CN107445952B - A kind of method and application of extracting grandisine and coptisine - Google Patents

Abstract

The invention belongs to the technical field of traditional Chinese medicine extraction, and discloses a method and application for extracting Coptisine and Coptisine. The method for extracting coptisine and grandisine avoids repeated column chromatography and uses a large amount of organic solvents and is cumbersome and time-consuming. The present invention also provides new uses of coptisine and coptisine in the prevention and treatment of senile dementia. The mechanism of action lies in the effect on acetylcholinesterase.

Description

A kind of method and application of extracting grandisine and coptisine

technical field

The invention relates to the technical field of pharmaceutical production, in particular to a method and application for extracting grandisine and coptisine.

Background technique

Coptis chinensis is one of the traditional Chinese medicinal materials and has a long history of medication. Coptis chinensis is rich in proberberine-type isoquinoline alkaloids, which have anti-tumor, anti-bacterial, anti-HIV, hypoglycemic and lipid-lowering properties, etc. Biological activity, low toxicity and low cost. In addition, previous studies have found that Coptis chinensis is rich in proberberine-type alkaloids such as rhizine, coptisine, and epiberberine. Because there are many isomers of these alkaloids and their structures are similar, it is relatively difficult to separate and purify (except berberine and palmatine), so they are generally only used as phytochemical reference substances in the market, and the separation and purification of coptisine and grandisin Most of the traditional system separation methods are used, using a variety of chromatographic materials, different solvent systems, and repeated application of various chromatographic materials for column chromatography, which leads to the use of a large number of organic solvents, resulting in long separation cycles and large amounts of organic solvents. Not only It is not conducive to environmental protection, but also time-consuming, laborious and costly, which leads to the high price of coptisine and grandisin, which are generally only used as phytochemical reference substances or reagents for scientific research in the market, so the later pharmacological activity research and its application are limited.

Contents of the invention

The technical problem to be solved in the present invention is to overcome the existing defects and provide a method for extracting Grandisine and Coptisine, which has high extraction efficiency and stable process, utilizes the insoluble properties of Coptisine Sulfate in water and alcohol, and other The solubility of alkaloids is relatively large, and recrystallization can be used to separate coptisine, and the phenolic alkaloids in coptis can be separated by reverse chromatography and hydrogen bond adsorption of polyamide, and further column chromatography can be used to obtain grandiose The invention greatly reduces the consumption of organic solvents, can greatly reduce the prices of grandisine and coptisine, reduces the burden on people, and can effectively solve the problems in the background technology.

In order to achieve the above object, the present invention provides the following technical solutions: a method for extracting grandisine and coptisine, comprising the steps of:

1) Coptidis or Coptis fibrous roots are crushed to extract the mother liquor: First, take out 1 kg of Coptidis Rhizoma and use a medicinal material grinder to crush it or cut and crush Coptis rhizome fibrous roots; add 10-20ml of concentrated hydrochloric acid to precipitate berberine, filter with diatomaceous earth, and use the mother liquor for later use;

2) Remove grease from the mother liquor; add ammonia water to the mother liquor to adjust the pH value to 11, filter, add ether or petroleum ether to the filtrate for extraction, and remove low-grade grease;

3) Crystallization: Utilizing the high solubility of coptisine in ethanol and relatively high content, its sulfate is insoluble in water and physical properties of ethanol. The solubility of coptisine sulfate in ethanol is 1:500. The solubility of sulfate in ethanol is 1:30. Add 90% sulfuric acid to the above alkali solution and add it drop by drop while stirring until a yellow precipitate is produced. The yellow precipitate is dissolved with 1L-2L of ethanol at 50°C by ultrasonic , add 5-10ml of sulfuric acid dropwise and recrystallize repeatedly two to three times to obtain more than 80% of coptisine sulfate precipitates, the HPLC detection purity is more than 85%, then add barium chloride solution with a molar ratio of 1:1 to reflux for half an hour, filter and concentrate , recrystallized from methanol acetone to obtain 400-800 mg of crystals of coptisine hydrochloride;

4) Concentration: Use the above remaining mother liquor, adjust the pH value of the filtrate to 10-11 with 5%-10% sodium hydroxide, free out phenolic alkaloids, and then filter through a filter, the filtrate is 5%-10% Dilute hydrochloric acid to adjust the pH value to 7, put macroporous resin D101 on the column for adsorption at a column volume rate of 0.5%-2.5% per minute, and then elute with 95% ethanol, and concentrate to obtain a crude extract. The extract is dissolved with 1mol/L ammonia water to obtain the upper column solution;

5) Column loading: When loading the column, apply the polyamide column at 0.5% to 1.0% of the column volume/min, and then use 0.8-1.5mol/L ammonia water to elute;

6) Elution collection: The column is eluted according to 0.5% to 1.0% of the column volume/min. When the component moves down to the bottom three-quarters of the column, the three colors of A, B, and C appear sequentially from top to bottom Band, collect B color band, and then use toluene, ethyl acetate, acetic acid and methanol according to the volume ratio of 4:2:1:1 developer and 1 part of ammonia double tank development for TLC identification, B color band components are The mixture of jatrorrhizine, tetrandrine, and grandisin was tested by thin-layer chromatography, and the same fractions were combined;

7) Preparation of Grandysin: adjust the pH of the components of the B ribbon group to 7, put on the macroporous resin D101, carry out the adsorption on the column at a column volume rate of 0.5%-2.5% per minute, and wash off the inorganic salt with water , and then eluted with 95% ethanol, concentrated to obtain a crude extract, recrystallized from acetone and methanol to obtain jatrorrhizine, the mother liquor is a mixture of jatrorrhizine, African tetrandrine, and grandisin, and the mother liquor is mixed with 100-200 mesh silica gel Sample, 300-400 mesh silica gel column chromatography, eluted with chloroform and methanol according to the volume ratio of 10:1 to 20:1, and then with toluene, ethyl acetate, acetic acid and methanol according to the volume ratio of 4:2: 1:1 developer and 1 part of ammonia were developed in double tanks, and TLC identification was carried out, and the same Rf value was combined to obtain Grandicin, with a purity of 95% by HPLC;

As a preferred technical solution of the present invention, after the pH value of the remaining mother liquor in step 2) is adjusted to 10 to 11, the solution needs to be left for 6-12 hours.

As a preferred technical solution of the present invention, when loading the column in the above step 5), stop loading the column when the volume of the column loading solution is 1/3 to 1/4 of the volume of the column.

As a preferred technical solution of the present invention, in the above steps 5) and 6), when the column is loaded, the polyamide column is loaded according to 0.5% to 1.0%/min of the column volume, and then 0.8-1.5mol/L of Ammonia elution;

Compared with the prior art, the beneficial effects of the present invention are: the method and process for extracting Grandisine and Coptisine, the process is simple and easy, the extraction efficiency is good, and the repeatability is good. The lipase inhibitory activity has not been reported in the literature. Through the activity screening of Coptisine and Coptisine, it is found that it has a good inhibitory activity of acetylcholinesterase, which can reduce the price of Coptisine and Coptisine, and provide the follow-up Research provides the material basis.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the implementation examples of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Embodiment one:

A method for extracting grandisine and coptisine, comprising the steps of:

1) Coptis chinensis is crushed to extract the mother liquor: first take out 1 kg of Coptis chinensis or Coptis chinensis must be crushed with a medicinal material grinder, add 10ml of concentrated hydrochloric acid to precipitate berberine, filter with diatomaceous earth, and use the mother liquor for later use;

2) Remove grease; add ammonia water to the mother liquor to adjust the pH value to 11, then add ether or petroleum ether for extraction to remove low-grade grease;

3) Crystallization: add 90% sulfuric acid to the remaining alkaline solution, add dropwise while stirring until a yellow precipitate is produced, and use 2L of ethanol to dissolve the yellow precipitate ultrasonically at 50°C, add 2-3ml dropwise and repeat repeatedly Crystallize two to three times to obtain more than 80% of berberine sulfate precipitates, and the HPLC detection purity is more than 85%, then dissolve with methanol or ethanol, then add barium chloride solution with a molar ratio of 1:1 and reflux for half an hour, and then pass through the filter. Filter and recrystallize from methanol acetone to obtain 400 mg of crystals of coptisine hydrochloride;

4) Concentration: Use the remaining mother liquor to free phenolic alkaloids with 5% sodium hydroxide, then adjust the pH value of the filtrate to 10 with 5% sodium hydroxide, and then free the remaining mother liquor after adjusting the pH value to 10. For phenolic alkaloids, the solution needs to be left for 6 hours, and then filtered through a filter. The filtrate is adjusted to a pH value of 7 with 5% dilute hydrochloric acid, and the macroporous resin D101 is loaded on the column at a column volume rate of 0.5% per minute. Adsorbed, and then eluted with 95% ethanol, concentrated to obtain a crude extract, which was dissolved with 1mol/L ammonia water to obtain the upper column liquid;

5) Column loading: When loading the column, load the polyamide column at 0.5%/min of the column volume, and stop loading the column when the volume of the column solution is one-third of the column volume, and then use 0.8mol/L ammonia water Elution;

6) Elution collection: The column is eluted at 0.5%/min of the column volume. When the component moves down to three quarters of the bottom of the column, three color bands of A, B, and C appear in sequence from top to bottom. Collect B color band, and then use toluene, ethyl acetate, acetic acid and methanol according to the volume ratio of 4:2:1:1 developer and 1 part of ammonia double tank development for TLC identification, the B color band component is the root The mixture of alkali, tetrandrine, and grandisin, the fractions were tested by thin-layer chromatography, and the same fractions were combined;

7) Preparation of Grandysin: adjust the pH value of the components of the B ribbon group to 7, apply the macroporous resin D101 to the column for adsorption at a column volume rate of 0.5% per minute, and then use 95% ethanol to elute. Concentrate to obtain crude extract, recrystallize from acetone and methanol to obtain jatrorrhizine, the mother liquor is a mixture of jatrorrhizine, tetrandrine and grandisine, the mother liquor is mixed with 100 mesh silica gel, 300 mesh silica gel column chromatography, and chloroform and methanol were eluted at a volume ratio of 20:1, and toluene, ethyl acetate, acetic acid, and methanol were developed with a volume ratio of 4:2:1:1 as a developing solvent and 1 part of ammonia water for TLC identification, and combined With the same Rf value, 300 mg of Grandisine was obtained, and the purity by HPLC was 95%.

Embodiment two:

A method for extracting grandisine and coptisine, comprising the steps of:

1) Coptis chinensis is crushed to extract the mother liquor: first take out 1 kg of Coptis chinensis or Coptis chinensis must be crushed with a medicinal material grinder, add 15ml of concentrated hydrochloric acid to precipitate berberine, filter with diatomaceous earth, and use the mother liquor for later use;

2) Remove grease; add ammonia water to the mother liquor to adjust the pH value to 11, then add ether or petroleum ether for extraction to remove low-grade grease;

3) Crystallization: Add 90% sulfuric acid to the remaining alkali solution, add dropwise while stirring until a yellow precipitate is produced, and use 1L of ethanol to ultrasonically dissolve the yellow precipitate at 50°C, add 10ml sulfuric acid dropwise and repeat Crystallize two to three times to obtain more than 80% of berberine sulfate precipitates, and the HPLC detection purity is more than 85%, then dissolve with methanol or ethanol, then add barium chloride solution with a molar ratio of 1:1 and reflux for half an hour, and then pass through the filter. Filter and recrystallize from methanol acetone to obtain 500 mg of crystals of coptisine hydrochloride;

4) Concentration: use the remaining mother liquor above, adjust the pH value of the filtrate to 11 with 8% sodium hydroxide, and release the phenolic alkaloid after the pH value of the remaining mother liquor is adjusted to 10. The solution needs to be placed for 8 hours. Filter through a filter again, the filtrate is adjusted to a pH value of 7 with 6% dilute hydrochloric acid, and the macroporous resin D101 is applied to the column for adsorption at a column volume rate of 1% per minute, and then eluted with 95% ethanol to This concentration obtains a crude extract, which is dissolved with 1mol/L ammonia water to obtain the upper column liquid;

5) Column loading: When loading the column, load the polyamide column at 0.7%/min of the column volume, stop loading the column when the volume of the column loading solution is one-third of the column volume, and then wash with 1.0mol/L ammonia water off;

6) Elution collection: The column is eluted at 0.7%/min of the column volume. When the component moves down to three quarters of the bottom of the column, three color bands of A, B, and C appear in sequence from top to bottom. Collect the color band B, and then carry out thin-layer identification with a developer with a volume ratio of toluene, ethyl acetate, isopropanol, methanol, and ammonia water at a volume ratio of 6:3:1.5:2:0.5. The components of the color band B are jatrorrhizine, The mixture of tetrandrine and grandiose new, use thin layer chromatography to test the fractions, and combine the same fractions;

7) Preparation of Grandysin: adjust the pH value of the components of the B ribbon group to 7, apply the macroporous resin D101 to the column for adsorption at a column volume rate of 1% per minute, and then use 95% ethanol to elute. Concentrate to obtain a crude extract, recrystallize from acetone and methanol to obtain jatrorrhizine, the mother liquor is a mixture of jatrorrhizine, tetrandrine and grandisine, the mother liquor is mixed with 100 mesh silica gel, and 300-400 mesh silica gel column chromatography, Elute with chloroform and methanol at a volume ratio of 10:1, then use toluene, ethyl acetate, acetic acid, and methanol for TLC identification at a volume ratio of 4:2:1:1, and combine the same Rf values To the new 50mg of grandiose, the HPLC detection purity is 95%.

Embodiment three:

A method for extracting grandisine and coptisine, comprising the steps of:

1) Coptis fibrous roots are cut to extract the mother liquor: first take out 1 kg of Coptis rhizome or Coptis chinensis and grind it with a medicinal material grinder, add 10-20ml of concentrated hydrochloric acid to precipitate berberine, filter with diatomaceous earth, and use the mother liquor for later use;

2) Remove grease; add ammonia water to the mother liquor to adjust the pH value to 11, then add ether or petroleum ether for extraction to remove low-grade grease;

3) Crystallization: Add 90% sulfuric acid to the remaining alkaline solution, and add dropwise while stirring until a yellow precipitate is produced. The yellow precipitate is dissolved with 1L ethanol at 50°C by ultrasound, and 6ml of concentrated sulfuric acid is added dropwise to recrystallize repeatedly. To three times, add dropwise, stir while adding, obtain more than 80% berberine sulfate precipitation, HPLC detection purity is more than 85%, then use 200ml methanol or ethanol, then add about molar ratio and be 1:1 (berberine sulfate: Barium chloride) was refluxed for half an hour, then filtered through a filter, and recrystallized from methanol and acetone to obtain 600 mg of crystals of coptisine hydrochloride;

4) Concentration: use the above remaining mother liquor, adjust the pH value of the filtrate to 11 with 10% sodium hydroxide, and release the phenolic alkaloid after the pH value of the remaining mother liquor is adjusted to 10. The solution needs to be placed for 10 hours. Filter through a filter again, the filtrate is adjusted to a pH value of 7 with 8% dilute hydrochloric acid, and the macroporous resin D101 is applied to the column for adsorption at a column volume rate of 2.0% per minute, and then eluted with 95% ethanol to This concentration obtains a crude extract, which is dissolved with 1mol/L ammonia water to obtain the upper column liquid;

5) Column loading: When loading the column, load the polyamide column at 0.8%/min of the column volume, stop loading the column when the volume of the column loading solution is one-third of the column volume, and then wash with 1.3mol/L ammonia water off;

6) Elution collection: The column is eluted at 0.8%/min of the column volume. When the component moves down to three quarters of the bottom of the column, three color bands of A, B, and C appear sequentially from top to bottom. Collect B color band, and then use toluene, ethyl acetate, acetic acid and methanol according to the volume ratio of 4:2:1:1 developer and 1 part of ammonia for TLC identification. The components of B color band are jatrorrhizine, African For the mixture of tetrandrine and grandisine, the fractions were tested by thin layer chromatography, and the same fractions were combined;

7) Preparation of Grandysin: adjust the pH value of the components of the B ribbon group to 7, apply the macroporous resin D101 to the column for adsorption at a column volume rate of 2.0% per minute, and then use 95% ethanol to elute. Concentrate to obtain crude extract, recrystallize with acetone and methanol to obtain jatrorrhizine, the mother liquor is a mixture of jatrorrhizine, African tetrandrine, and grandisin, the mother liquor is mixed with 60-100 mesh silica gel, and 300-400 mesh silica gel column layer analysis, eluted with chloroform and methanol according to the volume ratio of 20:1, and then used toluene, ethyl acetate, acetic acid and methanol according to the volume ratio of 4:2:1:1 developer and 1 part of ammonia for thin layer Identification, combined with the same Rf value to obtain Grandicin 40mg, the HPLC detection purity was 95%.

Embodiment four, grandisine and coptisine inhibit activity of acetylcholinesterase

Principle: The product of acetylcholinesterase and thioacetylcholine iodide generates a yellow substance with 5,5-dithiobisnitrobenzoic acid. This color reaction can be used to determine the inhibition of the test sample on acetylcholinesterase degree. For specific methods, please refer to the literature: Sun Qianyun, Yang Fumei. Comparative Study on Micro-screening Models of Acetylcholinesterase Inhibitors "Chinese Pharmacology Bulletin" 2008, 24(10): 1387-1392 5. Ellman method was used to test the inhibitory activity of AChE in vitro. Acetylcholine was used as the substrate, and chemically labeled dithionitrobenzoic acid was used as the chromogen, and the AChE inhibitory activity of the samples was determined on a 96-well plate. Tacrine was selected as the positive control drug. The inhibition rate of three different concentrations of acetylcholinesterase was obtained.

See Table 1

Table 1 The single-concentration inhibitory rate of coptisine and grandisine to acetylcholinesterase and butyrylcholinesterase

From the simple comparison in Table 1, Grandiseine and coptisine have obvious inhibitory effect on acetylcholinesterase, especially the inhibitory effect of Grandiseine on acetylcholinesterase at a single concentration of 1×10 ^-6 mol/L ^-1 The rate exceeds 50%, indicating that it has good selectivity for the two enzyme inhibitory activities, and may become a drug for the treatment of senile dementia, and its mechanism of action is mainly the inhibition of acetylcholinesterase.

The process of the invention is simple and easy, the repeatability is good, and the extraction efficiency is high, the price of the alkaloids containing grandisine and coptis can be greatly reduced, a material basis is provided for follow-up research, and the added value of the coptis is increased. There is no literature report on the inhibitory activity of coptisine and grandisine on acetylcholinesterase. Through the activity screening of coptisine and grandisine, it is found that it has the inhibitory activity of acetylcholinesterase. The present invention discloses that grandisine Coptisine has the inhibitory effect on acetylcholinesterase, and has the application prospect of being developed as an anti-senile dementia drug.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (3)

1. A method for extracting gladiolide and coptisine is characterized by comprising the following steps:

1) crushing coptis chinensis or coptis chinensis fibrous roots to extract mother liquor: firstly, taking out one kilogram of coptis and crushing the coptis by using a medicinal material crusher or cutting and crushing fibrous roots of the coptis; adding concentrated hydrochloric acid 10-20ml to precipitate berberine, filtering with diatomite, and keeping mother liquor for use;

2) removing grease from the mother liquor; adding ammonia water into the mother liquor to adjust the pH value to 11, filtering, adding diethyl ether or petroleum ether into the filtrate for extraction, and removing low-polarity oil;

3) and (3) crystallization: by utilizing the physical properties that coptisine has high solubility and relatively high content in ethanol, sulfate of coptisine is insoluble in water and ethanol, and the solubility of coptisine sulfate in ethanol is 1: 500, solubility of berberine sulfate in ethanol at room temperature is 1: 30, adding 90% sulfuric acid into the alkali solution, dropwise adding while stirring until a yellow precipitate is generated, ultrasonically dissolving the yellow precipitate at 50 ℃ by using 1L-2L of ethanol, dropwise adding 5-10ml of sulfuric acid, repeatedly recrystallizing for two to three times to obtain more than 80% coptisine sulfate precipitate, detecting the purity by HPLC (high performance liquid chromatography) to be more than 85%, then adding a barium chloride solution with the molar ratio of 1:1, refluxing for half an hour, filtering, concentrating, and recrystallizing by methanol acetone to obtain 400-800mg coptisine hydrochloride crystals;

4) concentration: using the residual mother liquor, adjusting the pH value of the filtrate to 10-11 by using 5-10% of sodium hydroxide, dissociating phenolic alkaloid, filtering by using a filter, adjusting the pH value of the filtrate to 7 by using 5-10% of dilute hydrochloric acid, adding macroporous resin D101, performing column loading adsorption at the column volume speed of 0.5-2.5% per minute, eluting by using 95% ethanol, concentrating to obtain a crude extract, and dissolving the crude extract by using 1mol/L ammonia water to obtain column loading liquid;

5) column mounting: when the polyamide is loaded on the column, the polyamide is loaded on the polyamide column according to 0.5 to 1.0 percent/min of the volume of the column, and then 0.8 to 1.5mol/L ammonia water is used for elution;

6) eluting and collecting: eluting the column according to the volume of 0.5-1.0%/min, sequentially presenting A, B, C color bands from top to bottom when the components move down to three quarters of the bottom of the column, collecting a B color band, performing thin-layer identification by using a developing agent and 1 part of ammonia water in a double-tank development manner according to the volume ratio of 4:2:1:1 of toluene, ethyl acetate, acetic acid and methanol, wherein the components of the B color band are a mixture of jatrorrhizine, african tetrandrine and gladiodine, detecting the flow by using thin-layer chromatography, and combining the same flow;

7) preparing gladiobin: adjusting the pH value of the component of the B color band to 7, loading the component on macroporous resin D101, loading the component on a column at the column volume speed of 0.5-2.5% per minute for adsorption, washing with water to remove inorganic salt, eluting with 95% ethanol, concentrating to obtain a crude extract, recrystallizing with acetone and methanol to obtain jateorhizine, wherein the mother liquor is a mixture of jateorhizine, african stephanine and glatirnew, mixing the mother liquor with 100-200-mesh silica gel, performing 300-400-mesh silica gel column chromatography, eluting with chloroform and methanol at the volume ratio of 10: 1-20: 1, developing with toluene, ethyl acetate, acetic acid and methanol at the volume ratio of 4:2:1:1 in a developing agent and 1 part of ammonia water, performing thin-layer identification, combining the same Rf value to obtain glatirnew, and detecting the purity of 95% by HPLC.

2. The method for extracting gladiolide and coptisine as claimed in claim 1, wherein: after the pH value of the residual mother liquor in the step 2) is adjusted to 11, the solution is required to be placed for 6-12 hours.

3. The method for extracting gladiolide and coptisine as claimed in claim 1, wherein: and (3) stopping the column feeding when the amount of the solution on the column is one third to one fourth of the volume of the column during the column feeding in the step 5).