CN105902583B - Chestnut shell extract and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a chestnut shell 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 chestnut shell extract is prepared by using chestnut shells as raw materials and ethanol water solution with the volume percentage concentration of 40-100% as a solvent, performing solvent extraction, and recovering the solvent. The chestnut shell extract has the function of inhibiting protein tyrosine phosphatase 1B (PTP1B), can be used as PTP1B inhibitor, and can be used for treating diabetes, obesity and other complications caused by diabetes, obesity and other complications.
Description
Technical Field
The invention relates to the technical field of natural product extraction and application, in particular to a chestnut shell extract and a preparation method and application thereof.
Background
At present, diabetes is clinically classified into insulin-dependent diabetes mellitus (IDDM, type I diabetes) and non-insulin-dependent diabetes mellitus (NIDDM, type II diabetes), wherein the type II diabetes accounts for 90% of diabetes. WHO predicts that by 2025 the number of diabetic patients will rise from 1.35 to 3 billion in 1995 due to aging of the population, obesity, unhealthy diet, and a lifestyle lacking exercise.
Type ii diabetes is characterized by the resistance of insulin-sensitive tissues, such as skeletal muscle, liver, adipose tissue, to the action of insulin. Although the specific mechanism is not clear, there must be a direct relationship between the attenuation or even the blocking of insulin signaling in its conduction pathway.
PTPases (protein tyrosine phosphatases) act at multiple links in the insulin signaling pathway, such as dephosphorylation of autophosphorylation-activated IR, thereby reducing receptor kinase activity; or dephosphorylating protein tyrosine residue in substrates of insulin receptors such as IRS-1, IRS-2, Shc and the like, thereby negatively regulating and controlling the pathway of insulin acting on the receptor. An imbalance in enzyme activity between specific PTPases and tyrosine kinases in the insulin pathway may be responsible for insulin resistance in type II diabetes.
Therefore, by finding inhibitors that act selectively on PTPases in this pathway, inhibit their activity, and potentiate and prolong insulin signaling, it has become an increasingly important new approach to the treatment of type II diabetes.
At present, the research on selective inhibitors of PTP1B (protein tyrosine phosphatase 1B) has been advanced, but most of the research is limited to peptide and peptoid compounds, such as: although the peptide compounds have high selectivity and strong inhibitory activity, the peptide compounds are difficult to penetrate cell membranes, and the structure of the peptide phosphate makes the peptide compounds difficult to become drug candidate compounds.
Recently, in reports of non-peptide PTP1B inhibitors, 2-carboxymethoxybenzoic acid compounds have strong inhibitory effects on PTP1B, and more importantly, (2S) -2- [ 4' - (2-benzyl-benzofuran) -3-biphenyl-4-oxo ] -3-phenyl-propionic acid compounds have strong selective inhibitory effects on PTP1B and also have significant effects on reducing glucose and insulin levels in the plasma of ob/ob mice. This is the first evidence that PTP1B inhibitors have direct pharmacological evidence of anti-diabetic activity (Malamas, M.S.et al.J.Med.chem.2000,43, 1293-. The method provides an opportunity for searching a high-efficiency and high-selectivity small-molecule non-peptide PTP1B inhibitor from natural resources.
Disclosure of Invention
Based on this, the present invention seeks to provide a natural ingredient having PTP1B inhibitory activity from natural sources.
A method for preparing chestnut shell extract comprises the following steps:
extraction: the chestnut shell extract is prepared by using chestnut shells as raw materials and ethanol water solution with the volume percentage concentration of 40-100% as a solvent, performing solvent extraction, and recovering the solvent.
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. The Chinese chestnut can be used as medicine, and comprises root, peel, leaf, flower, epicarp (chestnut shell), endocarp (chestnut shell), and bract (chestnut hair bulb or hull).
The chestnut shells are the epicarp of the chestnuts, are generally considered to be sweet, astringent and mild in property, and have the effects of lowering adverse qi and stopping bleeding.
After long-term experimental research, the inventor discovers that the ethanol extract of the chestnut epicarp (namely the chestnut shell) has the effect of inhibiting protein tyrosine phosphatase 1B (PTP1B), and the extract can be used as a PTP1B inhibitor and used for treating diabetes, obesity and other complications caused by the diabetes, the obesity and the other complications.
In one embodiment, in the extracting step, the solvent is 70-80% ethanol water solution by volume, preferably 75% ethanol water solution. The inventor finds that the chestnut shell extract extracted by 70-80% of ethanol water solution has better PTP1B activity inhibition effect through experiments.
In one embodiment, in the extraction step, the ratio of the mass of the raw materials to the volume of the solvent is 1g:5-10ml, and ultrasonic extraction is carried out for 2-4 times, and each time lasts for 30-60 min. 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 shell extract obtained in the extraction step onto macroporous adsorption resin, purifying, eluting with methanol-water solution with volume ratio of 1-3:8 (preferably 2:8), collecting eluate, and recovering solvent.
By further purification, the composition of the extract can be enriched for components that truly have inhibitory activity against PTP 1B. Furthermore, the present inventors have found, after numerous experiments, that the component having inhibitory activity against PTP1B is mainly present in the methanol-water eluate at a ratio of 1-3:8, and that the components of the eluate of other components are not as active as the component.
In one embodiment, the macroporous adsorbent resin is HP-20 in the purification step. The chestnut shell extract is purified by the macroporous adsorption resin of the type, so that the components which really have inhibitory activity to PTP1B in the composition of the extract can be better enriched.
In one embodiment, in the purification step, after the chestnut shell extract obtained in the extraction step is loaded on macroporous adsorption resin, gradient elution is sequentially carried out by using methanol-water solutions with volume ratios of 0:10, 0.5-2:9 and 1-3:8, the eluent of the methanol-water solutions with volume ratios of 1-3:8 is collected, and the solvent is recovered, so that the chestnut shell extract is obtained. Gradient elution is carried out by using water and methanol-water solution with the volume ratio of 0.5-2:9, partial impurity components are removed, and the method 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; the volume ratio of the components is 0.5-2:9 methanol-water solution elutes 1-3 column volumes; eluting with methanol-water solution at a volume ratio of 1-3:8 for 1-3 column volumes. Elution with the above column volume of eluent enables removal of impurity components to the maximum extent and retention of components having inhibitory activity against PTP 1B.
The invention also discloses the chestnut shell extract prepared by the preparation method of the chestnut shell extract. The chestnut shell extract has PTP1B activity inhibiting effect, is a natural PTP1B inhibitor, can be used as an inhibitor for inhibiting protein tyrosine phosphatase 1B and/or as an insulin sensitizer, and is used for preventing and treating diabetes, obesity and complications thereof.
The invention also discloses the application of the chestnut shell extract as an inhibitor for inhibiting protein tyrosine phosphatase 1B and/or as an insulin sensitizer.
In one embodiment, the inhibitor of protein tyrosine phosphatase 1B and/or the insulin sensitizer are used for preparing medicines for preventing and treating diabetes, obesity and complications thereof.
Compared with the prior art, the invention has the following beneficial effects:
the chestnut shell extract prepared by the method has the effect of inhibiting protein tyrosine phosphatase 1B (PTP1B), can be used as a PTP1B inhibitor, and is used for treating diabetes, obesity and other complications caused by the diabetes, the obesity and the other complications. Has important significance for developing and utilizing Chinese medicinal plant resources.
Moreover, the preparation method of the chestnut shell extract has the advantages of strong operability and suitability for industrial production.
Drawings
FIG. 1 is a graph showing the effect of the extracts of the examples on insulin sensitivity in STZ-hyperglycemic rats, respectively;
FIG. 2 is the effect of the extracts of the examples on glucose tolerance in STZ-hyperglycemic rats, respectively.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. 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, the macroporous resin (HP-20) chromatography used was Beijing green Baicao scientific development Co., Ltd; STZ (streptozotocin), a sanland product, analytically pure, prepared in a 0.05mol/ml citric acid pH4.5 solution in an ice bath at 4 ℃ before use, and used immediately; xiaoke Wan, Guangzhou Chinese medicine industry Co., Ltd., batch No.: GF 0085.
Example 1
A chestnut shell extract is prepared by the following method:
1. and (4) extracting.
Ultrasonically extracting dried and minced chestnut (Castanea mollissima Blume) shell with 100% ethanol (pure ethanol) at a ratio of 5-10 times (g/ml) of chestnut shell to ethanol for 2-4 times (30-60 min each time), and mixing extractive solutions. In this example, the chestnut shell is 500g, ethanol is 5L, and ultrasonic extraction is performed for 60min 3 times.
And then, carrying out reduced pressure concentration on the extracting solution to recover ethanol until the ethanol is difficult to concentrate, merging the concentrated solution, continuously heating in an open container to volatilize the ethanol as far as possible, cooling to obtain 32g of 100% ethanol extract, and drying to obtain the chestnut shell extract, namely the component I.
2. And (5) purifying.
Loading the chestnut shell extract obtained in the extraction step onto macroporous adsorption resin (HP-20), purifying, performing gradient elution by using methanol/water (v/v, 0:10, 1:9, 2:8, 10:0) as eluent, wherein 1L (equivalent to 2 column volumes) of each eluent is used for collecting a separation component of the methanol/water (v/v, 2:8), collecting the eluent (methanol/water is 2:8), concentrating the eluent under reduced pressure at normal temperature, and drying to obtain a refined component 4.3g of the chestnut shell extract, namely the component HI.
Example 2
A chestnut shell extract is prepared by the following method:
1. and (4) extracting.
Ultrasonically extracting dried and cut Chinese chestnut (Castanea mollissima Blume) shell with 75% ethanol water solution according to the material-liquid ratio (g/ml) of Chinese chestnut shell to ethanol of 5-10 times for 2-4 times, each time for 30-60 min, and mixing extractive solutions. In this example, the chestnut shell is 500g, ethanol is 5L, and ultrasonic extraction is performed for 60min 3 times.
And then, carrying out reduced pressure concentration on the extracting solution to recover ethanol until the ethanol is difficult to concentrate, merging the concentrated solution, continuously heating in an open container to volatilize the ethanol as far as possible, cooling to obtain 38.2g of 100% ethanol extract, and drying to obtain the chestnut shell extract, namely the component II.
2. And (5) purifying.
Loading the chestnut shell extract obtained in the extraction step onto macroporous adsorption resin (HP-20), purifying, performing gradient elution by using methanol/water (v/v, 0:10, 1:9, 2:8, 10:0) as eluent, wherein 1L (equivalent to 2 column volumes) of each eluent is used for collecting separation components of the methanol/water (v/v, 2:8), collecting eluent (methanol/water is 2:8), concentrating the eluent under reduced pressure at normal temperature, and drying to obtain refined components of the chestnut shell extract 6.2g, namely the component III.
In this example, the eluates of alcohol/water (v/v, 0:10, 1:9, 10:0) fractions were further concentrated at room temperature under reduced pressure and dried to obtain fractions a, B and C, respectively.
Example 3
A chestnut shell extract is prepared by the following method:
1. and (4) extracting.
Ultrasonically extracting dried and minced chestnut (Castanea mollissima Blume) shell with 50% ethanol water solution according to the material-liquid ratio (g/ml) of chestnut shell to ethanol of 5-10 times for 2-4 times, each time for 30-60 min, and mixing extractive solutions. In this example, the chestnut shell is 500g, ethanol is 5L, and ultrasonic extraction is performed for 60min 3 times.
And then, carrying out reduced pressure concentration on the extracting solution to recover ethanol until the ethanol is difficult to concentrate, merging the concentrated solution, continuously heating in an open container to volatilize the ethanol as far as possible, cooling to obtain 28.5g of 100% ethanol extract, and drying to obtain the chestnut shell extract, namely the component III.
2. And (5) purifying.
Loading the chestnut shell extract obtained in the extraction step onto macroporous adsorption resin (HP-20), purifying, performing gradient elution by using methanol/water (v/v, 0:10, 1:9, 2:8, 10:0) as eluent, wherein 1L (equivalent to 2 column volumes) of each eluent is used for collecting separation components of methanol/water (v/v, 2:8), collecting eluent (methanol/water is 2:8), concentrating the eluent under reduced pressure at normal temperature, and drying to obtain refined component 3.1g of the chestnut shell extract, namely component HIII.
Test example 1
The extracts obtained in the above examples were tested for their ability to inhibit PTP1B activity.
1. Experimental methods.
The protein tyrosine phosphatase PTP1B used for the screening was a GST fusion protein (source: U.S. Pat. No. A0230) expressed and purified from E.coli. Ultraviolet substrate pNPP is adopted to observe the activity inhibition of the recombinant enzyme at different concentrations so as to preliminarily evaluate the medicinal effect of the compound.
The product obtained as a result of hydrolysis of the phospholipid of the substrate pNPP by PTP1B has a strong light absorption at 410 nm. Therefore, the change of the light absorption at 410nm can be directly detected to observe the change of the activity of the enzyme and the inhibition of the enzyme by the compound. And sodium orthovanadate as positive control, IC thereof50It was 2.0. mu. mol/L. The results of the experiments are shown in the following table:
TABLE 1 IC of different extracts on PTP1B inhibitory Activity50(n＝10)
The experimental results show that: the ethanol extracts and active components with different concentrations have different effects on inhibiting the activity of PTP1B, wherein the component II and the component III (75% ethanol extract and 75% ethanol extract refined component) have the best effect on inhibiting the activity of PTP1B, and IC thereof5015.5 +/-1.5 mu g/ml and 11.3 +/-1.4 mu g/ml respectively.
Whereas fraction A, B, C was almost inactive compared to fraction III, indicating that the active ingredient having an inhibitory effect on PTP1B was present predominantly in the methanol/water (v/v, 2:8) eluate in the 75% ethanol extract.
Test example 2
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) are randomly divided into a blank group, a model group (Diabetic control), an experimental group (component I, component HI, component II, component HII, component III, component HIII) and a thirst-quenching pill 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 was intragastrically (200mg/kg) with component I, component HI, component II, component III, and component HIII, 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. After the last administration, blood was taken with broken tail (fasting for 6h before measurement). The experimental results are as follows:
TABLE 2 hypoglycemic conditions of STZ hyperglycemic rats with different extracts
The experimental results show that: the blood sugar of rats in the experimental group is reduced to different degrees, wherein the blood sugar reducing effect of the experimental group which is perfused with the stomach by the component II and the component III (75% ethanol extract and 75% ethanol extract refined component) is most obvious compared with the model group. Experimental data show that the hypoglycemic effect of the component IIis equivalent to that of the diabetes pill.
Test example 3
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 I, component HI, component II, component HII, component III, and component HIII (200mg/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 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 results of the experiment are shown in FIG. 1.
From the experimental results of fig. 1, it was shown that the ethanol extracts and the active ingredients at different concentrations have improved insulin sensitivity in rats with hyperglycemia caused by STZ, but the effects of the respective ingredients are different. Wherein, compared with the model group, the component II and the component III (75% ethanol extract and 75% ethanol extract refined component) have better insulin sensitivity enhancing effect, and the component III has the most obvious effect. Experimental data show that the hypoglycemic effect of the component IIis equivalent to that of the XZK group.
Test example 4
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 I, component HI, component II, component HII, component III, and component HIII (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 results of the experiment are shown in FIG. 2.
The experimental results in fig. 1 show that each group of drugs tends to improve the glucose tolerance of STZ-induced hyperglycemic rats, but the effects of each component are different. Wherein, the component II and the component III (75 percent ethanol extract and 75 percent ethanol extract refined component) have better effect, and the component III has the best effect. Experimental data show that the hypoglycemic effect of the component IIis superior to that of the XZK group.
Test example 5
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 I, component HI, component II, component HII, component III, and component HIII (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 3 different extracts on weight change in STZ hyperglycemic rats (n ═ 10)
The experimental results show that: the ethanol extracts with different concentrations and the active components have different effects on reducing the weight of rats with hyperglycemia caused by STZ. Wherein the fraction II and fraction III (75% ethanol extract and 75% ethanol extract refined fraction) have the best weight reduction effect compared with the model group. The experimental data show that the hypoglycemic effect of the component III is equivalent to that of the XKW groups.
According to the experimental results of the test examples, the analysis shows that the reduction of insulin sensitivity and the damage of the cell function of the islet β are main factors of the pathogenesis of type 2 diabetes, and the high expression of PTP1B can cause the reduction of the insulin sensitivity and the leptin resistance of the body, thereby causing type 2 diabetes and obesity.
The test data show that the chestnut shell extract of the invention can obviously inhibit PTP1B activity (test example 1). And through an STZ hyperglycemic rat blood sugar reduction experiment (test example 2), the blood sugar reduction effect of the extract on diabetes model animals is fully determined, the characteristics of relieving the clinical polydipsia and polyuria symptoms of diabetes of all groups of medicines are also prompted, and further, the study on the insulin sensitivity of STZ hyperglycemic rats shows that the extract can obviously improve the sensitivity of organisms to insulin and improve the bioavailability of insulin (test example 3). The glucose tolerance test of rats with hyperglycemia caused by STZ (test example 4) shows that the chestnut shell extract can obviously improve the glucose tolerance of organisms. The rat body weight change test data show that the extract has obvious effect on inhibiting body weight gain (test example 5).
The combination of the related test data shows that the 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 (4)
1. A preparation method of chestnut shell extract for inhibiting protein tyrosine phosphatase 1B is characterized by comprising the following steps:
extraction: using chestnut shells as raw materials, using ethanol water solution with the volume percentage concentration of 70-80% as a solvent, carrying out solvent extraction, and recovering the solvent to obtain a chestnut shell extract; the ratio of the mass of the raw materials to the volume of the solvent is 1g:5-10ml, and ultrasonic extraction is carried out for 2-4 times, each time for 30-60 min;
and (3) purification: loading the chestnut shell extract obtained in the extraction step to HP-20 type macroporous adsorption resin for purification, and sequentially eluting by using methanol-water solution with the volume ratio of 0:10 for 1-3 column volumes; the volume ratio of the components is 0.5-2:9 methanol-water solution elutes 1-3 column volumes; eluting with methanol-water solution at a volume ratio of 1-3:8 for 1-3 column volumes, and recovering solvent.
2. Chestnut shell extract prepared by the method for preparing chestnut shell extract for inhibiting protein tyrosine phosphatase 1B according to claim 1.
3. Use of chestnut shell extract according to claim 2 for the preparation of inhibitors of protein tyrosine phosphatase 1B and/or for the preparation of insulin sensitizers.
4. Use according to claim 3, characterized in that: the inhibitor of protein tyrosine phosphatase 1B and/or the insulin sensitizer are applied to the preparation of medicines for preventing and treating diabetes, obesity and complications thereof.
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| 板栗壳斗总酚对高脂糖尿病大鼠血糖和血脂水平的影响;陈思玉等;《心理医生杂志》;20120430(第214期);第456-457页 * |
| 板栗对糖尿病小鼠降血糖作用的研究;刘海鑫等;《数理医药学杂志》;20121231;第25卷(第1期);第57-61页 * |
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