CN114099574A

CN114099574A - Method for extracting coptis chinensis extract for reducing blood sugar and extract thereof

Info

Publication number: CN114099574A
Application number: CN202010900167.9A
Authority: CN
Inventors: 马云桐; 齐路明; 钟芙蓉; 严铸云
Original assignee: Chengdu University of Traditional Chinese Medicine
Current assignee: Chengdu University of Traditional Chinese Medicine
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2022-03-01
Anticipated expiration: 2040-08-31
Also published as: CN114099574B

Abstract

The invention belongs to the technical field of extraction of active ingredients of medicines, and particularly relates to a method for extracting active ingredients of a coptis chinensis medicinal material for reducing blood sugar and application of the extract. Aiming at the problem that the prior art lacks of optimization research of an extraction method aiming at effective components in specific Coptis plants with higher alkaloid content, the technical scheme of the invention is as follows: [1] taking dry powder of the coptis deltoidea medicinal material; [2] sequentially extracting the dried powder in the step [1] by adopting a plurality of solvents with different polarities, wherein the solvent comprises n-butyl alcohol; [3] and (3) collecting the extracting solution obtained by the n-butanol extraction in the step (2), and concentrating to obtain an extract, namely the extract. The method is used for extracting the active ingredients with the hypoglycemic effect in the coptis deltoidea, and the product can be further used for preparing hypoglycemic combination medicines.

Description

Method for extracting coptis chinensis extract for reducing blood sugar and extract thereof

Technical Field

The invention belongs to the technical field of extraction of active ingredients of medicines, and particularly relates to a method for extracting active ingredients of a coptis medicinal material for reducing blood sugar and application of the extract.

Background

Ranunculaceae Coptis plant is perennial herb and is distributed in the northern temperate zone. Ancient literature, famous medical records: the Chinese medicinal composition is mainly used for treating cold and heat in five storeys, chronic diarrhea , purulent blood, thirst quenching, convulsion, water and bone removal, stomach and intestine regulation, gallbladder benefiting and aphtha treatment. The record of "newly revised materia Medica" is: the coptis root, the Shudao person is thick and thick, the taste is extremely strong and bitter, and the thirst is treated most; jiangtong is like Lianzhu, and has good effect on treating dysentery. It is seen that ancient physicians have recognized that coptis has a better "thirst-quenching" effect in long-term clinical practice. The research of plant chemistry and pharmacology shows that the alkaloid component in the coptis chinensis medicinal material is the main active substance for playing the role of reducing the blood sugar. In the prior art, few researches are made on the content difference of alkaloid components in coptis chinensis medicinal materials based on different basic sources. And research for optimizing the extraction method of effective components in specific coptis plants with high alkaloid content is lacked.

Wherein the coptis deltoidea is mainly distributed in the eyebrow and Hongya zone in Sichuan China. The wild plants are frequently cultivated in mountain forests with the altitude of 1600-. It has been reported in the prior art that the coptis deltoidea contains alkaloids such as berberine, coptisine, methyl coptisine, tetrandrine and the like, and can treat symptoms such as acute conjunctivitis, acute bacillary dysentery, acute gastroenteritis, hematemesis, carbuncle, furuncle, sore and ulcer and the like. However, reports related to the hypoglycemic effect of the coptis deltoidea are lacked at present.

Disclosure of Invention

Aiming at the problems that the prior art lacks of content difference of alkaloid components in different original coptis medicinal materials and lacks of extraction optimization research of effective components in specific coptis plants with higher alkaloid content. The invention provides an extraction method of coptis chinensis medicinal material active ingredients for reducing blood sugar and application of the extract. The purpose is as follows: the variety of the coptis medicinal material is optimized, the extraction method is optimized, and the hypoglycemic effect of the coptis medicinal material extract is enhanced.

A method for extracting active ingredients of a coptis chinensis medicinal material for reducing blood sugar comprises the following steps:

[1] taking dry powder of the coptis deltoidea medicinal material;

[2] sequentially extracting the dried powder in the step (1) by adopting petroleum ether, ethyl acetate and n-butanol solvent; the three solvents are solvents with different polarities, so that different active ingredients can be extracted by different solvents when the coptis deltoidea is extracted;

[3] and (3) collecting the n-butanol part extracting solution in the step (2), and concentrating to obtain an extract, namely the extract.

Preferably, the Coptis triangularis is dried rhizome of Coptis C.deltoidea belonging to Coptis of Ranunculaceae; preferably, the content of glatiramer in the coptidis rhizoma is higher than that of other coptidis plants.

Preferably, the preparation method of the dried medicinal powder in the step [1] comprises the following steps: the rhizome of the coptis deltoidea is cleaned, dried in the air, dried to constant weight and then ground into powder of 100 meshes.

Preferably, the petroleum ether in step [2] has a boiling range of 60 to 90.

Preferably, the extraction process in step [2] is carried out by using a rapid solvent extractor.

Further preferably, the step [2] specifically comprises the following steps:

[2-1] sequentially pumping a plurality of solvents with different polarities in a rapid solvent extraction instrument to prepare extraction parts with different polarities, wherein the solvents comprise n-butyl alcohol;

[2-2] sequentially arranging a gasket and glass fiber filter paper at the bottom of an extraction tank of a rapid solvent extraction instrument, then placing a sample into the extraction tank, and then filling quartz sand;

and [2-3] starting a solvent extractor for extraction.

Further preferably, in the step [2-2], the mass of the dried powder of the coptis deltoidea medicinal material added into the extraction tank is 0.5-2.0g, the specification of the extraction tank is 40ml, and the amount of the quartz sand added into the extraction tank is 40-60 g.

Further preferably, in the step [2-3], the temperature during the extraction is 90 ℃ and the pressure is 100 bar.

Preferably, in the step [2-3], n-butanol is used as an extraction solvent to circulate for four times, wherein the 1 st circulation process is heating for 1-2min, keeping for 0min and releasing for 5min, and the 2 nd-4 th circulation process is heating for 1-2min, keeping for 2-4min and releasing for 5 min; the extraction solvent except n-butanol is circulated for three times, and each circulation process comprises heating for 1-2min, maintaining for 2-4min and releasing for 5 min.

Further preferably, in the step [2-3], the solvent washing time is 2min and the gas washing time is 3min after the extraction of each extraction solvent is completed.

The invention also provides an extract obtained by the extraction method.

The invention also provides application of the extract of the active ingredients of the coptis chinensis medicinal material for reducing the blood sugar in preparing a blood sugar reducing medicinal composition.

After the technical scheme is adopted, n-butyl alcohol is selected to extract the coptis deltoidea to obtain the extract. According to the results of alpha-glucosidase inhibition experiments, compared with other types of coptis chinensis medicinal materials or extracts obtained by extracting coptis deltoidea with other solvents, the extracts have more excellent alpha-glucosidase inhibition activity, and therefore have better values of being used as hypoglycemic drugs or being used for preparing hypoglycemic combination drugs. According to the results of ultrafiltration rapid sieve experiments and molecular docking simulation, the extract of the n-butanol part of the coptis deltoidea contains alkaloids such as magnoflorine, gladiobin, coptisine, berberine and the like as potential alpha-glucosidase inhibitors. Especially, the content of gladiolide in the coptis deltoidea is obviously higher than that of other three coptis medicinal materials, so that the alpha-glucosidase inhibition effect of the n-butyl alcohol part extract of the coptis deltoidea is further enhanced. Furthermore, the invention also provides an optimization scheme of various process parameters when the active ingredients in the coptis deltoidea are extracted, so that the extraction rate can be further improved, and the content of the active ingredients in the extract can be improved.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows that the petroleum ether and n-butanol fractions of the respective coptis chinensis medicinal materials in the embodiment of the invention inhibit the activity of alpha-glucosidase IC₅₀A value;

FIG. 2 shows the inhibition curves of the petroleum ether and n-butanol fraction extracts of the respective Coptidis rhizoma materials in the examples of the present invention for inhibiting the activity of α -glucosidase;

FIG. 3 shows an ultrafiltration chromatogram of an embodiment of the invention.

Detailed Description

The technical solution of the present application is further described below by specific examples.

Example 1 extraction of extracts of different Coptis species and their different polar solvents

This example extracts active ingredients from four Coptis plants, Coptis chinensis (Coptis chinensis), Coptis deltoidea, coptidis anthriscus (c.omeiensis), and Coptis yunnanensis (c.teeta), and analyzes the inhibitory effect of their extracts on α -glucosidase activity.

The chemicals used in this example were as follows:

phosphate buffer tablets and alpha-glucosidase (EC:3.2.1.20) were purchased from Sigma Aldrich. p-nitrophenyl-beta-D-glucopyranoside (PNPG) and acarbose are from Tokyo chemical industries, Inc. Sodium carbonate was obtained from Shanghai Tantake technologies, Inc. The chemical standards of coptisine, jateorhizine, magnoflorine, epiberberine, gladiolide, palmatine and african tetrandrine are purchased from the company Duokema Biotech, Inc., and berberine is purchased from the State food and drug administration of China. Chromatographic grade acetonitrile and formic acid were purchased from Saimer Fei technologies. Analytical grade organic reagents used for the preparation of the extracts were all from Chengdu Colon GmbH. Deionized water is produced from a millipore water purification system.

The specific process for extracting the active ingredient in this example is as follows:

(1) separating the rhizome of 4 kinds of coptis, washing with clear water, drying in air, drying in an oven at 60 ℃ until the weight is constant, and grinding into 100-mesh powder by using a grinder. Finally, these powders were collected in plastic bags of the corresponding sample names and kept dry.

(2) Weighing Coptidis rhizoma dry powder 0.5-2.0g each, and extracting with E-916 rapid extractor. Pumping petroleum ether, ethyl acetate and n-butanol in sequence to prepare three extraction parts with different polarities. Firstly, putting a piece of glass fiber filter paper and a metal gasket at the bottom of a 40mL extraction tank, putting a sample, and then filling 40-60g of quartz sand. During the extraction, the temperature and pressure were continuously set at 90 ℃ and 100 bar. The extraction parameters are detailed in table 1.

TABLE 1E-916 fast extractor parameter settings

(3) After extraction, the corresponding fractions were collected and the solvent was spun off using a rotary evaporator.

After the four coptis medicinal materials are extracted according to the method, extracts of petroleum ether, ethyl acetate and n-butyl alcohol parts of the four coptis medicinal materials are obtained. The petroleum ether and ethyl acetate extracts were dissolved in 10% dimethyl sulfoxide respectively to prepare a solution with a concentration of 0.2g/mL, and the n-butanol extract was dissolved in 50% methanol to prepare a solution with a concentration of 0.2 g/mL. Finally, these solutions were passed through a filter with a pore size of 0.45 μm and were ready for use as sample solutions for subsequent testing.

Example 2 blood sugar lowering Activity test of extracts of different Coptis species and different polar solvents

The following experiment was conducted to inhibit α -glucosidase with respect to the glucose lowering effect of each sample obtained in example 1. The specific process of the experiment is as follows:

(1) a piece of the phosphate buffer sheet was placed in 200mL of water to prepare a phosphate buffer solution having a pH of 7.4 and a concentration of 10 mmol/L. Adding 20 mu L of sample solution, 20 mu L of alpha-glycosidase solution with the concentration of 0.3U/mL and 100 mu L of phosphate buffer solution into a 96-well plate, placing the mixture in a thermostat at 37 ℃ for incubation for 15 minutes, and taking out the mixture;

(2) adding 20 mu L of PNPG solution with the concentration of 10mmol/L, incubating again for 15 minutes, and taking out;

(3) the reaction was stopped by adding 80. mu.L of a 0.2mol/L sodium carbonate solution and the absorbance was measured at 405nm in a SpectraMax iD3 enzyme-labeled meter.

The inhibition (%) was calculated as: inhibition rate (%) ([ 1- (A) ]_SG-A_BAG)/(A_CG-A_BLG)]X 100% (wherein A)_SGAbsorbance for adding the sample solution and alpha-glucosidase; a. the_BAGThe absorbance of the sample solution to which no alpha-glucosidase was added; a. the_CGThe absorbance of the alpha-glucosidase when no sample solution is added; a. the_BLGAbsorbance when neither the sample solution nor the α -glucosidase was added. The missing solution was replaced with phosphate buffer). All analyses were repeated three times. Half Inhibitory Concentration (IC) was calculated using Origin 9.1 software₅₀) And an inhibition curve was plotted.

Half Inhibitory Concentration (IC) for each sample₅₀) As shown in fig. 1, the inhibition curves are shown in fig. 2. The ethyl acetate site is not shown in the figure because it has no inhibitory activity. By comparing IC's at two other sites₅₀Value, IC found for the extracts of four Coptis chinensis Franch in the Petroleum Ether site₅₀The value is about 2 to 7 times that of the n-butanol fraction. Therefore, it was confirmed that the n-butanol fraction had the strongest inhibitory effect on α -glucosidase and was used as a highly active extraction site.

Acarbose is a potent alpha-glucosidase inhibitor, IC, in the prior art₅₀The value was 4.24. + -. 0.04mg/mL (see FIG. 2). Although acarbose shows better inhibitory activity than the n-butyl alcohol extract of the coptis, the maximum inhibitory rate is lower than 90 percent, and the n-butyl alcohol extracts of the four coptis are all higher than 95 percent. It can be seen that acarbose and therefore, it is of interest to find potential α -glucosidase inhibitors from the n-butanol fraction of coptis deltoidea. Extract of n-butanol fraction of Coptis triangularisIt is also valuable to prepare hypoglycemic drug compositions together with other active substances.

Further observing the four n-butanol extract with best blood sugar reducing activity of the coptis chinensis, finding that the n-butanol extract of the coptis chinensis has the highest inhibition activity on alpha-glucosidase from the inhibition rate curves of the four coptis chinensis, and then the coptis chinensis, the Yunnan coptis chinensis and the meadowrue coptis chinensis, and the IC of the coptis chinensis, the Yunnan coptis chinensis and the meadowrue coptis chinensis₅₀The values were 24.95. + -. 2.14, 28.24. + -. 0.12, 28.64. + -. 0.21 and 29.11. + -. 0.02mg/mL, respectively.

It can be seen that the extract of n-butanol fraction of coptidis triangularis has the highest inhibitory activity among the extracts of petroleum ether, ethyl acetate and n-butanol fractions of the four coptidis rhizoma medicinal materials prepared in example 1.

Example 3 identification of the hypoglycemic active ingredient and its content in Coptis deltoidea

To further identify the active ingredient inhibiting alpha-glucosidase in the extract of example 1, a rapid ultrafiltration screen experiment was performed below. The specific process of the experiment is as follows:

(1) preparing an ultrafiltration sample solution: diluting sample solution prepared from n-butanol fraction of Coptidis rhizoma to 6mg/mL, and mixing the sample solution for use.

(2) An experimental group was prepared by mixing 100. mu.L of 1U/mL alpha-glucosidase with an equal volume of the mixed sample solution. After incubation at 37 ℃ for 30min, the cells were transferred to a 30kDa ultrafiltration tube and centrifuged at 10000g for 10 min. Then 200. mu.L of phosphate buffer (pH 7.4) was added, centrifuged at 10000g for 10min, and unbound compound was washed off, and this operation was repeated twice. A ligand-enzyme complex is obtained.

(3) Subsequently, 200. mu.L of methanol-water (50:50, v/v, pH 3.30) was added to the ligand-enzyme complex, and after standing for 10min, the mixture was centrifuged at 10000g for 10min, and the procedure was repeated 2 times. And finally, collecting the eluent to perform UPLC-Q-TOF-MS/MS analysis. The sample solution was reacted with denatured enzyme instead of active enzyme as a blank control. The preparation process of the denatured enzyme comprises the following steps: the inactivated enzyme can be obtained by putting 1U/mL alpha-glucosidase into a 60 ℃ dry thermostat for 3 min.

The above experiment was repeated three times.

The resulting ultrafiltration chromatogram is shown in FIG. 3, in which the solid line (experimental group) represents compounds that specifically bind to the enzyme and the dotted line is a blank control group for monitoring compounds that non-specifically bind to the enzyme. And identifying the potential activity inhibitor by taking the peak area of the experimental group higher than the peak area of the blank control group as a screening standard.

As can be seen from the figure, the 8 peaks show differences and are used as potential inhibitors, and the compounds all belong to alkaloid compounds, namely magnoflorine, gladiobin, jateorhizine, epiberberine, african tetrandrine, coptisine, palmatine and berberine. The research of predecessors has been carried out, and jateorhizine, epiberberine, coptisine, palmatine and berberine are screened from coptis root as alpha-glucosidase inhibitor by off-line ultrafiltration technology. In the present study, magnoflorine, gladiolide and african tetrandrine were first screened from coptis deltoidea and were also potential inhibitors. Wherein, the content of gladiolide in the coptis deltoidea is obviously higher than that of other three coptis medicinal materials. Namely, in the extract obtained by the method, the variety of the components with the hypoglycemic activity in the coptis deltoidea and the content of part of the components (gladiobin) are higher, which further proves that the extract obtained by the extraction method has higher hypoglycemic activity.

The experiments show that the extract of the n-butanol fraction of the coptis deltoidea provided by the embodiment is rich in alkaloid compounds and high in content, so that the extract can be used as a potential alpha-glucosidase inhibitor. Particularly, the content of gladiobin in the coptis deltoides is obviously higher than that of other three coptis medicinal materials, so that the better alpha-glucosidase inhibition effect of the extract of the n-butyl alcohol part of the coptis deltoides is further enhanced.

Claims

1. The extraction method of the coptis chinensis extract for reducing the sugar is characterized by comprising the following steps of:

[1] taking dry powder of the coptis deltoidea medicinal material;

[2] sequentially extracting the dried powder in the step (1) by adopting petroleum ether, ethyl acetate and n-butanol solvent;

[3] and (3) collecting the n-butanol extraction part extract in the step (2), and concentrating to obtain an extract, namely the extract.

2. The extraction method of the active ingredients of the coptis chinensis medicinal material for reducing the sugar as claimed in claim 1, is characterized in that: the Coptis triangularis is dried rhizome of Coptis C.deltoidea belonging to Coptis of Ranunculaceae; preferably, the content of glatiramer in the coptidis rhizoma is higher than that of other coptidis plants.

3. The method for extracting active ingredients of coptis chinensis medicinal material for reducing blood sugar according to claim 1 or 2, characterized in that the preparation method of the dry medicinal powder in the step [1] is as follows: the rhizome of the coptis deltoidea is cleaned, dried in the air, dried to constant weight and then ground into powder of 100 meshes.

4. The method for extracting active ingredients of coptis chinensis medicinal material for reducing blood sugar according to claim 1, wherein the boiling range of petroleum ether in the step [2] is 60-90.

5. The method for extracting the active ingredients of the coptis chinensis medicinal material for reducing the sugar according to the claim 1 or 4, which is characterized by comprising the following steps: and (3) carrying out the extraction process in the step (2) by adopting a rapid solvent extractor.

6. The extraction method of the active ingredients of the coptis chinensis medicinal material for reducing the sugar according to claim 4, wherein the step [2] specifically comprises the following steps:

[2-3] starting a solvent extractor for extraction;

preferably, in the step [2-3], n-butanol is used as an extraction solvent to circulate for four times, wherein the 1 st circulation process is heating for 1-2min, keeping for 0min and releasing for 5min, and the 2 nd-4 th circulation process is heating for 1-2min, keeping for 2-4min and releasing for 5 min; circulating other extraction solvents except n-butanol for three times, wherein each circulation process comprises heating for 1-2min, maintaining for 2-4min and releasing for 5 min; the solvent washing time is 2min and the gas washing time is 3min after the extraction of each extraction solvent is finished.

7. The extraction method of the active ingredients of the coptis chinensis medicinal material for reducing the sugar according to claim 6, is characterized by comprising the following steps: in the step (2-2), the mass of the dried powder of the coptis deltoidea medicinal material added into the extraction tank is 0.5-2.0g, the specification of the extraction tank is 40ml, and the amount of the quartz sand added into the extraction tank is 40-60 g.

8. The extraction method of the active ingredients of the coptis chinensis medicinal material for reducing the sugar according to claim 6, is characterized by comprising the following steps: in the step (2-3), the temperature in the extraction process is 90 ℃, and the pressure is 100 bar.

9. An extract prepared by the extraction method of any one of claims 1 to 8.

10. Use of the extract of claim 9 for the preparation of a hypoglycemic medicament.