored in adipose tissues in the form of TG, and the excessive TG can be deposited in tissues such as islet β cells and liver, and finally diseases such as obesity and diabetes are caused.

TG is mainly synthesized in the liver, adipose tissue and small intestine. TG synthesis has two pathways: the glycerol triphosphate pathway (major pathway) and the monoacylglycerol pathway (minor pathway). First, glycerol-3-phosphate reacts with 2 molecules of fatty acyl-CoA to form phosphatidic acid, which is dephosphorylated by a phosphatase to form Diacylglycerol (DAG). DAG can also be produced via the secondary pathway monoacylglycerols and 1 molecule fatty acyl-CoA. While diacylglycerol acyltransferase (DGAT) serves as the key last step in catalyzing TG synthesis: transfer of acyl-CoA acyl groups to DAG to form TG is the rate-limiting enzyme in TG synthesis.

DGAT is an endoplasmic reticulum membrane integrin and has two subtypes: DGAT1 and DGAT 2. DGAT1 and DGAT2 are widely expressed in all tissues of mammals, have undergone the convergent evolution in function, can catalyze the synthesis of TG from sn-1, 2-diacylglycerol and fatty acyl CoA, and have no specificity to the substrate fatty acyl CoA. DGATl belongs to the cholesterol acyltransferase gene family, and DGAT2 belongs to the Mramanniana gene family. It was found that DGAT was administered either on a normal diet or on a high fat diet^-/-The volume of mouse fat cells is all compared with DGAT^+/+Mice were used. Notably, DGAT1 was administered after a high fat diet^+/+The mouse adipocyte volume increased two-fold, while DGATl^-/-The size of the lipofectcells of the mice did not change significantly. At the same time, DGAT1^-/-The leptin activity of the mice is also improved. The DGAT2 function is down-regulated, so that the content of TG in the liver of an obese mouse can be obviously reduced, and the formation of fatty liver can be improved. These studies show that the DGAT activity inhibitor has important pharmaceutical research value in the aspects of preventing obesity, regulating lipid metabolism disorder, preventing hepatic steatosis and the like.

In 2006, the DGAT1 inhibitor BAY74-4113 of pfeiy was in phase I clinical trials; in 2009, norwalk's DGAT1 inhibitor has entered clinical trials in II. DGAT1 has become the focus of research on drugs for obesity, diabetes and other diseases. At present, no natural lipid-lowering drug taking DGAT1 as a target point is on the market, which provides an opportunity for searching a high-efficiency and high-selectivity small-molecule DGAT1 inhibitor from natural resources.

Disclosure of Invention

Based on this, the present invention seeks to provide a natural ingredient having DGAT1 inhibitory activity from natural sources.

A preparation method of chestnut pulp extract comprises the following steps:

extraction: the Chinese chestnut pulp is used as a raw material, ethanol water solution with the volume percentage concentration of 40-80% is used as a solvent, solvent extraction is carried out, and the solvent is recovered, so that the Chinese chestnut pulp extract is obtained.

The Chinese chestnut (Castanea mollissima Blume) belongs to Fagaceae chestnut nut plants, namely chestnut, big chestnut and the like, is a special plant in China and is mainly produced in Guangdong, Guangxi, Yunnan, Jiangxi and the like. Mainly used for preventing and treating hypertension, coronary heart disease, arteriosclerosis, osteoporosis and other diseases, and is a good tonic product with anti-aging and life prolonging effects; chestnut is sweet in taste and warm in nature, and enters spleen, stomach and kidney meridians; has the functions of nourishing stomach, invigorating spleen, invigorating kidney, strengthening muscle and tendon, promoting blood circulation and stopping bleeding.

After long-term experimental research based on modern medical theories and technologies, the inventor discovers that the ethanol extract of the Chinese chestnut pulp has the function of inhibiting diacylglycerol acyltransferase (DGAT), and can use the extract as a DGAT inhibitor to treat obesity and other complications caused by obesity.

In one embodiment, in the extracting step, the solvent is an ethanol aqueous solution with a concentration of 45% to 55% by volume. Preferably 50% ethanol in water. The inventor finds that the chestnut pulp extract extracted by 45% -55% of ethanol water solution has better DGAT inhibiting activity through experiments.

In one embodiment, in the extraction step, the ratio of the mass of the raw materials to the volume of the solvent in the extraction step is 1g:10-20ml, and ultrasonic extraction is carried out for 2-4 times, wherein each time is 90-120 min;

the specific method for recovering the solvent comprises the following steps: concentrating under reduced pressure to recover ethanol, concentrating to relative density of 1.08-1.10 at 75-80 deg.C, adding 2-3 times of water, stirring, standing at room temperature for 12-36 hr, precipitating, and collecting precipitate to obtain semen Castaneae pulp extract. The extraction method has the advantages of high extraction efficiency, strong operability and good activity of the obtained product.

In one embodiment, the method further comprises a purification step, wherein the purification step comprises:

loading the chestnut pulp extract obtained in the extraction step to a reverse silica gel chromatographic column for purification, performing gradient elution by using an ethanol-water solution system, collecting part of eluent with the volume ratio of ethanol to water of 3.5-4.5:5.5-6.5, and recovering the solvent to obtain the chestnut pulp extract.

By further purification, the composition of the extract can be enriched for components that truly have inhibitory activity against DGAT. Moreover, after a large number of experiments, the inventor finds that the component with the activity of inhibiting DGAT mainly exists in the ethanol-water eluent with the ratio of 3.5-4.5:5.5-6.5, and the activity of the components in the eluent of other components is not as good as that of the component.

In one embodiment, in the purification step, the reverse silica gel is a octadecylsilyl-bonded silica gel having a particle size of 100-. The Chinese chestnut pulp extract is purified by the reverse silica gel of the type, so that the part which really has inhibitory activity to DGAT in the composition of the extract can be better enriched.

In one embodiment, in the purification step, the chestnut pulp extract obtained in the extraction step is loaded on a reverse silica gel chromatographic column, and then is subjected to gradient elution by using ethanol-water solutions with volume ratios of 0:10 and 3.5-4.5:5.5-6.5, the eluates of the ethanol-water solutions with volume ratios of 3.5-4.5:5.5-6.5 are collected, and the solvent is recovered, so that the Chinese chestnut pulp extract is obtained. The method is characterized by firstly eluting with water to remove part of impurity components in the product, and has good purification effect.

In one embodiment, in the purification step, 1-3 column volumes are eluted with a methanol-water solution in a volume ratio of 0: 10; eluting with methanol-water solution at volume ratio of 4:6 for 1-3 column volumes; the eluate of the ethanol-water solution was collected in a volume ratio of 4: 6. Eluting with the eluent with the column volume to remove impurity components to the maximum extent and retain components with DGAT inhibiting activity.

The invention also discloses the Chinese chestnut pulp extract prepared by the preparation method of the Chinese chestnut pulp extract. The semen Castaneae pulp extract has DGAT inhibiting activity, is a natural DGAT inhibitor, can be used as inhibitor for inhibiting diacylglycerol acyltransferase, and can be used for preventing and treating obesity and its complications.

The application of the Chinese chestnut pulp extract as an inhibitor for inhibiting diacylglycerol acyltransferase and/or as an insulin sensitizer is also disclosed.

In one embodiment, the inhibitor for inhibiting diacylglycerol acyltransferase and/or the use thereof as an insulin sensitizer is used for preparing medicaments for preventing and treating diabetes, obesity and complications thereof.

Compared with the prior art, the invention has the following beneficial effects:

the Chinese chestnut pulp extract prepared by the method has the function of inhibiting diacylglycerol acyltransferase (DGAT), can be used as a DGAT inhibitor or an insulin sensitizer and is used for treating diabetes, obesity and other complications caused by diabetes and obesity. Has important significance for developing and utilizing Chinese medicinal plant resources.

In addition, the preparation method of the Chinese chestnut pulp extract has the advantages of strong operability and suitability for industrial production.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In the following examples, C18 reverse phase silica gel was used, manufactured by Merck, inc, cat # M2014441; STZ (streptozotocin), a sanland product, is analytically pure, is prepared in an ice bath at 4 ℃ with 0.05mol/ml citric acid pH4.5 solution before use and is used immediately; xuezhikang, produced by Beijing Beidawei Biotech Co., Ltd, batch No. 2015055, human insulin, produced by Novonide (China) pharmaceutical Co., Ltd, batch No. 2014223. Xiaoke Wan, produced by Guangzhou Zhongyiyao pharmaceutical Co., Ltd, batch No.: GF 0085.

Example 1

A chestnut pulp extract is prepared by the following steps:

1. and (4) extracting.

Extracting dried and minced chestnut pulp with 40% volume percentage concentration ethanol for 2-4 times (90-120 min each time) according to 10-20 times of chestnut pulp to ethanol feed-liquid ratio (g/ml), and mixing extractive solutions. In this example, the chestnut pulp is 500g, ethanol is 5L-10L, and ultrasonic extraction is performed for 3 times, each time for 120 min.

Then, decompressing and concentrating the extracting solution to recover the ethanol, concentrating the extracting solution to the relative density of 1.08 to 1.10 (at the temperature of 75 to 80 ℃), cooling the extracting solution, adding 2 to 3 times of water into the extracting solution, stirring the extracting solution, standing the extracting solution for 24 hours at normal temperature, separating out the precipitate, filtering or centrifuging the precipitate, placing the precipitate in an open container, heating the precipitate to volatilize the ethanol as far as possible, cooling the precipitate to obtain 28.1g of 40 percent ethanol extract, and drying the extract to obtain the Chinese chestnut pulp extract, namely the component A1.

2. And (5) purifying.

Loading the chestnut pulp extract obtained in the extraction step to a reversed phase silica gel chromatography (C-18; particle size 150 mu m), purifying, performing gradient elution by using ethanol/water (v/v, 0:10, 4:6, 5:5, 10:0) as eluent, wherein each eluent is 1L (equivalent to 2 column volumes), collecting separation components of the ethanol/water (v/v, 4:6), collecting eluent (ethanol/water is 4:6), concentrating the eluent under reduced pressure at normal temperature, and drying to obtain refined components of the chestnut pulp extract, namely the component A2.

Example 2

A chestnut pulp extract is prepared by the following steps:

1. and (4) extracting.

Extracting dried and minced chestnut pulp with 50% volume percentage concentration ethanol for 2-4 times (90-120 min each time) according to 10-20 times of chestnut pulp to ethanol feed-liquid ratio (g/ml), and mixing extractive solutions. In this example, the chestnut pulp is 500g, ethanol is 5L-10L, and ultrasonic extraction is performed for 3 times, each time for 120 min.

Then, decompressing and concentrating the extracting solution to recover the ethanol, concentrating the extracting solution to the relative density of 1.08 to 1.10 (at the temperature of 75 to 80 ℃), cooling the extracting solution, adding 2 to 3 times of water, stirring the water, standing the extracting solution for 24 hours at normal temperature, separating out the precipitate, filtering or centrifuging the precipitate, putting the precipitate into an open container, heating the precipitate to volatilize the ethanol as far as possible, cooling the precipitate to obtain 37.5g of 50 percent ethanol extract, and drying the extract to obtain the Chinese chestnut pulp extract, namely the component B1.

2. And (5) purifying.

Loading the chestnut pulp extract obtained in the extraction step to a reversed phase silica gel chromatography (C-18; particle size 150 mu m), purifying, performing gradient elution by using ethanol/water (v/v, 0:10, 4:6, 5:5, 10:0) as eluent, wherein each eluent is 1L (equivalent to 2 column volumes), collecting separation components of the ethanol/water (v/v, 4:6), collecting eluent (ethanol/water is 4:6), concentrating the eluent at normal temperature under reduced pressure, and drying to obtain a refined component of the chestnut pulp extract of 5.3g, namely a component B2.

In this example, the eluate of the alcohol/water (v/v, 0:10, 5:5, 10:0) fractions was concentrated at room temperature under reduced pressure and dried to obtain fraction BI, fraction BII and fraction BIII, respectively.

Example 3

A chestnut pulp extract is prepared by the following steps:

1. and (4) extracting.

Extracting dried and minced chestnut pulp with 80 vol% ethanol for 2-4 times (90-120 min each time) at a material-to-liquid ratio (g/ml) of chestnut pulp to ethanol of 10-20 times, and mixing extractive solutions. In this example, the chestnut pulp is 500g, ethanol is 5L-10L, and ultrasonic extraction is performed for 3 times, each time for 120 min.

Then, decompressing and concentrating the extracting solution to recover the ethanol, concentrating the extracting solution to the relative density of 1.08 to 1.10 (at the temperature of 75 to 80 ℃), cooling the extracting solution, adding 2 to 3 times of water, stirring the water, standing the extracting solution for 24 hours at normal temperature, separating out the precipitate, filtering or centrifuging the precipitate, putting the precipitate into an open container, heating the precipitate to volatilize the ethanol as far as possible, cooling the precipitate to obtain 33.1g of 80 percent ethanol extract, and drying the extract to obtain the Chinese chestnut pulp extract, namely the component C1.

2. And (5) purifying.

Loading the chestnut pulp extract obtained in the extraction step to a reversed phase silica gel chromatography (C-18; particle size 150 mu m), purifying, performing gradient elution by using ethanol/water (v/v, 0:10, 4:6, 5:5, 10:0) as eluent, wherein each eluent is 1L (equivalent to 2 column volumes), collecting separation components of the ethanol/water (v/v, 4:6), collecting eluent (ethanol/water is 4:6), concentrating the eluent under reduced pressure at normal temperature, and drying to obtain refined components of the chestnut pulp extract, namely the component C2.

Test example 1

The activity of the extract obtained in the above examples was tested for inhibiting DGAT 1.

1. Experimental methods.

In vitro detection of DGATl inhibitors human DGATl expressed on insect cell membranes was used as the enzyme source (Proc. Natl. Acad. sci.1998,95, 13018-13023). Sf9 cells (source: ProSpec, cat # CYT-421) were infected with a recombinant baculovirus containing the human DGATl coding sequence (source: Givone Biotech Co., Ltd., cat # BL10097) and harvested after 48 hours.

Cells were lysed by sonication and centrifuged at 28000rpm in 40% sucrose solution for 1 hour at 4 ℃ to give the DGAT1 enzyme. Intercellular septal debris were collected, washed and stored in liquid nitrogen.

Detection of DGAT1 activity: by the method described by modified Coleman (Coleman) (methodsin enzymology1992,209, 98-102).

The reaction system comprises: 100. mu.g/ml sample, 0.4. mu.g of DGAT1 enzyme obtained as described above, 5mM MgCl₂And 1.2mM sn-1, 2-diacylglycerol-glycerol, adjusted to a total assay volume of 200. mu.L with distilled water, and cultured in a test tube.

By adding 100 mu mol/L into the system¹⁴Glycerol acyl-coenzyme A (0.05. mu. Ci) was reacted with gentle brief shaking at 25 ℃. After 30min the reaction was stopped by adding 1.5mL 2-propanol, heptane, water (80:20: 2). After the reaction had stopped, l.0mL of heptane and 0.5mL of 0.1M carbonate buffer (pH9.5) were added to separate the radiotriacylglycerol product into an organic phase, and 2.0mL of an alkaline ethanol solution (ethanol: 0.5N NaOH: H) was added₂O50: 10:40) was washed once. DGAT1 activity was quantified by liquid scintillation photography by detecting radioactivity in the upper heptane layer. The experimental results are as follows:

TABLE 1 IC of different extracts on the inhibitory Activity of DGAT1₅₀(n＝10)

The experimental results show that: the ethanol extracts and refined components with different concentrations have different effects on inhibiting the activity of DGAT1, wherein the component B1 and the component B2 (50% ethanol extract and 50% ethanol extract refined component) have the best inhibiting activity on DGAT 1.

Whereas, the inferior activity of component BI, BII and BIII compared to component B2 indicates that the active ingredient having the effect of inhibiting DGAT1 is mainly present in the ethanol/water (v/v, 4:6) eluate in the 50% ethanol extract.

Test example 2

The extracts obtained in the above examples were used for weight loss experiments in hyperlipidemic rats, respectively.

1. Experimental methods.

50 healthy and clean Kunming male rats are taken, 40 rats with the weight of 160-220 g are screened out after basic feed is fed for 7 days in a balanced manner, and are randomly grouped into a blank group, a model group, an experimental group (a component A1 and a component A2) and a Xuezukang control group (XZK).

Rats form an animal model of hyperlipidemia by feeding high-fat diet. From the start to the end of the experiment, normal group (i.e., blank group) and high-fat model group (i.e., model group) were given an equal volume of physiological saline, and the administration group (i.e., experimental group) and control group (Xuezhikang control group) were gavaged with A1 fraction and A2 fraction (200mg/kg) and Xuezhikang (200mg/kg), respectively, 1 time/d, and gavaged continuously for 30d, and body weight was measured daily. The experimental results are as follows:

TABLE 2 weight loss in hyperlipidemic rats by different extracts

The experimental results show that compared with the model group, the active components A1 and A2 have obvious weight reducing effect on the hyperlipidemic rats caused by high-fat diet, wherein the weight reducing effect of the component A2 is equivalent to that of the hyperlipidemic control group.

Test example 3

The extracts obtained in the above examples were used for the blood glucose lowering test in STZ hyperglycemic rats, respectively.

1. Experimental methods.

100 Kunming rats (male) were randomly divided into a blank group, a model group (Diabetic control), an experimental group (A1 component, A2 component) and a Xiaokewan control group (XKW).

Except for the blank group, each group was fasted for 1d and was intraperitoneally injected with 60mg/kg of STZ (prepared with 0.05mol/ml citric acid pH4.5 solution in 4 ℃ ice bath, immediately used) to create a diabetes model. Blood glucose was measured 72h after injection by cutting off the tail and taking blood (fasting for 6h before measurement), blood glucose value was higher than 11.1mmol/L for experiment (10/group).

The experimental group is respectively drenched with A1 component and A2 component (200 mg/kg); XKW group is administered by XIAOKE pill (250 mg/kg); the blank group and the model group are filled with physiological saline with the same volume as the stomach. Dosing was for 30d, weighed daily, and dosed once. After the last administration, blood was taken with broken tail (fasting for 6h before measurement). The experimental results are as follows:

TABLE 3 hypoglycemic conditions of STZ hyperglycemic rats with different extracts

The experimental results show that: the rats in the experimental group all have different blood sugar reduction degrees, wherein the blood sugar reduction effect of the component A2 is most obvious. Experimental data show that the component A2 has better blood sugar lowering effect than diabetes treating pills.

Test example 4

The effect of the extract obtained in the above example on insulin sensitivity in STZ hyperglycemic rats was examined.

1. Experimental methods.

The experimental groups were gavaged with component A1, component A2 (300mg/kg), respectively; the blank group and the model group are filled with physiological saline with the same volume as the stomach. Dosing was for 30d, weighed daily, and dosed once. The long-acting human insulin is injected into the abdominal cavity of each group within 30 days of administration for 1IU/kg every day. After 30 days, the rats of each group are respectively injected with 0.05, 0.5, 1.0 and 2.5IU/kg of quick-acting human insulin intraperitoneally every day within four days, the blood sugar before and after the quick-acting human insulin is given is measured, and the ratio is calculated. The experimental results are as follows:

TABLE 4 insulin sensitivity of different extracts in STZ hyperglycemic rats

The experimental results show that different components have improved insulin sensitivity on rats with hyperglycemia caused by STZ, but the effects of the components are different, and the effect of enhancing the insulin sensitivity is most obvious when the component A2 is compared with a model group.

Test example 5

The extracts obtained in the above examples were each tested for their effect on glucose tolerance in STZ-induced hyperglycemic rats.

1. Experimental methods

The experimental groups were gavaged with component A1, component A2 (300mg/kg), respectively; XKW group is administered by XIAOKE pill (250 mg/kg); equal volume of saline (10 per group) was perfused into the stomach for the blank and model groups. Dosing was for 30d, weighed daily, and dosed once. After administration for 30 days, the above groups are fasted for 4h (9: 00-13: 00), glucose (300mg/kg) is perfused, and blood sugar before (0min) and 30min, 60min and 120min after perfusion is measured. The experimental results are as follows:

TABLE 5 glucose tolerance of different extracts in STZ-hyperglycemic rats

The experimental results show that each component has the tendency of improving the sugar tolerance of the rat with hyperglycemia caused by STZ, but the effect of each component is different. Experimental data show that the blood sugar reducing effect of the component A2 is equivalent to that of the XZK group.

Test example 6

The extracts obtained in the above examples were used for the body weight change test of STZ-hyperglycemic rats, respectively.

The experimental groups were gavaged with component A1, component A2 (300mg/kg), respectively; XKW group is administered by XIAOKE pill (250 mg/kg); the blank group and the model group are filled with physiological saline with the same volume as the stomach. Dosing was for 30d, weighed daily, and dosed once. The experimental results are as follows:

TABLE 6 different extracts on weight change in STZ hyperglycemic rats (n ═ 10)

The experimental results show that: the different components have different effects on the weight gain of the rats with hyperglycemia caused by STZ. Wherein the component A2 has the best effect of increasing body weight.

The above test data show that the chestnut extract fractions of the present invention are all capable of inhibiting DGAT activity (test example 1). Through a hyperlipidemia rat weight reducing experiment (test example 2), the Chinese chestnut pulp extract disclosed by the invention is fully determined to have a weight reducing effect on hyperlipidemia model animals, and further, an STZ hyperglycemia-induced hyperglycemia rat blood sugar reducing experiment (test example 3) shows that the Chinese chestnut pulp extract disclosed by the invention can obviously reduce blood sugar of model animals, improve the sensitivity of organisms to insulin and improve the bioavailability of insulin (test example 4). The sugar tolerance test of rats with hyperglycemia caused by STZ (test example 5) shows that the Chinese chestnut pulp extract can obviously improve the sugar tolerance of organisms. The weight change test data of rats show that (test example 6) the Chinese chestnut pulp extract has obvious effect of improving the weight average of the body of a diabetes model animal.

The relevant test data are combined, and the Chinese chestnut extract has potential practical significance for treating type 2 diabetes, obesity and complications.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for preparing a chestnut pulp extract for inhibiting diacylglycerol acyltransferase, comprising the steps of:

extraction: performing solvent extraction by taking Chinese chestnut pulp as a raw material and an ethanol water solution with the volume percentage concentration of 45-55% as a solvent, wherein the ratio of the mass of the raw material to the volume of the solvent is 1g:10-20ml, and performing ultrasonic extraction for 2-4 times, and each time lasts for 90-120 min; concentrating under reduced pressure to recover ethanol, concentrating to relative density of 1.08-1.10 at 75-80 deg.C, adding 2-3 times of water, stirring, standing at room temperature for 12-36 hr, precipitating, and collecting precipitate to obtain semen Castaneae pulp extract;

and (3) purification: loading the Chinese chestnut pulp extract obtained in the extraction step to a octadecylsilyl bonded silica gel chromatographic column with the particle size of 100-; eluting with methanol-water solution at volume ratio of 4:6 for 1-3 column volumes; collecting the eluent of the ethanol-water solution with the volume ratio of 4:6, and recovering the solvent to obtain the compound.

2. A chestnut pulp extract prepared by the method for preparing a chestnut pulp extract according to claim 1.

3. Use of the chestnut pulp extract of claim 2 for the preparation of an inhibitor for inhibiting diacylglycerol acyltransferase and/or for the preparation of an insulin sensitizer.

4. Use of the chestnut pulp extract of claim 2 for the preparation of a medicament for the prevention and treatment of diabetes, obesity and complications thereof.