Efficient and green separation and purification method of sophora flavescens alkaloid

Abstract

The invention provides an efficient and green separation and purification method of sophora flavescens alkaloid, which specifically comprises the following steps: 1) mixing sophora flavescens powder with an extract liquor, heating, stirring, condensing, refluxing, standing, and filtering to remove sophora flavescens powder residues to obtain a crude extract liquor; and 2) extracting the crude extract by using a C18 solid-phase extraction column, and eluting by using an eluent to respectively obtain matrine and oxymatrine. Compared with the prior art, the method has the advantages that firstly, the C18 solid-phase extraction column is used for extraction, then the matrine and the oxymatrine are separated from the C18 solid-phase extraction column through the eluent, and meanwhile, the effects of removing impurities, purifying and enriching the sophora flavescens alkaloid are achieved. The method lays a foundation for wide-range use of high-extraction-amount and high-purity sophora flavescens alkaloid in the later period.

Description

Technical Field

[0001] The invention relates to the technical field of alkaloid separation and purification, and in particular to an efficient and green method for separating and purifying sophora flavescens alkaloids. Background Art

[0002] Sophora flavescens Ait. is the dried root of Sophora flavescens, a plant of the genus Sophora in the Leguminosae family. It has a cylindrical root that is usually bifurcated at the bottom, 10-30 cm long, 1-2.5 cm in diameter, and is brown or gray-brown in appearance. The cork is very thin and often breaks and bends outward, easily falling off in pieces to reveal a yellow, slightly shiny inner bark. It is very hard and not easy to break. The cross section is yellow-white and fibrous, and the wood has radial textures. Other names include water sophora, ground sophora, and white stem. It is cold in nature and bitter in taste. It enters the heart, spleen, and kidney meridians and is a traditional Chinese medicine.

[0003] The main chemical components of Sophora flavescens are quinolizidine alkaloids and flavonoids. In addition, there are other compounds such as volatile oils, a small amount of quinones, saponins and amino acids. Sophora flavescens has the effects of clearing away heat and dampness, and killing insects. It can be used for heat dysentery, blood in the stool, leucorrhea, eczema, wet sores, skin itching and other symptoms. In addition, the Sophora flavescens alkaloids in Sophora flavescens have diuretic, anti-diarrhea, anti-ulcer, lipid-lowering, anti-arrhythmic, anti-hepatitis B and anti-tumor effects. Nowadays, the clinical application scope of this traditional Chinese medicine is constantly expanding. It has been confirmed that its alkaloids can not only inhibit tumors, but also have the effects of inhibiting and killing various microorganisms, and also have a wide range of pharmacological effects on the immune system, cardiovascular system, and central nervous system. In addition, Sophora flavescens can also be used as botanical insecticides.

[0004] There are many reports on the extraction process of Sophora flavescens alkaloids on the market, and its extraction process has reached maturity, but the separation and purification process of Sophora flavescens alkaloids still has many obvious defects. For example, the principle of purification by macroporous adsorption resin method is to rely on van der Waals force to adsorb organic matter, or rely on hydrogen bonds to form semi-chemical adsorption, and have molecular sieve effect, and finally selectively adsorb organic matter without interference from inorganic salts. However, the separation and purification effect of macroporous adsorption resin method is poor and the recovery rate is low. Due to the above reasons, the consumption of eluent is large during separation and purification, and most of the chemical reagents used pollute the environment, with more impurities, further purification is required, and a lot of time is wasted. In addition, in the extraction process of Sophora flavescens alkaloids, the sublimation method has a low yield and is accompanied by decomposition; the solvent method has a low leaching rate, and the raw materials and extracts are easily contaminated and corroded, and there are also disadvantages such as time-consuming.

[0005] Therefore, it is very necessary to provide an efficient and green method for separating and purifying Sophora flavescens alkaloids. Summary of the invention

[0006] The purpose of the present invention is to provide an efficient and green method for separating and purifying matrine alkaloids. First, a C18 solid phase extraction column is used for extraction, and then matrine and oxymatrine are separated from the C18 solid phase extraction column by an eluent, thereby achieving the effect of removing impurities, purifying and enriching matrine alkaloids.

[0007] The specific technical solutions of the present invention are as follows:

[0008] An efficient and green method for separating and purifying Sophora flavescens alkaloids comprises the following steps:

[0009] 1) mixing the sophora flavescens powder and the extract, heating and stirring to condense and reflux, standing, filtering out the sophora flavescens powder residue, and obtaining a crude extract;

[0010] 2) extracting the crude extract using a C18 solid phase extraction column, and then eluting with an eluent to obtain matrine and oxymatrine, respectively.

[0011] In step 1), the particle size of the sophora flavescens powder is 90-100 mesh;

[0012] In step 1), the preparation method of the sophora flavescens powder is as follows: soaking the sophora flavescens with intact and full epidermis in distilled water, performing ultrasonic cleaning, and drying; and then grinding into the sophora flavescens powder. The ultrasonic cleaning includes low-frequency ultrasonic cleaning and high-frequency ultrasonic cleaning; the frequency range of the low-frequency ultrasonic cleaning is 33KHz-40KHz, and the cleaning time is 25min, and the frequency range of the high-frequency ultrasonic cleaning is 60KHz-80KHz, and the cleaning time is 15min.

[0013] In step 1), the solid-liquid ratio of Sophora flavescens powder to the extract is 1 g: 10 ml;

[0014] In step 1), the extract is an ethanol aqueous solution with a volume concentration of 65%;

[0015] In step 1), the mixture is heated, stirred, condensed and refluxed, and the heating temperature ranges from 80° C. to 90° C. The heating, stirring and condensation reflux time are both 2 h.

[0016] In step 1), the standing time is in the range of 60 min to 80 min;

[0017] In step 1), filtering out the Sophora flavescens powder residue is performed by filtering out the Sophora flavescens powder residue through a Buchner funnel, and the filtration accuracy of the qualitative filter paper used is 80-120 μm;

[0018] In step 2), the specific method of extracting the crude extract with the C18 solid phase extraction column is: continuously adding the crude extract to the C18 solid phase extraction column at a flow rate of 3 BV / h; until the content of sophora flavescens alkaloids in the crude extract is the same as the content of sophora flavescens alkaloids in the effluent.

[0019] In step 2), eluting with an eluent means eluting the C18 solid phase extraction column with an eluent at a flow rate of 2-4 BV / h; collecting the obtained eluent to obtain matrine and oxymatrine; the preferred flow rate is 3 BV / h.

[0020] The extraction and elution of the present invention are both carried out at room temperature, and the eluent is an aqueous solution of 3.3wt% hydrochloric acid-20% ethanol by volume concentration (elution object: matrine, optimal dosage: 25ml) and an aqueous solution of 3.3wt% hydrochloric acid-60% ethanol by volume concentration (elution object: oxymatrine, optimal dosage: 25ml), and the flow rate is 2BV / h; or, the eluent is an aqueous solution of 20% ethanol by volume concentration (elution object: matrine, optimal dosage: 15ml) and an aqueous solution of 60% ethanol by volume concentration (elution object: oxymatrine, optimal dosage: 15ml), and the flow rate is 3BV / h; or, the eluent is an aqueous solution of sodium hydroxide with a concentration of 0.05wt%-0.2wt% (elution object: matrine, optimal dosage: 40ml) and an aqueous solution of 4% hydrochloric acid (elution object: oxymatrine, optimal dosage: 40ml), and the flow rate is 4BV / h;

[0021] Preferably, when using a 3.3wt% hydrochloric acid-20vol% ethanol aqueous solution and a 3.3wt% hydrochloric acid-60vol% ethanol aqueous solution as eluents, the two can be used together or one after the other; the other two groups of eluents are also applicable.

[0022] Preferably, the eluent is an ethanol aqueous solution with a volume concentration of 20% to elute matrine, and an ethanol aqueous solution with a volume concentration of 60% to elute oxymatrine, and the flow rate is 3 BV / h.

[0023] Solid-phase extraction (SPE) is a pretreatment technology that combines liquid-solid extraction and liquid chromatography. It is a physical extraction process that includes liquid and solid phases. It has the characteristics of high carbon loading, strong hydrophobicity, full coverage and end-capping, which can greatly reduce the interference of alkaline and polar compounds and can withstand high pH environments. Based on the theory of liquid-solid chromatography, it can selectively adsorb the target or impurities, and can achieve good recovery rate and precision, greatly improve the elution efficiency, reduce the amount of impurities, and greatly increase the concentration of the target compound.

[0024] Compared with the prior art, the present invention firstly uses C18 solid phase extraction column for extraction, and then separates matrine and oxymatrine from the C18 solid phase extraction column by eluent, and simultaneously achieves the effect of removing impurities, purifying and enriching matrine alkaloids. In order to reduce the impurity content in matrine alkaloids under the premise of green, safe and efficient, the present invention proposes to separate and purify matrine alkaloid components by green and efficient C18 solid phase extraction method, laying a foundation for the large-scale use of high-extraction and high-purity matrine alkaloids in the later stage. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0027] The matrine alkaloids described in the present invention include matrine and oxymatrine. The present invention provides an efficient and green method for separating and purifying matrine alkaloids, comprising the following steps:

[0028] S1. Select a batch of Sophora flavescens with complete and full epidermis, soak the Sophora flavescens in distilled water, perform ultrasonic cleaning and then dry. During ultrasonic cleaning, perform ultrasonic cleaning twice on Sophora flavescens. First, remove Sophora flavescens from sewage after low-frequency ultrasonic cleaning, and soak Sophora flavescens in clean distilled water and perform high-frequency ultrasonic cleaning. After cleaning, remove Sophora flavescens and dry it. Use the cavitation effect of ultrasound to impact and peel off the dirt on the surface of Sophora flavescens to achieve the purpose of cleaning. Among them, the frequency range of low-frequency ultrasonic cleaning is 33KHz-40KHz, and the cleaning time is 25min. The frequency range of high-frequency ultrasonic cleaning is 60KHz-80KHz, and the cleaning time is 15min. First, the surface of Sophora flavescens can be cleaned as a whole by low-frequency ultrasonic cleaning to remove impurities attached to the surface of Sophora flavescens, while high-frequency ultrasonic cleaning has a good cleaning effect on the wrinkles on the surface of Sophora flavescens, and the cleaning effect of Sophora flavescens is ensured by low-frequency and high-frequency coordination.

[0029] S2. Grind the Sophora flavescens into powder with a fineness range of 90-100 mesh. The finer the Sophora flavescens, the larger the contact area with the extract, which is beneficial to the extraction of the components in Sophora flavescens. Then mix the Sophora flavescens powder with the extract and heat, stir, condense and reflux. The heating, stirring and condensing and reflux time are both 2 hours. After standing, filter out the Sophora flavescens residue to produce a crude extract. The extract is 65% ethanol by volume, and the solid-liquid ratio of the sophora flavescens powder to the extract is 1g:10ml, usually 200g of the sophora flavescens powder is mixed with 2000ml of 65% ethanol aqueous solution by volume, the 65% ethanol aqueous solution by volume is a solvent for sophora flavescens alkaloids, and can effectively extract the sophora flavescens alkaloids, the above heating temperature range is 80°C-90°C, and the standing treatment time range is 60min-80min, the heating temperature can improve the extraction efficiency of the extract for the effective components of sophora flavescens, and after sufficient standing to facilitate the sedimentation of the sophora flavescens powder residue, the sophora flavescens powder residue is filtered out through a Buchner funnel, and the filtration accuracy of the qualitative filter paper used is 80-120μm;

[0030] S3. Use high performance liquid chromatography according to the instrument reference conditions: a. Chromatographic column C18 column, column length 150mm, inner diameter 4.6mm, filler particles 5μm, or equivalent; b. Mobile phase: potassium dihydrogen phosphate solution (0.05mol / L)-acetonitrile (potassium dihydrogen phosphate solution and acetonitrile volume ratio 94:6); c. Flow rate: 1.2ml / min; d. Column temperature: 40℃; e. Detection wavelength: 230nm; f. Injection volume: 15μl, to detect the total concentration of matrine and total concentration of oxymatrine in the crude extract. The liquid chromatography parameter setting can detect matrine and oxymatrine in matrine alkaloids separately, and accurately detect the contents of matrine and oxymatrine respectively. Then accurately weigh 60 ml of octadecyl silica gel, and use the wet column method: 2wt% NaOH solution for elution, the amount is 2BV, the flow rate is 1.5BV / h; then use distilled water to wash at the same flow rate until neutral; 5wt% HCl solution for elution, the amount is 2BV, the flow rate is 1.5BV / h; then use distilled water to wash at the same flow rate until neutral; 95% volume concentration ethanol aqueous solution for elution, the amount is 2BV, the flow rate is 1.5BV / h; then use distilled water to wash at the same flow rate until there is no alcohol smell; finally, make 2 30ml C18 solid phase extraction columns, namely C18 solid phase extraction column No. 1 and C18 solid phase extraction column No. 2. The above wet column loading method can remove the impurities present in the octadecyl silica gel, ensure that low-impurity and high-purity matrine and oxymatrine are obtained later, and improve the purification efficiency;

[0031] S4, dynamic adsorption: continuously add the crude extract obtained in step S2 to the No. 1 C18 solid phase extraction column at a flow rate of 3BV / h, collect the effluent, and under the condition of a flow rate of 3BV / h, the C18 solid phase extraction column can be guaranteed to adsorb as much matrine alkaloids as possible; then perform a qualitative test by thin layer chromatography (TLC), and the appearance of a spot at the same position as the matrine reference substance is the end point of dynamic adsorption. The thin layer chromatography method can more accurately obtain the maximum volume of a certain weight of octadecyl silica gel adsorbing the crude extract, and finally obtain the maximum adsorption volume of 30ml of octadecyl silica gel on the crude extract, and calculate the maximum adsorption amount of 30ml of octadecyl silica gel on matrine alkaloids by the maximum adsorption amount formula. Maximum adsorption amount of matrine (mg) = maximum adsorption volume (ml) × total concentration of matrine (mg / ml); Maximum adsorption amount of oxymatrine (mg) = maximum adsorption volume (ml) × total concentration of oxymatrine (mg / ml).

[0032] According to the same method and parameters, multiple groups of experiments were repeated to obtain multiple groups of No. 1 C18 solid phase extraction columns after elution for subsequent experiments.

[0033] The thin layer chromatography method is as follows: Take matrine and oxymatrine reference substances, add anhydrous ethanol to make a solution containing 0.2 mg per 1 mL, as the reference substance solution. Pipette the collected sample and the reference substance and spot them on the same silica gel G thin layer plate, use chloroform-methanol-25wt% ammonia water with a volume ratio of 5:1:0.5 as the developing agent, develop, dry, and spray bismuth potassium iodide test solution. Bismuth potassium iodide reaction (Dra-gendoff reaction): Alkaloid precipitation reaction, which can be used for the identification of alkaloids (test tube reaction or thin layer chromatography color developer) yellow, orange-red amorphous precipitate.

[0034] S5. Static adsorption: 3BV of the crude extract obtained in step S2 was added to the No. 2 C18 solid phase extraction column at a flow rate of 2BV / h, and the effluent was collected and named as effluent 1; then 2BV of deionized water was used to rinse the No. 2 C18 solid phase extraction column, and the effluent was collected and named as effluent 2. Effluent 1 and effluent 2 were combined to obtain a combined effluent. The combined effluent was tested by high performance liquid chromatography in step S3 to detect the concentration of unabsorbed matrine and the concentration of unabsorbed oxidized matrine, and then the adsorption rate of matrine (%) = (total amount of matrine - unabsorbed amount of matrine) / total amount of matrine (mg) × 100%; the adsorption rate of oxidized matrine (%) = (total amount of oxidized matrine - unabsorbed amount of oxidized matrine) / total amount of oxidized matrine (mg) × 100%; total amount of matrine (mg) = volume of crude extract used (ml) × total concentration of matrine (mg / ml); total amount of oxidized matrine (mg) = volume of crude extract used (ml) × total concentration of oxidized matrine (mg / ml); amount of unabsorbed matrine (mg) = volume of combined effluent (ml) × concentration of unabsorbed matrine (mg / ml); amount of unabsorbed oxidized matrine (mg) = volume of combined effluent (ml) × concentration of unabsorbed oxidized matrine (mg / ml).

[0035] S6. Elute the No. 1 C18 solid phase extraction column obtained by the same method in step S4 respectively with eluents of different properties at a flow rate of 2-4 BV / h, collect the eluents respectively, and measure the volume of each eluent respectively. The eluents of different properties can elute the matrine and oxymatrine adsorbed in the octadecyl silica gel into the eluents respectively, thereby obtaining low-impurity and high-purity matrine and oxymatrine. The concentration of matrine in the eluent and the concentration of oxidized matrine in the eluent are detected by high performance liquid chromatography in step S3, so that the resolution of matrine and oxidized matrine can be calculated; the calculation method is: the amount of eluted matrine (mg) = the volume of the eluent (ml) × the concentration of matrine in the eluent (mg / ml); the amount of eluted oxidized matrine (mg) = the volume of the eluent (ml) × the concentration of oxidized matrine in the eluent (mg / ml); the theoretical resolution rate of matrine (%) = the amount of eluted matrine (mg) / the total amount of matrine (mg) × 100%, the theoretical resolution rate of oxidized matrine (%) = the amount of eluted oxidized matrine (mg) / the total amount of oxidized matrine (mg) × 100%. The collected eluent contains purified low-impurity high-purity matrine and oxidized matrine.

[0036] The eluents used were: 3.3wt% hydrochloric acid-20% ethanol aqueous solution, 3.3wt% hydrochloric acid-60% ethanol aqueous solution, 20% ethanol aqueous solution, 60% ethanol aqueous solution, 0.05wt%-0.2wt% sodium hydroxide aqueous solution, and 4% hydrochloric acid aqueous solution.

[0037] In the present invention, the cleaning effect of Sophora flavescens is ensured by ultrasonic cleaning combined with high and low frequencies, and the components in Sophora flavescens are extracted by extracting liquid after grinding Sophora flavescens, and after filtering out the residue, the content of Sophora flavescens alkaloids (oxymatrine and matrine) in the crude extract is first detected by high performance liquid chromatography, and then two extraction columns are made with a certain volume of octadecyl silica gel, and the No. 1 C18 solid phase extraction column controls the flow rate to continuously add the crude extract, and the effluent is qualitatively detected by thin layer chromatography (TLC), and the maximum adsorption amount of a certain volume of octadecyl silica gel is calculated. The No. 2 C18 solid phase extraction column controls the flow rate to add a certain volume of crude extract, and the effluent is subjected to the same high performance liquid chromatography to detect the content of Sophora flavescens alkaloids, and the adsorption rate of matrine and oxymatrine is calculated. Then, the No. 1 C18 solid phase extraction column was eluted with eluents of different properties, and the eluents were subjected to the same high performance liquid chromatography to detect the content of matrine alkaloids, thereby calculating the resolution rate of matrine and oxymatrine, and the eluents collected respectively contained purified low-impurity and high-purity matrine and oxymatrine.

[0038] According to the differences in process parameters during the separation and purification process, the novel, efficient and green method for separating and purifying Sophora flavescens alkaloids provided by the present invention is further described through the following specific examples.

[0039] Example 1

[0040] An efficient and green method for separating and purifying Sophora flavescens alkaloids comprises the following steps:

[0041] S1. Select a batch of Sophora flavescens with complete and plump epidermis, soak the Sophora flavescens in distilled water, perform ultrasonic cleaning and then air-dry. During ultrasonic cleaning, perform ultrasonic cleaning on the Sophora flavescens twice, first with a low-frequency ultrasonic cleaning at a frequency of 37KHz for 25 minutes, remove the Sophora flavescens from the sewage, and soak the Sophora flavescens in clean distilled water and perform high-frequency ultrasonic cleaning at a frequency of 70KHz for 15 minutes. After cleaning, remove the Sophora flavescens and air-dry.

[0042] S2, grinding Sophora flavescens into Sophora flavescens powder with a powder fineness of 95 mesh, then mixing the Sophora flavescens powder with a volume concentration of 65% ethanol aqueous solution extract at a solid-liquid ratio of 1g:10ml, heating and stirring at 85°C, condensing and refluxing for 2h, and standing for 70min, filtering out the Sophora flavescens powder residue through a Buchner funnel, and the filtration accuracy of the qualitative filter paper used is 100μm; obtaining a crude extract;

[0043] S3. Detect the total concentration of matrine and oxymatrine in the crude extract by high performance liquid chromatography, and the detection conditions are set according to the above method; accurately weigh 60 ml of octadecyl silica gel, and wet pack it into a column according to the above method: finally make two 30 ml C18 solid phase extraction columns; respectively, C18 solid phase extraction column No. 1 and C18 solid phase extraction column No. 2;

[0044] Then, the crude extract was continuously added to the No. 1 C18 solid phase extraction column at a flow rate of 3BV / h, the effluent was collected, and a thin layer chromatography (TLC) qualitative test was performed according to the above method; 3BV of the crude extract was added to the No. 2 C18 solid phase extraction column at a flow rate of 2BV / h to collect the effluent 1; 2BV of deionized water was used to rinse, and the effluent 2 was collected, and the collected effluent 1 and effluent 2 were combined, and the content of matrine and oxymatrine was detected according to the same high performance liquid chromatography method as above;

[0045] Multiple groups of No. 1 C18 solid phase extraction columns and No. 2 C18 solid phase extraction columns were treated separately according to the same method as above for subsequent experiments;

[0046] S4, using 5BV 25ml of 3.3wt% hydrochloric acid-volume concentration 20% ethanol aqueous solution and 3BV 25ml of 3.3wt% hydrochloric acid-volume concentration 60% ethanol aqueous solution to elute the No. 1 C18 solid phase extraction column in step S3 at a flow rate of 2BV / h, collecting the eluates respectively, and measuring the volume of each eluate respectively, using the same high performance liquid chromatography method to detect the content of matrine alkaloids in the eluates respectively, and the collected eluates contain purified low-impurity high-purity matrine and oxymatrine.

[0047] Example 2

[0048] An efficient and green method for separating and purifying Sophora flavescens alkaloids comprises the following steps:

[0049] S1, select a batch of Sophora flavescens with complete and full epidermis, and soak the Sophora flavescens in distilled water and then perform ultrasonic cleaning and then dry. During ultrasonic cleaning, the Sophora flavescens is ultrasonically cleaned twice, first with a frequency of 37KHz low-frequency ultrasonic cleaning for 25min, then the Sophora flavescens is fished out from the sewage, and the Sophora flavescens is soaked in clean distilled water and then subjected to a frequency of 70KHz high-frequency ultrasonic cleaning for 15min, and after completing the cleaning, the Sophora flavescens is fished out and dried;

[0050] S2, grinding Sophora flavescens into Sophora flavescens powder with a powder fineness of 95 mesh, then mixing the Sophora flavescens powder with a volume concentration of 65% ethanol aqueous solution extract at a solid-liquid ratio of 1g:10ml, heating to 85°C with stirring, condensing and refluxing for 2h, and standing for 70min, filtering out the Sophora flavescens powder residue through a Buchner funnel, and the filtration accuracy of the qualitative filter paper used is 100μm; obtaining a crude extract;

[0051] S3. Measure the total concentration of matrine and oxymatrine in the crude extract by high performance liquid chromatography in the same manner, accurately weigh 60 ml of octadecyl silica gel, wet-pack the column in the same manner, and prepare two 30 ml C18 solid phase extraction columns, namely C18 solid phase extraction column No. 1 and C18 solid phase extraction column No. 2; continuously add the crude extract to C18 solid phase extraction column No. 1 at a flow rate of 3 BV / h, collect the effluent, and then perform a qualitative test by thin layer chromatography (TLC); add 3 BV of the crude extract to C18 solid phase extraction column No. 2 at 2 BV / h to collect effluent 1; rinse with 2 BV of deionized water, collect effluent 2, and combine the collected effluent 1 and effluent 2, and perform a content test of matrine alkaloids by the same high performance liquid chromatography;

[0052] Treat multiple groups of No. 1 C18 solid phase extraction columns and No. 2 C18 solid phase extraction columns respectively according to the same method as above;

[0053] S4. Elute the No. 1 C18 solid phase extraction column in step S3 with 5BV 15ml of 20% ethanol aqueous solution and 4.5BV 15ml of 60% ethanol aqueous solution at a flow rate of 3BV / h, collect the eluates, measure the volume of each eluate, and detect the content of matrine alkaloids in the eluates by the same high performance liquid chromatography. The eluates collected contain purified low-impurity and high-purity matrine and oxymatrine.

[0054] Example 3

[0055] An efficient and green method for separating and purifying Sophora flavescens alkaloids comprises the following steps:

[0056] S1, select a batch of Sophora flavescens with complete and full epidermis, and soak the Sophora flavescens in distilled water and then perform ultrasonic cleaning and then dry. During ultrasonic cleaning, the Sophora flavescens is ultrasonically cleaned twice, and the Sophora flavescens is first removed from the sewage after low-frequency ultrasonic cleaning, and the Sophora flavescens is soaked in clean distilled water and then subjected to high-frequency ultrasonic cleaning. After cleaning, the Sophora flavescens is removed and dried; wherein, the frequency during low-frequency ultrasonic cleaning is 37KHz, and the time is 25min; the frequency during high-frequency ultrasonic cleaning is 70KHz, and the time is 15min;

[0057] S2, grinding Sophora flavescens into Sophora flavescens powder with a powder fineness of 95 mesh, then mixing the Sophora flavescens powder with a volume concentration of 65% ethanol aqueous solution extract at a solid-liquid ratio of 1g:10ml, heating and stirring at 85°C, condensing and refluxing for 2h, and standing for 70min, filtering out the Sophora flavescens powder residue through a Buchner funnel, and the filtration accuracy of the qualitative filter paper used is 100μm; obtaining a crude extract;

[0058] S3, using the same high performance liquid chromatography method as above to measure the content of Sophora flavescens alkaloids in the crude extract, accurately weighing 60 ml of octadecyl silica gel, and wet-packing the column using the same method as above to prepare two 30 ml C18 solid phase extraction columns, namely C18 solid phase extraction column No. 1 and C18 solid phase extraction column No. 2;

[0059] The crude extract was continuously added to the No. 1 C18 solid phase extraction column at a flow rate of 3 BV / h, the effluent was collected, and then a thin layer chromatography (TLC) qualitative test was performed, and 3 BV / h of the crude extract was added to the No. 2 C18 solid phase extraction column at a flow rate of 2 BV / h to collect the effluent 1; the effluent was rinsed with 2 BV of deionized water, and the effluent 2 was collected, and the collected effluent 1 and effluent 2 were combined, and the content of Sophora flavescens alkaloids was detected according to the same high performance liquid chromatography method;

[0060] Treat multiple groups of No. 1 C18 solid phase extraction columns and No. 2 C18 solid phase extraction columns respectively according to the same method as above;

[0061] S4, using 6BV 40ml of 0.15wt% sodium hydroxide aqueous solution and 1BV 40ml of 4% hydrochloric acid aqueous solution to elute the No. 1 C18 solid phase extraction column in step S3 at a flow rate of 4BV / h, collecting the eluates respectively, and measuring the volume of each eluate respectively, using the high performance liquid chromatography in S3 to detect the content of matrine alkaloids in the eluates respectively, and the eluates collected respectively contain the purified low-impurity high-purity matrine and oxymatrine.

[0062] The present invention adopts high performance liquid chromatography to measure the contents of matrine and oxymatrine in the crude extract, and the measured content of matrine is 1.44803 mg / ml, and the content of oxymatrine is 0.88681 mg / ml.

[0063] The present invention uses two eluents to elute the same C18 solid phase extraction column to elute purified low-impurity high-purity matrine and oxymatrine respectively. Since matrine and oxymatrine have different effects and can be effectively separated, they are described separately.

[0064] The following Table 1 shows the resolution of matrine in Examples 1-3.

[0065] Table 1 Resolution of matrine in Examples 1-3

[0066]

[0067] As shown in Table 1, when the eluent volume is different, the eluent type for eluting matrine is different, and the eluent elution flow rate is different, the resolution rate of matrine is also different. When 15ml of 20% ethanol is used as the eluent for eluting matrine, and the flow rate is 3BV / h, the resolution rate of matrine is the largest, which is 85.936%; when 25ml of 3.3% acid water-20% ethanol is used as the eluent for eluting matrine, and the flow rate is 2BV / h, the resolution rate of matrine is second, which is 72.478%; when 40ml of 0.15% sodium hydroxide aqueous solution is used as the eluent for eluting matrine, and the flow rate is 4BV / h, the resolution rate of matrine is the smallest, which is 62.319%; therefore, it can be obtained from the comparison of the above table that when 15ml of 20% ethanol is used as the eluent, and the flow rate is 3BV / h, the resolution rate of matrine is the largest, reaching 85.936%.

[0068] As shown in Table 1, the HPLC method measured the matrine concentration in the crude extract after passing through the column to be 0.25387 mg / ml (Example 1), 0.19202 mg / ml (Example 2), and 0.32358 mg / ml (Example 3), and the matrine concentration in the eluate to be 1.64821 mg / ml (Example 1), 3.14676 mg / ml (Example 2), and 0.93888 mg / ml (Example 3). The following Table 2 shows the resolution of oxidized matrine in Examples 1-3.

[0069] Table 2 Resolution of oxymatrine in Examples 1-3

[0070]

[0071] It can be seen from Table 2 that when the eluent volume used is different, the eluent type used to elute oxymatrine is different, and the elution flow rate of the eluent is different, the resolution of oxymatrine is also different. When 15 ml of 60% ethanol was used as the eluent for eluting oxidized matrine and the flow rate was 3BV / h, the resolution rate of oxidized matrine was the highest, which was 84.367%; when 25 ml of 3.3% acid water-60% ethanol was used as the eluent for eluting oxidized matrine and the flow rate was 2BV / h, the resolution rate of oxidized matrine was the second highest, which was 76.289%; when 40 ml of 4% acid water was used as the eluent for eluting oxidized matrine and the flow rate was 4BV / h, the resolution rate of oxidized matrine was the lowest, which was 60.523%; therefore, from the comparison in the above table, it can be concluded that when 15 ml of 60% ethanol was used as the eluent and the flow rate was 3BV / h, the resolution rate of oxidized matrine was the highest, reaching 84.367%.

[0072] As can be seen from Table 2, the HPLC method measured the concentration of oxidized matrine in the crude extract after passing through the column to be 0.26571 mg / ml (Example 1), 0.19792 mg / ml (Example 2), and 0.34587 mg / ml (Example 3), and the HPLC method measured the concentration of matrine in the eluate to be 0.92751 mg / ml (Example 1), 1.72417 mg / ml (Example 2), and 0.45845 mg / ml (Example 3).

[0073] According to the experimental method in the literature "Study on the Separation Properties of Matrine Using 7 Macroporous Adsorption Resins", the maximum adsorption capacity of D101 macroporous resin for matrine and oxymatrine under static conditions, adsorption rate and resolution rate under dynamic conditions were measured. Table 3 shows the maximum adsorption capacity of C18 solid phase extraction column and D101 macroporous resin for matrine under static conditions.

[0074] Table 3 Maximum adsorption of matrine under static conditions by C18 solid phase extraction column and D101 macroporous resin

[0075]

[0076] The formula for calculating the maximum adsorption amount of matrine is: the maximum adsorption volume of crude extract × the concentration of matrine

[0077] As shown in Table 3, when the filling volume is the same, both are 30ml, under static conditions, the C18 solid phase extraction column made of octadecyl silica gel can adsorb a maximum crude extract volume of 86.5ml, and the maximum adsorption amount of matrine is 125.255mg; the chromatography column made of D101 macroporous resin can adsorb a maximum crude extract volume of 70.4ml, and the maximum adsorption amount of matrine is 101.941mg; the comparison between the two shows that when the filling volume is the same, under static conditions, the maximum adsorption amount of matrine by the C18 solid phase extraction column is greater than that by the chromatography column made of D101 macroporous resin. Table 4 shows the maximum adsorption amount of oxidized matrine by the C18 solid phase extraction column and D101 macroporous resin under static conditions.

[0078] Table 4 Maximum adsorption of oxymatrine under static conditions by C18 solid phase extraction column and D101 macroporous resin

[0079]

[0080] As shown in Table 4, when the filling volume is the same, both are 30ml, under static conditions, the C18 solid phase extraction column made of octadecyl silica gel can adsorb a maximum crude extract volume of 86.5ml, and the maximum adsorption of oxymatrine is 76.709mg; the chromatography column made of D101 macroporous resin can adsorb a maximum crude extract volume of 70.4ml, and the maximum adsorption of oxymatrine is 62.431mg; the comparison between the two shows that when the filling volume is the same, under static conditions, the maximum adsorption of oxymatrine by the C18 solid phase extraction column is greater than that by the chromatography column made of D101 macroporous resin. Table 5 shows the adsorption rate of matrine under dynamic conditions by the C18 solid phase extraction column and D101 macroporous resin.

[0081] Table 5 Adsorption rate of matrine under dynamic conditions by C18 solid phase extraction column and D101 macroporous resin

[0082]

[0083] It can be seen from Table 5 that when the filling volume is the same, both are 30 ml, the filling materials are different, and the flow rate of the crude extract through the column is different, the C18 solid phase extraction column made of octadecyl silica gel passes 60 ml of the crude extract through the column at a flow rate of 2BV / h, and 52 ml of the effluent is collected. The adsorption rate of matrine by the C18 solid phase extraction column is 88.507%; when the chromatography column made of D101 macroporous resin passes 60 ml of the crude extract through the column at a flow rate of 3BV / h, and 49 ml of the effluent is collected, the adsorption rate of matrine by the D101 macroporous resin is 74.365%; by comparing the two, it can be obtained that when the filling volume is the same, the filling materials are different, and the flow rate of the crude extract through the column is different, under dynamic conditions, the adsorption rate of matrine by the C18 solid phase extraction column is greater than that by the chromatography column made of D101 macroporous resin.

[0084] High performance liquid chromatography measured that the concentration of matrine in the effluent after the crude extract was filtered through a C18 solid phase extraction column was 0.19202 mg / ml, and the concentration of matrine in the effluent after the crude extract was adsorbed by a macroporous resin was 0.45453 mg / ml.

[0085] Table 6 shows the adsorption rate of oxymatrine under dynamic conditions by C18 solid phase extraction column and D101 macroporous resin.

[0086] Table 6 Adsorption rate of oxymatrine under dynamic conditions by C18 solid phase extraction column and D101 macroporous resin

[0087]

[0088] It can be seen from Table 6 that when the filling volume is the same, both are 30 ml, the filling materials are different, and the flow rate of the crude extract through the column is different, the C18 solid phase extraction column made of octadecyl silica gel passes 60 ml of the crude extract through the column at a flow rate of 2BV / h, and when 52 ml of the effluent is collected, the adsorption rate of oxymatrine by the C18 solid phase extraction column is 80.657%; when the chromatography column made of D101 macroporous resin passes 60 ml of the crude extract through the column at a flow rate of 3BV / h, and when 49 ml of the effluent is collected, the adsorption rate of oxymatrine by the D101 macroporous resin is 72.964%; by comparing the two, it can be seen that when the filling volume is the same, the filling materials are different, and the flow rate of the crude extract through the column is different, under dynamic conditions, the adsorption rate of oxymatrine by the C18 solid phase extraction column is greater than that by the chromatography column made of D101 macroporous resin.

[0089] The results of high performance liquid chromatography showed that the concentration of oxymatrine in the effluent of the crude extract after filtration through a C18 solid phase extraction column was 0.19792 mg / ml, and the concentration of oxymatrine in the effluent of the crude extract after adsorption through a macroporous resin was 0.29358 mg / ml.

[0090] Table 7 shows the resolution of matrine under dynamic conditions using C18 solid phase extraction column and D101 macroporous resin.

[0091] Table 7 Resolution of matrine under dynamic conditions by C18 solid phase extraction column and D101 macroporous resin

[0092]

[0093] It can be seen from Table 7 that when the filling volume is the same, all 30 ml, the filling materials are different, the flow rate of the crude extract through the column is different, and the flow rate of the eluent through the column is different, the C18 solid phase extraction column made of octadecyl silica gel passes 60 ml of the crude extract through the column at a flow rate of 2BV / h, and 52 ml of the effluent is collected, and then the eluent is passed through the column at a flow rate of 3BV / h to obtain 21 ml of the eluent, and the resolution rate of matrine by the C18 solid phase extraction column is 85.936%; when the chromatography column made of D101 macroporous resin passes 60 ml of the crude extract through the column at a flow rate of 3BV / h, 49 ml of the effluent is collected, and then the eluent is passed through the column at a flow rate of 2BV / h to obtain 32.4 ml of the eluent, the resolution rate of matrine by the D101 macroporous resin is 72.341%.

[0094] High performance liquid chromatography measured that the concentration of matrine in the crude extract after passing through a C18 solid phase extraction column was 0.19202 mg / ml, and the concentration of matrine in the eluate was 3.14676 mg / ml; the concentration of matrine in the crude extract after passing through a macroporous resin adsorption was 0.45453 mg / ml, and the concentration of matrine in the eluate was 1.44257 mg / ml.

[0095] Table 8 shows the resolution of oxymatrine under dynamic conditions using C18 solid phase extraction column and D101 macroporous resin.

[0096] Table 8 Resolution of oxymatrine under dynamic conditions by C18 solid phase extraction column and D101 macroporous resin

[0097]

[0098] It can be seen from Table 8 that when the filling volume is the same, both are 30 ml, the filling materials are different, the flow rate of the crude extract through the column is different, and the flow rate of the eluent through the column is different, the C18 solid phase extraction column made of octadecyl silica gel passes 60 ml of the crude extract through the column at a flow rate of 2BV / h, and 52 ml of the effluent is collected, and then the eluent is passed through the column at a flow rate of 3BV / h to obtain 21 ml of the eluent, and the resolution rate of the C18 solid phase extraction column for matrine is 84.367%; when the chromatography made of D101 macroporous resin passes 60 ml of the crude extract through the column at a flow rate of 3BV / h, 49 ml of the effluent is collected, and then the eluent is passed through the column at a flow rate of 2BV / h to obtain 32.4 ml of the eluent, the resolution rate of D101 macroporous resin for matrine is 76.518%.

[0099] High performance liquid chromatography measured that the concentration of oxymatrine in the crude extract after passing through a C18 solid phase extraction column was 0.19792 mg / ml, and the concentration of oxymatrine in the eluate was 1.72417 mg / ml; the concentration of oxymatrine in the crude extract after passing through a macroporous resin adsorption was 0.29358 mg / ml, and the concentration of oxymatrine in the eluate was 0.91687 mg / ml.

[0100] From the comparison of Tables 3 to 8 above, it can be seen that compared with the macroporous adsorption resin method, the solid phase extraction technology is higher than the macroporous adsorption resin method in terms of the maximum adsorption amount of matrine and oxymatrine under static conditions, as well as the adsorption rate and resolution rate of matrine and oxymatrine under dynamic conditions. The eluate collected by the solid phase extraction method is not only green and safe, but also can efficiently separate matrine and oxymatrine from matrine alkaloids to obtain low-impurity and high-purity matrine and oxymatrine solutions.

[0101] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An efficient and green method for separating and purifying Sophora flavescens alkaloids, characterized in that: The highly efficient and green method for separating and purifying Sophora flavescens alkaloids comprises the following steps: 1) mixing the sophora flavescens powder and the extract, heating and stirring to condense and reflux, standing, filtering out the sophora flavescens powder residue, and obtaining a crude extract; 2) extracting the crude extract using a C18 solid phase extraction column, and then eluting with an eluent to obtain matrine and oxymatrine, respectively.

2. The method for separating and purifying the efficient and green Sophora flavescens alkaloids according to claim 1, characterized in that: In step 1), the preparation method of the sophora flavescens powder is as follows: soaking the sophora flavescens with intact and plump epidermis in distilled water, performing ultrasonic cleaning, and drying; and then grinding into the sophora flavescens powder.

3. The method for separating and purifying the efficient and green Sophora flavescens alkaloids according to claim 2, characterized in that: Ultrasonic cleaning includes low-frequency ultrasonic cleaning and high-frequency ultrasonic cleaning; the frequency range of the low-frequency ultrasonic cleaning is 33KHz-40KHz, and the frequency range of the high-frequency ultrasonic cleaning is 60KHz-80KHz.

4. The method for separating and purifying the efficient and green Sophora flavescens alkaloids according to claim 1, characterized in that: In step 1), the extract is an ethanol aqueous solution with a volume concentration of 65%.

5. The method for separating and purifying the efficient and green Sophora flavescens alkaloids according to claim 1 or 4, characterized in that: In step 1), the solid-liquid ratio of Sophora flavescens powder to the extract is 1 g:10 ml.

6. According to the efficient and green method for separating and purifying Sophora flavescens alkaloids as described in claim 1 or 4, in step 1), heating, stirring, condensing and reflux means that the heating temperature ranges from 80°C to 90°C.

7. The method for separating and purifying the highly efficient and green Sophora flavescens alkaloids according to claim 1, characterized in that: In step 2), the specific method of extracting the crude extract with the C18 solid phase extraction column is: continuously adding the crude extract to the C18 solid phase extraction column at a flow rate of 3BV / h.

8. The method for separating and purifying the highly efficient and green Sophora flavescens alkaloids according to claim 1, characterized in that: In step 2), eluting with an eluent means eluting the C18 solid phase extraction column with an eluent at a flow rate of 2-4 BV / h; collecting the obtained eluent to obtain matrine and oxymatrine.

9. The method for separating and purifying the efficient and green Sophora flavescens alkaloids according to claim 1, characterized in that: The eluent is an aqueous solution of 3.3wt% hydrochloric acid-20% ethanol by volume concentration and an aqueous solution of 3.3wt% hydrochloric acid-60% ethanol by volume concentration; or, the eluent is an aqueous solution of 20% ethanol by volume concentration and an aqueous solution of 60% ethanol by volume concentration; or, the eluent is an aqueous solution of 0.05wt%-0.2wt% sodium hydroxide and an aqueous solution of 4% hydrochloric acid.

10. The efficient and green method for separating and purifying Sophora flavescens alkaloids according to claim 9, characterized in that: The eluent is a 20% by volume ethanol aqueous solution and a 60% by volume ethanol aqueous solution, and the flow rate is 3 BV / h.

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