阅读说明：本技术 一种木麻黄鞣宁的制备方法及其降糖用途 (Preparation method and blood sugar reducing application of casuarina equisetifolia tannate ) 是由 陈卫 徐阳 艾哈迈德·K·拉什旺 于 2021-07-07 设计创作，主要内容包括：本发明公开了一种木麻黄鞣宁的制备方法及其降糖用途,该制备方法包括醇提浓缩、乙酸乙酯萃取、高压制备液相色谱纯化及高速逆流色谱分离,通过将高压制备液相色谱与高速逆流色谱结合,再通过对工艺参数的优化,从物质组成复杂的地稔原料中分离制备得到高纯度的木麻黄鞣宁单体；经活性试验发现,该木麻黄鞣宁单体能够显著抑制α-葡萄糖苷酶的活性,可用于制备α-葡萄糖苷酶抑制剂,控制餐后血糖水平。(The invention discloses a preparation method of casuarina equisetifolia tannin and a hypoglycemic application thereof, wherein the preparation method comprises alcohol extraction concentration, ethyl acetate extraction, high-pressure preparation liquid chromatography purification and high-speed countercurrent chromatography separation, and a high-purity casuarina equisetifolia tannin monomer is obtained by separating and preparing a herba melastomae dodecandri raw material with complex composition of substances by combining the high-pressure preparation liquid chromatography with the high-speed countercurrent chromatography and optimizing process parameters; activity tests show that the casuarina tannine monomer can obviously inhibit the activity of alpha-glucosidase, and can be used for preparing an alpha-glucosidase inhibitor and controlling the postprandial blood sugar level.)

1. The preparation method of the casuarina tannine is characterized by comprising the following steps:

(1) crude extraction: using melastoma dodecandrum lour fruit as a raw material, and performing alcohol extraction concentration, ethyl acetate extraction and nitrogen blowing to obtain melastoma dodecandrum lour ethyl acetate extract powder;

(2) high pressure preparative liquid chromatography separation: dissolving the melastoma dodecandrum lour ethyl acetate extract powder, injecting into a high-pressure preparative liquid chromatography, performing gradient elution by using a C18 chromatographic column through a mobile phase, and performing aftertreatment to obtain the ephedrine hydrochloride tannin extract freeze-dried powder;

the mobile phase is as follows: the phase A is pure acetonitrile, and the phase B is a formic acid-water system with the volume percentage concentration of formic acid of 1.5-5%;

the procedure for the gradient elution was: keeping the volume percentage concentration of the phase A unchanged within 0-5 min, increasing the volume percentage concentration of the phase A from 5% to 60% within 5-30 min, and collecting the eluate for 24-26 min;

(3) high-speed countercurrent chromatographic separation: dissolving the casuarina equisetifolia tannin extract freeze-dried powder with a mobile phase by using ethyl acetate-water-trifluoroacetic acid as a two-phase solvent system, injecting the dissolved casuarina equisetifolia tannin extract freeze-dried powder into a high-speed counter-current chromatography for separation, and performing separation and freeze-drying to obtain casuarina equisetifolia tannin powder;

the volume ratio of the ethyl acetate to the water to the trifluoroacetic acid is 1: 1: 0.01 to 0.1.

2. The method for preparing casuarinins according to claim 1, wherein in step (1), the alcohol extraction is concentrated, specifically:

mixing the cleaned melastoma dodecandrum fruit with an acidic ethanol solution, filtering after complete ultrasonic extraction, collecting filtrate, carrying out vacuum rotary evaporation on the filtrate at 40-50 ℃ to remove ethanol, and concentrating to obtain a crude extract of the melastoma dodecandrum extract;

the acidic ethanol solution is an ethanol solution with the acid volume percentage concentration of 0.1-1.5%;

the acid is at least one of hydrochloric acid, formic acid, acetic acid and citric acid;

the volume percentage concentration of the ethanol solution is 50-95%;

the mass volume ratio of the melastoma dodecandrum to the acidic ethanol solution is 1: 5-12 g/mL.

3. The method for preparing casannin according to claim 1, wherein in the step (1), ethyl acetate is extracted for several times by using ethyl acetate as an extracting agent, an ethyl acetate phase is collected, vacuum rotary evaporation is carried out at 40-50 ℃, and nitrogen is blown to obtain melastoma dodecandrum ethyl acetate extract powder.

4. The method for preparing casannins according to claim 1, wherein in step (2), the high pressure preparative liquid chromatography is specifically:

redissolving the melastoma dodecandrum ethyl acetate extract powder by DMSO, and then injecting into a high-pressure preparative liquid chromatograph for purification;

the concentration after re-dissolution is 10-60 mg/mL, and the sample injection amount is 1-3 mL;

the specification of the C18 chromatographic column is 20mm multiplied by 250mm, and the temperature is 25-30 ℃.

5. The method for preparing casannins according to claim 1, wherein in step (3), the high-speed countercurrent chromatography is specifically performed by:

preparing the two-phase solvent system, wherein an upper phase is a stationary phase, a lower phase is a mobile phase, pumping the stationary phase into a high-speed counter-current chromatograph at a flow rate of 20-30 mL/min, pumping the mobile phase at a flow rate of 1-8 mL/min under the conditions of 25-35 ℃ and a main engine rotation speed of 700-1000 r/min, dissolving the casuarina equisetifolia tannin extract freeze-dried powder with the mobile phase after the two phases are balanced, injecting a sample, collecting an effluent liquid containing a target product after liquid phase detection, and concentrating under reduced pressure to obtain a casuarina tannin concentrated solution.

6. The method of preparing casannins according to claim 5, wherein the volume ratio of ethyl acetate, water and trifluoroacetic acid in the biphasic solvent system is 1: 1: 0.01;

pumping the stationary phase into a high-speed counter-current chromatograph at a flow rate of 20-30 mL/min, and pumping the mobile phase at a flow rate of 3-4 mL/min under the conditions of 25-35 ℃ and a main engine rotation speed of 850-950 r/min;

the concentration of the casuarina equisetifolia tannine extract freeze-dried powder after being dissolved by the mobile phase is 10-30 mg/mL, and the sample injection volume is 1-15 mL.

7. Use of casuarinins in the preparation of alpha-glucosidase inhibitors, characterized in that it is prepared according to the method of any one of claims 1 to 6.

Technical Field

The invention relates to the field of separation and purification of natural products and application of reducing blood sugar, in particular to a preparation method of casuarina equisetifolia tannine and application of casuarina equisetifolia tannine tannase in preparation of an alpha-glucosidase inhibitor.

Background

Diabetes is one of chronic diseases which are common worldwide, and diabetics are usually accompanied by hyperglycemia symptoms, and long-term hyperglycemia can cause damage to tissues and organs such as nerves, hearts, blood vessels, kidneys and the like, and cause various acute and chronic complications. According to the statistics of the World Health Organization (WHO), 4.22 billion diabetic patients are in the world in 2014, wherein the type 2 diabetes is the most common. In recent years, with the change of dietary habits and life styles of people and the acceleration of aging process, the prevalence rate of diabetes in China is rapidly increased, and the number of diabetes patients in China reaches 1.1 hundred million by the end of 2016. Therefore, the research on the prevention and treatment of diabetes is of great significance.

Alpha-glucosidase (EC 3.2.1.20), also known as alpha-D-glucosidase hydrolase, is a membrane-bound enzyme in the glycoside hydrolase GH31 family, including sucrase, maltase, isomaltase, etc., and is mainly present in small intestine villus mucosa brush border cells. After eating, alpha-glucosidase can hydrolyze carbohydrates in food into glucose, and the glucose is absorbed and enters blood circulation to cause blood sugar to rise, so that the alpha-glucosidase is one of the main target enzymes for controlling postprandial blood sugar. At present, alpha-glucosidase inhibitors on the market in China mainly comprise acarbose, voglibose and miglitol, and adverse reactions of gastrointestinal tracts, such as abdominal distension, gas exhaustion and the like, are usually caused by using the inhibitors. Therefore, the development of safe and effective food functional factors and the targeted inhibition of the activity of alpha-glucosidase are effective strategies for reducing the incidence rate of diabetes and improving the health condition of diabetic patients. At present, a great deal of researchers at home and abroad have focused on food-derived functional factors and try to screen out alpha-glucosidase inhibitors which have excellent activity and do not cause side effects on human bodies from foods. At present, flavonoids, alkaloids, phenolic compounds, curcumin compounds and terpenoids from food sources have been reported to have strong alpha-glucosidase inhibitory activity in vitro, and the activity of the flavonoids, the alkaloids, the phenolic compounds, the curcumin compounds and the terpenoids is superior to that of a positive drug acarbose, but the alpha-glucosidase inhibitory activity of a melastoma dodecandrum extract is rarely reported.

Melastoma dodecandrum Lour (Melastoma dodecandrum Lour.) is a Melastoma dodecandrum Lour, fructus vitis viniferae, fructus punica granati, herba euphorbiae humifusae, solani dioica and the like, is a common herb used by she nationalities and is recorded in 2015 edition "Zhejiang province Chinese medicine processing standard". Melastoma dodecandrum lour is one of the main medicinal materials of 3 Chinese patent medicine prescriptions, namely Zi Di Ning Xue san, Gong Yan Ping dripping pill and Gong Yan Ping pian which are collected from Chinese pharmacopoeia of 2015 edition. Melastoma dodecandrum is produced in southern China, is mainly distributed under hillside grasses and forests with elevation below 1300m in provinces such as Jiangxi, Fujian and Zhejiang in the south of the Yangtze river, is favored by acid soil, has strong vitality, and has the characteristics of cold resistance, drought resistance, rapid growth and the like. Melastoma dodecandrum lour is a plant with ornamental, medicinal and edible functions. The whole herbal medicine is sweet, astringent and cool in taste, has the effects of clearing heat and removing toxicity, promoting blood circulation and stopping bleeding and the like, and is clinically applied to epidemic cerebrospinal meningitis, hemorrhoids, pelvic inflammation, metrorrhagia and other diseases. The patent inventors find for the first time that the melastoma dodecandrum extract has significant alpha-glucosidase inhibitory activity through a large amount of studies, and further screen a natural product casuarinin having significant alpha-glucosidase inhibitory activity from the melastoma dodecandrum extract through an activity-mediated separation and purification strategy. At present, the preparation method of casuarina equisetifolia tannine and the alpha-glucosidase inhibitory activity and the hypoglycemic application thereof are not reported.

Disclosure of Invention

The invention discloses a preparation method of casuarina equisetifolia tannine and a hypoglycemic application thereof.

The specific technical scheme is as follows:

a preparation method of casuarina tannine comprises the following steps:

(1) crude extraction: taking fresh melastoma dodecandrum lour fruits as raw materials, and obtaining melastoma dodecandrum lour ethyl acetate extract powder after ethanol extraction concentration and ethyl acetate extraction;

(2) high pressure preparative liquid chromatography separation: dissolving the melastoma dodecandrum lour ethyl acetate extract powder, injecting into a high-pressure preparative liquid chromatography, performing gradient elution by using a C18 chromatographic column through a mobile phase, and performing aftertreatment to obtain the ephedrine hydrochloride tannin extract freeze-dried powder;

the mobile phase is as follows: the phase A is pure acetonitrile, and the phase B is a formic acid-water system with the volume percentage concentration of formic acid of 1.5-5%;

the procedure for the gradient elution was: keeping the volume percentage concentration of the phase A unchanged within 0-5 min, increasing the volume percentage concentration of the phase A from 5% to 60% within 5-30 min, and collecting the eluate for 24-26 min;

(3) high-speed countercurrent chromatographic separation: dissolving the casuarina equisetifolia tannin extract freeze-dried powder with a mobile phase by using ethyl acetate-water-trifluoroacetic acid as a two-phase solvent system, injecting the dissolved casuarina equisetifolia tannin extract freeze-dried powder into a high-speed counter-current chromatography for separation, and performing separation and freeze-drying to obtain casuarina equisetifolia tannin powder;

the volume ratio of the ethyl acetate to the water to the trifluoroacetic acid is 1: 1: 0.01 to 0.1;

all percentages of starting materials present in the present invention are by volume unless otherwise specified.

In the step (1), the alcohol extraction and concentration specifically comprises the following steps:

mixing the cleaned melastoma dodecandrum fruit with an acidic ethanol solution, filtering after complete ultrasonic extraction, collecting filtrate, carrying out vacuum rotary evaporation on the filtrate at 40-50 ℃ to remove ethanol, and concentrating to obtain melastoma dodecandrum coarse extract;

the acidic ethanol solution is an ethanol solution with the acid volume percentage concentration of 0.1-1.5%;

the acid is at least one of hydrochloric acid, formic acid, acetic acid and citric acid;

the volume percentage concentration of the ethanol solution is 50-95%;

the mass volume ratio (namely the material-liquid ratio) of the melastoma dodecandrum to the acidic ethanol solution is 1: 5-12 g/mL.

Preferably, the ultrasonic extraction time is 60-240 min, and the process is carried out at 25-49 ℃ under the condition of keeping out of the sun.

In order to ensure complete extraction of melastoma dodecandrum lour, the filter residue obtained after primary extraction is repeatedly extracted for a plurality of times according to the same conditions.

Preferably, the volume percentage concentration of the ethanol solution is 60-70%, and the mass volume ratio of the melastoma dodecandrum to the acidic ethanol is 1: 8 g/mL.

In the step (1), the organic solvent is used for extraction, ethyl acetate is used as an extractant, extraction is carried out for 3-5 times, an ethyl acetate phase is collected, vacuum rotary evaporation is carried out at 40-50 ℃, and nitrogen is blown to obtain melastoma dodecandrum ethyl acetate extract powder.

In the step (2), the high-pressure preparative liquid chromatography separation specifically comprises:

redissolving the melastoma dodecandrum ethyl acetate extract powder by DMSO, and then injecting into a high-pressure preparative liquid chromatograph for purification;

the concentration after re-dissolution is 10-60 mg/mL, and the sample injection amount is 1-3 mL;

the specification of the C18 chromatographic column is 20mm multiplied by 250mm, and the temperature is 25-30 ℃;

in the step (3), the high-speed counter-current chromatography separation specifically comprises:

preparing the two-phase solvent system, wherein an upper phase is a stationary phase, a lower phase is a mobile phase, pumping the stationary phase into a high-speed counter-current chromatograph at a flow rate of 20-30 mL/min, pumping the mobile phase at a flow rate of 1-8 mL/min under the conditions of 25-35 ℃ and a main engine rotation speed of 700-1000 r/min, dissolving the casuarina equisetifolia tannin extract freeze-dried powder with the mobile phase after the two phases are balanced, injecting a sample, collecting an effluent liquid containing a target product after liquid phase detection, and concentrating under reduced pressure to obtain a casuarina tannin concentrated solution.

Preferably, in the biphasic solvent system, the volume ratio of ethyl acetate, water and trifluoroacetic acid is 1: 1: 0.01;

pumping the stationary phase into a high-speed counter-current chromatograph at a flow rate of 20-30 mL/min, and pumping the mobile phase at a flow rate of 3-4 mL/min under the conditions of 25-35 ℃ and a main engine rotation speed of 850-950 r/min;

the concentration of the casuarina equisetifolia tannine extract freeze-dried powder after being dissolved by the mobile phase is 10-30 mg/mL, and the sample injection volume is 1-15 mL.

After the preparation process is optimized, the purity of the casuarina equisetifolia tannine monomer obtained by separation and purification reaches 98 percent.

Further activity tests show that the casuarina tannine monomer obtained by separation and purification has obvious inhibition effect on alpha-glucosidase and IC₅₀The value is 0.22 mu M, which is obviously superior to that of a positive medicineSubstance acarbose (IC)₅₀0.52mM), can be used as a novel alpha-glucosidase inhibitor of natural sources for controlling postprandial blood sugar.

Compared with the prior art, the invention has the following advantages:

the invention combines the high-speed counter-current chromatography and the high-pressure preparative liquid chromatography for the first time, and then separates and prepares the high-purity casuarina equisetifolia tannine monomer from the melastoma dodecandrum fruit by optimizing the process parameters, wherein the purity of the casuarina equisetifolia tannine monomer can reach 98 percent at most; the separation method has the advantages of large sample processing capacity, good repeatability and the like, and can be used for preparing high-purity casuarina equisetifolia tannine in a large scale, so that the industrial production can be realized; further activity tests show that the casuarina equisetifolia tannine can obviously inhibit the activity of alpha-glucosidase, can be used for reducing postprandial blood sugar, and no research report exists at present on a preparation method of the casuarina equisetifolia tannine and the alpha-glucosidase inhibition activity and glucose reduction application of the casuarina equisetifolia tannine.

Drawings

FIG. 1 is a liquid chromatogram of the ethyl acetate fraction of Melastoma dodecandrum in example 1;

FIG. 2 is a liquid chromatogram of casuarina tannine purified by preparative liquid chromatography in example 1;

FIG. 3 is a high performance liquid chromatogram of casuarina tannine purified by high-speed countercurrent chromatography in example 1;

FIG. 4 is a mass spectrum of casuarinin and its fragmentation pattern in example 1;

FIG. 5 is a high performance liquid chromatogram of the final product casuarinin in comparative example 1;

FIG. 6 is a graph showing the α -glucosidase inhibitory activity of the ethyl melastoma acetate extract prepared in example 1;

FIG. 7 is a graph showing the α -glucosidase inhibitory activity of casuarinin prepared in example 1.

Detailed Description

The invention will be further described with reference to specific examples, which are given below only by way of illustration, without limiting the scope of the invention:

example 1

Cleaning 1kg of melastoma dodecandrum, mixing the raw materials according to a material-liquid ratio of 1: 8(w/v, g/mL) is added with 70 percent (the volume ratio of ethanol to water is 70: 30) ethanol water solution containing 0.1 percent (v/v) hydrochloric acid (the volume ratio of ethanol to water is 70: 30), and the mixture is ultrasonically extracted for 60min at 40 ℃ in the dark, then the vacuum filtration is carried out, the extraction of filter residues is repeated once, the filtrates are combined, and the ethanol is removed by vacuum rotary evaporation at 45 ℃ to obtain the crude extract of the melastoma dodecandrum extract.

Adding the herba Melastomatis Dodecandri extract crude extract into ethyl acetate with the same volume, extracting for 4 times, collecting ethyl acetate phase, vacuum rotary evaporating at 45 deg.C for concentrating, and blowing nitrogen to obtain herba Melastomatis Dodecandri ethyl acetate extract powder with liquid chromatogram shown in FIG. 1.

The powder of the ethyl acetate extract of Melastoma dodecandrum is dissolved in DMSO to a concentration of 20mg/mL using a preparative liquid phase column, C1820 mm X250 mm, and injected into preparative liquid chromatography in a sample volume of 4mL, mobile phase: phase A: pure acetonitrile, phase B: formic acid (5%): water (95%); the column temperature was 30 ℃, the flow rate was 10mL/min, and the gradient was: 5% of phase A for 0-5 min, and 5% -60% of phase A for 5-30 min. The Melastoma dodecandrum lour ethyl acetate extract is injected into the preparation liquid phase for separation, and the single sample injection amount is 4 mL. Detecting with ultraviolet detector, collecting peak of 24-26 min, concentrating under reduced pressure, and freeze drying to obtain lyophilized powder of MAHUANGMA TANNING extract, wherein the liquid chromatogram of the lyophilized powder is shown in FIG. 2.

Ethyl acetate, water and trifluoroacetic acid in a volume ratio of 1: 1: placing in a separating funnel at a ratio of 0.01, shaking thoroughly, standing for 30min, separating the upper and lower phases, and ultrasonic degassing for 30 min. The upper phase was used as stationary phase and the lower phase as mobile phase. Starting a high-speed counter-current chromatograph, preheating for 30min, setting a circulating water bath at 25 ℃, pumping the stationary phase into the instrument at a flow rate of 30mL/min, positively rotating, and starting the instrument to enable the rotating speed of a host to reach 900 r/min. Pumping the mobile phase at a flow rate of 3mL/min after the rotation speed is stable, dissolving 200mg of casuarina equisetifolia tannin extract freeze-dried powder in 15mL of the mobile phase after the two phases are balanced in a pipeline, injecting a sample and detecting under an ultraviolet detector, collecting target peak components, concentrating under reduced pressure, and freeze-drying to obtain high-purity casuarina equisetifolia tannin, wherein a liquid chromatogram of the casuarina equisetifolia tannin is shown in figure 3.

As can be seen from comparing the high performance liquid chromatograms in fig. 1-3, after the melastoma dodecandrum is subjected to extraction and concentration, ethyl acetate extraction and high pressure preparative liquid chromatography purification, the obtained casuarina equisetifolia tannate freeze-dried powder mainly contains casuarina equisetifolia tannate and a small amount of other impurities, and is further subjected to high speed counter current chromatography purification, a casuarina equisetifolia tannate monomer can be obtained, and the purity is 99.07%.

Example 2

Cleaning 5kg of melastoma dodecandrum, mixing the raw materials according to a material-liquid ratio of 1: 8(w/v, g/mL) is added with 70 percent (the volume ratio of ethanol to water is 70: 30) ethanol water solution containing 0.1 percent (v/v) hydrochloric acid (the volume ratio of ethanol to water is 70: 30), and the mixture is ultrasonically extracted for 60min at 40 ℃ in the dark, then the vacuum filtration is carried out, the extraction of filter residues is repeated once, the filtrates are combined, and the ethanol is removed by vacuum rotary evaporation at 45 ℃ to obtain the crude extract of the melastoma dodecandrum extract.

Adding the herba Melastomatis Dodecandri extract crude extract into ethyl acetate with the same volume, extracting for 4 times, collecting ethyl acetate phase, vacuum rotary evaporating at 45 deg.C for concentration, and blowing with nitrogen to obtain herba Melastomatis Dodecandri ethyl acetate extract powder.

The powder of the ethyl acetate extract of Melastoma dodecandrum is dissolved in DMSO to a concentration of 40mg/mL using a preparative liquid phase column, C1820 mm X250 mm, injected into preparative liquid chromatography in a sample volume of 4mL, mobile phase: phase A: pure acetonitrile, phase B: formic acid (5%): water (95%); the column temperature was 30 ℃, the flow rate was 10mL/min, and the gradient was: 5% of phase A for 0-5 min, and 5% -60% of phase A for 5-30 min. The Melastoma dodecandrum lour ethyl acetate extract is injected into the preparation liquid phase for separation, and the single sample injection amount is 4 mL. Detecting under an ultraviolet detector, collecting peaks of 24-26 min, concentrating under reduced pressure, and freeze-drying to obtain the casuarina equisetifolia tannin extract freeze-dried powder.

Ethyl acetate, water and trifluoroacetic acid in a volume ratio of 1: 1: placing in a separating funnel at a ratio of 0.01, shaking thoroughly, standing for 30min, separating the upper and lower phases, and ultrasonic degassing for 30 min. The upper phase was used as stationary phase and the lower phase as mobile phase. Starting a high-speed counter-current chromatograph, preheating for 30min, setting a circulating water bath at 25 ℃, pumping the stationary phase into the instrument at a flow rate of 30mL/min, positively rotating, and starting the instrument to enable the rotating speed of a host to reach 900 r/min. Pumping the mobile phase at a flow rate of 3mL/min after the rotation speed is stable, dissolving 300mg of casuarina equisetifolia tannin extract freeze-dried powder in the 15mL of mobile phase after the two phases are balanced in a pipeline, injecting a sample and detecting under an ultraviolet detector, collecting target peak components, concentrating under reduced pressure, and freeze-drying to obtain the casuarina equisetifolia tannin with high purity of 98.64%.

Example 3

Washing 10kg of melastoma dodecandrum, and mixing the raw materials according to a material-liquid ratio of 1: 8(w/v, g/mL) is added with 70 percent (the volume ratio of ethanol to water is 70: 30) ethanol water solution containing 0.1 percent (v/v) hydrochloric acid (the volume ratio of ethanol to water is 70: 30), and the mixture is ultrasonically extracted for 60min at 40 ℃ in the dark, then the vacuum filtration is carried out, the extraction of filter residues is repeated once, the filtrates are combined, and the ethanol is removed by vacuum rotary evaporation at 45 ℃ to obtain the crude extract of the melastoma dodecandrum extract.

Adding the herba Melastomatis Dodecandri extract crude extract into ethyl acetate with the same volume, extracting for 4 times, collecting ethyl acetate phase, vacuum rotary evaporating at 45 deg.C for concentration, and blowing with nitrogen to obtain herba Melastomatis Dodecandri ethyl acetate extract powder.

The powder of the ethyl acetate extract of Melastoma dodecandrum is dissolved in DMSO to a concentration of 60mg/mL using a preparative liquid phase column, C1820 mm X250 mm, injected into preparative liquid chromatography in a sample volume of 4mL, mobile phase: phase A: pure acetonitrile, phase B: formic acid (5%): water (95%); the column temperature was 30 ℃, the flow rate was 10mL/min, and the gradient was: 5% of phase A for 0-5 min, and 5% -60% of phase A for 5-30 min. The Melastoma dodecandrum lour ethyl acetate extract is injected into the preparation liquid phase for separation, and the single sample injection amount is 4 mL. Detecting under an ultraviolet detector, collecting peaks of 24-26 min, concentrating under reduced pressure, and freeze-drying to obtain the casuarina equisetifolia tannin extract freeze-dried powder.

Ethyl acetate, water and trifluoroacetic acid in a volume ratio of 1: 1: placing in a separating funnel at a ratio of 0.01, shaking thoroughly, standing for 30min, separating the upper and lower phases, and ultrasonic degassing for 30 min. The upper phase was used as stationary phase and the lower phase as mobile phase. Starting a high-speed counter-current chromatograph, preheating for 30min, setting a circulating water bath at 25 ℃, pumping the stationary phase into the instrument at a flow rate of 30mL/min, positively rotating, and starting the instrument to enable the rotating speed of a host to reach 900 r/min. Pumping the mobile phase at a flow rate of 3mL/min after the rotation speed is stable, dissolving 300mg of casuarina equisetifolia tannin extract freeze-dried powder in the 15mL of mobile phase after the two phases are balanced in a pipeline, injecting a sample and detecting under an ultraviolet detector, collecting target peak components, concentrating under reduced pressure, and freeze-drying to obtain the casuarina equisetifolia tannin with high purity of 98.12%.

Comparative example 1

The preparation process is the same as in example 1, except that the solvent system of the high-speed countercurrent chromatography separation is replaced by: n-butanol: methyl tert-butyl ether: methanol: water: trifluoroacetic acid was prepared as follows: 3: 1: 5: mixing at a volume ratio of 0.01. FIG. 5 is a high performance liquid chromatogram of the final product casuarinin in comparative example 1; tests show that the purity of the obtained casuarina tannine monomer is only 76.02 percent and is far lower than 98 percent.

Comparative example 2

In comparison with example 1, the purification step of preparative liquid chromatography was omitted, and the other steps were not changed, and it was tested that the monomer of casuarina tannine with high purity could not be obtained.

Comparative example 3

In comparison with example 1, the collection time in the preparative liquid chromatography purification step was adjusted to 22-24min or 26-28min, and other steps were unchanged, and high purity casuarinin monomer could not be obtained by the test.

Application example 1

Casuarinins prepared in example 1 were dissolved in DMSO to prepare a series of concentrations (2. mu.g/mL, 3. mu.g/mL, 4. mu.g/mL, 5. mu.g/mL, 8. mu.g/mL, 10. mu.g/mL) as inhibitors. Alpha-glucosidase was diluted with 0.1mol/L phosphate buffer (PBS, pH 6.9) to an enzyme activity of 0.5U/mL. Substrate p-nitrophenyl- α -D-glucopyranoside (PNPG) was formulated with 0.1mol/L phosphate buffer (PBS, pH 6.9) to a concentration of 1mmol/L. enzyme reaction system 20 μ L was mixed with 10 μ L inhibitor, 130 μ L buffer and 40 μ L substrate were added, and reacted at 37 ℃ for 30min, followed by addition of 200 μ L of 1mol/L sodium carbonate solution, and absorbance was measured at 405 nm. The blank group replaced the inhibitor with buffer. The enzyme activity inhibition rate is according to the following formulaAnd (3) calculating: inhibition ratio%_{Blank space}-A_{Experiment of})/A_{Blank space}100%. IC of casuarina tannine measured under the reaction system₅₀The value was 0.22. mu. mol/L and the positive control acarbose was 0.52 mmol/L. By comparison, the casuarina tannine prepared by separation has the effect of inhibiting alpha-glucosidase (IC)₅₀0.22 μmol/L) significantly better than the positive control acarbose.

FIG. 7 shows the α -glucosidase inhibitory activity curve of casuarinin prepared in example 1, and the α -glucosidase inhibitory activity curve of casuarina extract prepared in example 1 was further tested for α -glucosidase inhibitory activity, as shown in FIG. 6. As can be seen from the comparison between FIG. 6 and FIG. 7, the alpha-glucosidase inhibitory activity of the casuarina equisetifolia tannine extracted by the method of the invention is significantly higher than that of the melastoma dodecandrum lour ethyl acetate extract. Therefore, the casuarina tannine separated and prepared from the melastoma dodecandrum can be used as a novel alpha-glucosidase inhibitor from natural sources.