CN105311117B

CN105311117B - Method for removing bicolor from rhizoma corydalis decumbentis total alkaloids, product and application

Info

Publication number: CN105311117B
Application number: CN201410379747.2A
Authority: CN
Inventors: 杨义芳; 谢欣辛; 孙百玲
Original assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Current assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Priority date: 2014-08-04
Filing date: 2014-08-04
Publication date: 2020-08-21
Anticipated expiration: 2034-08-04
Also published as: CN105311117A

Abstract

The invention discloses a method for removing bicolor in total alkaloids of corydalis amabilis, a product and application. The invention provides a method for removing bicolor from total alkaloids of corydalis decumbens, which comprises the following steps: step (1): in a solvent, carrying out hydrolysis reaction on a supercritical extract of corydalis amabilis and alkali to obtain corydalis amabilis total alkaloids; step (2): separating the total alkaloids of corydalis amabilis obtained in step (1) by high-speed centrifugal partition chromatography to obtain total alkaloids of corydalis amabilis from which bicuculline is removed; the separation is carried out in a high-speed centrifugal partition chromatograph. The method for removing the bicuculline in the total alkaloids of the decumbent corydalis tuber has the advantages of simple steps, short time and high removal rate, is suitable for large-scale industrial production, and the obtained total alkaloids of the decumbent corydalis tuber has low content of the bicuculline and is suitable for clinical application.

Description

Method for removing bicolor from rhizoma corydalis decumbentis total alkaloids, product and application

Technical Field

The invention relates to a method for removing bicolor in total alkaloids of corydalis amabilis, a product and application.

Background

Cerebrovascular diseases are very serious health and social problems, have the characteristics of high morbidity, high disability rate, high recurrence rate, high mortality rate, high medical expense ratio and the like, and still show an ascending trend. Cerebrovascular disease is the third leading cause of death following tumor and heart disease in the european and american countries; in china, cerebrovascular disease is the second leading cause of death following tumors. Cerebrovascular disease is represented by stroke. About 80% of cerebral apoplexy belongs to ischemic cerebral apoplexy, which means that cerebral blood supply is obstructed, or arterial blood perfusion is reduced, or blood supply is stopped completely due to sudden blood flow interruption, so that cerebral tissue ischemia and hypoxic necrosis are caused, and irreversible damage is caused to local cerebral tissue.

With the intensive research on acute cerebral apoplexy, the importance of antioxidant therapy in the treatment of acute ischemic cerebral apoplexy is more and more concerned.

Corydalis decumbens is tuber of Corydalis decumbens (Thunb.) pers of Corydalis of Papaveraceae. Also named as YILIANJINDAN, YEYANHU, DONGLIANSHENXIAN, FUDIYANGSHU, and SHUIZHU. The medicine (Yili gold pill) is recorded as early as Qing dynasty Zhao Zhimin Ben Cao gang mu Shi Yi (supplement to compendium of materia Medica): "one kind of cave-in immortal, another name of wild Yanghu, Jiangnan Huilai peony".

The decumbent corydalis tuber plants grow on hills or low mountain slopes and grasslands, and are favored to grow on the sand texture with warm and humid climate, sunny exposure, good drainage and deep soil layer. Mainly produced in Jiangxi province, Hunan province, Fujian province, Zhejiang province, Anhui province and the like, and the Jiangxi province is the best in terms of production quality. Rhizoma corydalis Decumbentis is a common folk Chinese medicine, has analgesic and antiinflammatory effects, and can be used for treating stomach ache, cancer pain, ischialgia, rheumarthritis, and sequela of poliomyelitis. Recent pharmacological research finds that rhizoma corydalis Decumbentis shows good curative effect on cardiovascular and cerebrovascular diseases, such as treating senile dementia, resisting arrhythmia, inhibiting platelet aggregation, etc.

The active ingredient of rhizoma corydalis Decumbentis is alkaloid. Alkaloid is also used as qualitative and quantitative index of rhizoma corydalis Decumbentis in the section of Chinese pharmacopoeia 2010 edition.

The decumbent corydalis tuber total alkaloids can inhibit cerebral thrombosis, and the protective effect of the decumbent corydalis tuber total alkaloids on neuronal cell injury caused by cerebral ischemia/reperfusion is also proved exactly. Meanwhile, the total alkaloids of corydalis amabilis can obviously increase the myocardial trophism blood flow volume and can obviously improve the hypoxia tolerance of mice by increasing the blood and oxygen supply of important organs such as heart, brain and the like. The multidirectional and multi-target cerebral apoplexy treatment effect provides a solid theoretical and experimental support for developing decumbent corydalis tuber total alkaloids into a traditional Chinese medicine innovative medicine for treating cerebral apoplexy.

However, the convulsion-causing toxicity of the total alkaloids of corydalis amabilis is not beneficial to expanding the dosage range in clinical use. Bifunoline, also known as dicentrine, is a well-established convulsant in total alkaloids of corydalis amabilis. The screening shows that the convulsion medicine has a strong convulsion effect and has the characteristics of quick action and short duration compared with a classical convulsion agent. And the current research proves that the bicuculline damages neuron cells through various ways in the convulsion-causing process, and the improvement of the tolerance and resistance of the neurons to ischemia and hypoxia in the treatment of cerebral apoplexy is one of the treatment means. Reducing the content of the bicuculline in the total alkaloids of the decumbent corydalis is an effective way for improving the safety of the total alkaloids of the decumbent corydalis and improving the neuroprotective effect of the decumbent corydalis.

The subject group has established a supercritical extraction process of rhizoma corydalis decumbentis total alkaloids, and can quickly and efficiently obtain rhizoma corydalis decumbentis total alkaloids extract with high alkaloid content. On the basis of the work, a convenient and easy process method is established, the content of the Bifunoline in the total alkaloid of the corydalis amabilis is reduced, the total alkaloid of the corydalis amabilis with reduced toxicity and basically unchanged protective effect on neurons is obtained, and the technical problem which needs to be solved at present is urgently solved.

Disclosure of Invention

The invention aims to overcome the defects that the content of bicuculline in the total alkaloids of corydalis amabilis is high, and the total alkaloids of corydalis amabilis are not suitable for clinical application and industrial production, and provides a method for removing bicuculline in the total alkaloids of corydalis amabilis, a product and application thereof. The invention applies the new FCPC technology to the removal of the Bifunoline from the total alkaloids of the corydalis amabilis for the first time, and successfully carries out the amplification experiment of the FCPC technology on the obtained process, so that the obtained product has high removal rate and good repeatability. The method for removing the bicuculline in the total alkaloids of the decumbent corydalis tuber has the advantages of simple steps, easy operation, short time and high removal rate, is suitable for large-scale industrial production, and the obtained total alkaloids of the decumbent corydalis tuber has low content of the bicuculline and is suitable for clinical application.

The supercritical extraction method of corydalis amabilis has high extraction rate of total alkaloids, is energy-saving and environment-friendly, but brings a large amount of volatile oil and fatty acid in the extraction process, so that the water solubility of the extract is poor, the extract is difficult to dry completely, and the storage and the application of the subsequent preparation development are not facilitated.

The invention provides a method for removing bicolor in total alkaloids of corydalis decumbens, which comprises the following steps:

step (1): in a solvent, carrying out hydrolysis reaction on a supercritical extract of corydalis amabilis and alkali to obtain total alkaloids of corydalis amabilis;

step (2): separating the total alkaloids of corydalis amabilis obtained in step (1) by high-speed centrifugal partition chromatography to obtain total alkaloids of corydalis amabilis from which bicuculline is removed; the separation is carried out in a high-speed centrifugal partition chromatograph.

The bicuculline in the supercritical extract of corydalis amabilis can be converted into the carboxylic salt of the bicuculline in the step (1), so that the total alkaloid of corydalis amabilis containing the carboxylic salt of the bicuculline is obtained.

In the step (1), the supercritical extract of corydalis amabilis can be obtained by adopting a supercritical extraction method of corydalis amabilis reported in patent CN101058576A, or can be prepared by a first method.

The first method comprises the following steps:

step 1: pulverizing rhizoma corydalis Decumbentis (corydalis decumbens (lour.) Merr. of Papaveraceae), and alkalifying to obtain alkalified rhizoma corydalis Decumbentis; in the alkalization, the mass ratio of the alkali to the corydalis amabilis is 0.001-1, preferably 0.001-0.1.

Step 2: the alkalized corydalis amabilis obtained in the step 1 is put in supercritical CO₂Extracting and separating in an extractor coupled with membrane separation, and drying to obtain rhizoma corydalis Decumbentis total alkaloid extract.

In the step 1, the rhizoma corydalis Decumbentis (corydalis decumbens (lour.) Merr) is pulverized preferably until the particle size of rhizoma corydalis Decumbentis (corydalis decumbens (lour.) Merr) of Papaveraceae is 10-60 mesh (i.e. 1.74-0.273 mm). The alkalization is preferably performed by using an inorganic base, the inorganic base is preferably one or more of an ammonia water solution, calcium hydroxide, sodium carbonate and sodium bicarbonate, and is further preferably an ammonia water solution, the mass concentration of the ammonia water solution is preferably 1% -10%, and the mass concentration refers to the mass percentage of ammonia gas in the total mass of the ammonia water solution. The ammonia water solution can be obtained by mixing an ammonia water reagent and water, the volume ratio of the ammonia water reagent to the water is preferably 5-10%, and the volume ratio refers to the percentage of the volume of the ammonia water reagent to the volume of the water. The ammonia water reagent can be a conventional commercial ammonia water reagent in the field, and the concentration of the ammonia water reagent is preferably 2-30%. The inorganic base is preferably used in the form of an aqueous solution thereof, the concentration of the aqueous solution of the inorganic base is preferably 1-10%, and the concentration refers to the percentage of the mass of the inorganic base in the total mass of the aqueous solution of the inorganic base.

In step 2, the supercritical CO₂The time for extraction and separation is preferably 0.5-5 h; the supercritical CO₂The entrainer adopted during extraction and separation is preferably methanol and/or ethanol, and further preferably ethanol, the ethanol is preferably ethanol water solution with the mass percentage of 95%, and the mass percentage refers to the mass percentage of the ethanol in the total mass of the ethanol water solution. Said "supercritical CO₂The membrane in the extraction instrument coupled with the membrane separation is preferably an ultrafiltration membrane, a nanofiltration membrane, a reverse osmosis membrane or a hollow fiber membrane, and is further preferably a nanofiltration membrane; the aperture of the nanofiltration membrane is preferably 1-3 nanometers. The aperture of the ultrafiltration membrane is preferably 10-40 nm. The aperture of the reverse osmosis membrane is preferably 1-5 nm. The pore diameter of the hollow fiber membrane is preferably 1 to 40 nanometers.

In step 2, the supercritical CO₂The extraction apparatus coupled with extraction and membrane separation is assembled according to a schematic diagram recorded in the book of New technology for extracting and separating traditional Chinese medicine (4 months in 2010, published by chemical industry publishers, P353), and the supercritical CO is used for extracting and separating the traditional Chinese medicine₂The extraction apparatus coupled with the membrane separation comprises the following components: an extraction device, a separation device, a high-pressure pump, a membrane device, a cooler and a gas storage tank.

In the present invention, the method for preparing total alkaloid extract of decumbent corydalis tuber preferably further comprises step 3, more preferably further comprises step 3 and step 4, and still more preferably further comprises step 3, step 4 and step 5.

And step 3: and (3) adjusting the pH value of the corydalis total alkaloid extract obtained in the step (2) to 2-5, extracting by adopting ultrasonic waves, and then filtering by adopting an ultrafiltration or microfiltration membrane to obtain a purified solution of the corydalis total alkaloid extract.

And 4, step 4: adjusting the pH value of the solution of the total alkaloid extract of corydalis decumbens obtained in the step 3 to 8-12, filtering, and washing a solid phase with water to be neutral to obtain a further purified total alkaloid extract of corydalis decumbens;

and 5: recrystallizing the further purified total alkaloid extract of corydalis decumbens obtained in the step 4 to obtain the recrystallized total alkaloid extract of corydalis decumbens.

In the step 3, the frequency of the ultrasonic extraction is preferably 30 Hz-80 Hz, and the time of the ultrasonic extraction is preferably 0.5-3 hours. The aperture of the ultrafiltration or microfiltration membrane is preferably 0.3-0.8 micron; the pH is preferably adjusted by using an acid, the acid is preferably an inorganic acid and/or an organic acid, the inorganic acid is preferably hydrochloric acid, and the organic acid is preferably tartaric acid. The acid is preferably used in the form of an aqueous solution thereof, and when the acid is used in the form of an aqueous solution thereof, the mass percentage concentration of the aqueous solution of the acid is preferably 1% to 10%, and more preferably 1% to 5%, and the mass percentage refers to the percentage of the mass of the acid to the total mass of the aqueous solution of the acid. The volume ratio of the aqueous acid solution to the extract of the total alkaloids of corydalis decumbens obtained in step 2 is preferably 5-10, and more preferably 5-8. Step 3 preferably comprises a drying step, wherein the purified solution of the total alkaloid extract of corydalis decumbens is dried to obtain a purified total alkaloid extract of corydalis decumbens; the drying may be carried out according to methods and conditions conventional in the art for such procedures.

In the step 4, the pH is preferably 9-11. The pH is preferably adjusted by using an inorganic base, the inorganic base is preferably sodium hydroxide or potassium hydroxide, the inorganic base can be used in the form of an aqueous solution of the inorganic base, when the inorganic base is used in the form of an aqueous solution of the inorganic base, the mass concentration of the aqueous solution of the inorganic base is preferably 3% to 15%, more preferably 4% to 10%, and the mass concentration refers to the percentage of the mass of the inorganic base to the total mass of the aqueous solution of the inorganic base. Step 4 preferably comprises a drying step, which may be carried out according to methods and conditions conventional in the art for such procedures.

In step 5, said recrystallization can be carried out by methods and conditions conventional in the art for such procedures. The solvent adopted by recrystallization is preferably an alcohol solvent and/or a halogenated hydrocarbon solvent, the alcohol solvent is preferably methanol and/or ethanol, the halogenated hydrocarbon solvent is preferably a chlorinated hydrocarbon solvent, and the chlorinated hydrocarbon solvent is preferably chloroform. Step 5 preferably comprises a drying step, which may be carried out according to methods and conditions conventional in the art for such procedures.

The corydalis amabilis total alkaloid extract prepared by the first method comprises the following components in percentage by weight: protopine: 15% -40%; tetrahydropalmatine: 10% -40%; buckling device: 0 to 10% (excluding 0); palmatine hydrochloride: 0.01 to 2 percent; violin: 2% -10%; xiawuning alkali: 15% -30%; other alkaloids and extracts: 1 to 5 percent.

In the corydalis decumbens total alkaloid extract, the content of total alkaloid is 60 to 100 percent by HPLC method, wherein the total content of six major alkaloids accounts for more than 95 percent of the corydalis decumbens total alkaloid extract.

In the step (1), if the supercritical extract of corydalis amabilis is obtained by the supercritical extraction method of corydalis amabilis reported in patent CN101058576A, the mass percentage content of the total alkaloid of corydalis amabilis is 50-100%, and the mass percentage content refers to the mass percentage of the total alkaloid in the supercritical extract of corydalis amabilis to the mass of the supercritical extract of corydalis amabilis. The main alkaloids in the rhizoma corydalis Decumbentis total alkaloids have the following composition ratio: 15 to 35 percent of protopine, 10 to 25 percent of tetrahydropalmatine, 5 to 15 percent of Bixatole, 0.05 to 0.2 percent of palmatine hydrochloride, 5 to 15 percent of corydalmine, 15 to 30 percent of xiaolingnine and 10 to 25 percent of other alkaloids and extracts.

In the step (1), the solvent can adopt a conventional solvent for the reaction hydrolysis reaction in the field, and in the invention, an alcohol solvent and/or a ketone solvent are particularly preferred, and the alcohol solvent is preferably methanol and/or ethanol, and is further preferably ethanol; the ketone solvent is preferably acetone.

In the step (1), the mass-to-volume ratio of the supercritical extract of corydalis amabilis to the solvent is preferably 0.002 g/mL-0.2 g/mL, and more preferably 0.02 g/mL-0.01 g/mL.

In the step (1), the base may be a conventional base for such hydrolysis reaction in the art, and in the present invention, an inorganic base is particularly preferred, the inorganic base is preferably sodium hydroxide or potassium hydroxide, the inorganic base preferably participates in the reaction in the form of an aqueous solution thereof, the molar volume concentration of the aqueous solution of the inorganic base is preferably 0.1mol/L to 2mol/L, and more preferably 0.5mol/L to 0.8mol/L, and the molar volume concentration refers to the ratio of the number of moles of the inorganic base to the volume of the aqueous solution of the inorganic base.

In the step (1), the molar ratio of the alkali to the supercritical extract of corydalis amabilis is preferably 1: 3-1: 6.

in step (1), the temperature of the hydrolysis reaction may be a temperature conventional in the art, and in the present invention, 20 ℃ to 60 ℃ is particularly preferred, and 20 ℃ to 30 ℃ is further preferred.

In the step (1), the progress of the hydrolysis reaction can be monitored by a conventional monitoring method (such as HPLC) of the reaction in the field, the time when the biconazole disappears is taken as the end point of the reaction, and the reaction time is preferably 1 h-2 h.

In step (2), the high-speed centrifugal partition chromatograph may be a high-speed centrifugal partition chromatograph conventional in the art, preferably a high-speed centrifugal partition chromatograph manufactured by Rousselet Robatel of france and having a model number of FCPC a or FCPC C.

In the step (2), the rotating speed of the high-speed centrifugal distribution chromatograph is preferably 1200 rpm/min-2000 rpm/min.

In the step (2), the flow rate of the high-speed centrifugal distribution chromatograph is preferably 2.0 mL/min-6.0 mL/min; the flow rate refers to the flow rate of the mixed solution formed by the supercritical extract of corydalis amabilis and the solvent system in the high-speed centrifugal distribution chromatograph.

In the step (2), the solvent system of the high-speed centrifugal partition chromatography preferably adopts a mixed solvent of ethyl acetate and water or a mixed solvent of chloroform and water; the volume ratio of ethyl acetate to water in the mixed solvent of ethyl acetate and water is preferably 1: 1-5: 1; the volume ratio of chloroform to water in the mixed solvent of chloroform and water is preferably 1: 1-5: 1.

in the step (2), the sample loading concentration of the high-speed centrifugal distribution chromatograph is preferably 20 mg/mL-120 mg/mL; the sample loading concentration refers to the ratio of the mass of the supercritical extract of corydalis amabilis to the volume of the solvent system.

In the method for removing bicolor from total alkaloids of corydalis decumbens, the removal rate of bicolor is more than 98%, and the recovery rate of other alkalis is as high as more than 90%.

In the present invention, the step (2) preferably comprises the following specific steps:

the method comprises the following steps: fully mixing the solvent system, and respectively taking an upper phase and a lower phase;

step two: removing the betuline carboxylate in the total alkaloids of corydalis decumbens obtained in the step (1) by taking the lower phase as a stationary phase and the upper phase as a mobile phase;

step three: collecting rhizoma corydalis Decumbentis total alkaloids.

In the third step, the collected sample is preferably collected at 282nm detection wavelength.

In the third step, the solvent of the collected and prepared sample is preferably removed to obtain the total alkaloid of corydalis decumbens, the solvent removal is preferably carried out under the condition of reduced pressure, and the pressure of the solvent removal is preferably-0.06 Mpa to-0.1 Mpa; the temperature for removing the solvent is preferably 30 to 60 ℃.

The invention also provides the decumbent corydalis tuber total alkaloid obtained by the method for removing the bicolor in the decumbent corydalis tuber total alkaloid.

The invention also provides application of the corydalis total alkaloids obtained by the method for removing the bicolor in the corydalis total alkaloids in preparing a medicine for treating and/or preventing diseases of neuronal cell injury caused by cerebral ischemia or reperfusion.

In the present invention, the diseases of neuronal cell damage caused by cerebral ischemia or reperfusion include cerebral thrombosis and cerebral apoplexy.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the invention optimizes the process of removing the Bifunoline in the rhizoma corydalis decumbentis total alkaloids by the FCPC method, determines reasonable process parameters by combining actual production conditions, aims to provide material basis for the subsequent activity research of the rhizoma corydalis decumbentis alkaloids, and simultaneously makes the industrialized preparation of the bulk drug of the rhizoma corydalis decumbentis total alkaloids with low toxicity and high activity possible. The technique "FCPC" used in the present invention refers to high-speed centrifugal partition chromatography, which is liquid-liquid partition chromatography. The method for removing the bicolor in the total alkaloid of the decumbent corydalis tuber has the advantages of simple steps, short time and high removal rate, and the obtained total alkaloid of the decumbent corydalis tuber has low content of the bicolor, thereby being suitable for clinical application and large-scale industrial production.

Drawings

FIG. 1 is an FCPC (high speed centrifugation partition chromatography) chromatogram of decumbent corydalis tuber total alkaloid with bicuculine removed in example 4.

FIG. 2 is an HPLC (high performance liquid chromatography) chromatogram of five alkaloid (Bifuuling, xiawuning alkali, Protoline, tetrahydropalmatine and palmatine) reference substances in rhizoma corydalis total alkaloids.

FIG. 3 is HPLC (high performance liquid chromatography) spectrum of supercritical extract of rhizoma corydalis Decumbentis.

FIG. 4 is a HPLC (high performance liquid chromatography) chromatogram of decumbent corydalis total alkaloid with bicuculline removed obtained in example 4.

Fig. 5 is a graph of the effect of XTW-SFE and XTW-SFE-RB on LDH release rate from PC12 cells, wherein P < 0.05 and P < 0.01, compared to model group.

Fig. 6 is a graph of the effect of XTW-SFE and XTW-SFE-RB on the apoptosis rate of PC12, wherein P < 0.05 and P < 0.01 compared to model group.

FIG. 7 is a graph showing the effect of XTW-SFE and XTW-SFE-RB on the viability (%) of PC12 cells, wherein P < 0.05 and P < 0.01 in comparison with the model group and P < 0.05 and P # 0.01 in comparison with the negative group.

FIG. 8 shows CO₂A schematic diagram of an extraction process coupling supercritical extraction and membrane separation.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Raw materials and reagents

The supercritical extraction instrument HA121-50-01-C (Jiangsu Nantong Huaan supercritical Co., Ltd.), Agilent HP1260 type high performance liquid chromatograph, quaternary gradient pump and HP cheme chromatographic workstation. MSU224S-100-Duz balance (Sartorius); high speed centrifugal partition chromatography (FCPC) (Kromaton, Rousselet Robatel); a SHIMDZU LC-10AD pump; HD-21-2 ultraviolet detector (Shanghai Jiapeng science and technology, Inc.); SHIMDZU-7725i sample injector.

Preparation of total alkaloids from corydalis decumbens used in the following examples 1 to 13:

taking dried tubers of corydalis amabilis, and crushing into medicinal material powder of 20-100 meshes; taking a certain amount of medicinal powder, adding ammonia water, and stirring until the pH is 8-10; putting the medicinal materials into an extraction kettle, extracting under the extraction pressure of 30 MPa-50 MPa when the extraction temperature is 40-75 ℃, the temperature of a resolving kettle I is 30-70 ℃ and the temperature of a resolving kettle II is 30-70 ℃, continuously adding ethanol as an entrainer in the extraction process, and adding the ethanol at the flow rate of 260 mL/h; and stopping extracting after 2-3 hours, and obtaining the total alkaloid extract from the resolution kettle I, namely the total alkaloid extract of corydalis amabilis. All of the following examples were conducted using the same batch of samples with a bicolor content of 7.81%, a marronine content of 19.21%, a protopine content of 5.43%, a tetrahydropalmatine content of 18.12%, and a palmatine content of 0.12%.

Detection method

Each alkaloid control was quantitatively weighed and compared with mobile phase a (%): b (%) ═ 88: 12 (the% indicates volume percent) and preparing control solutions of the following concentrations of bicolor 0.0532mg/mL, xiaolingnine 0.1202mg/mL, protopine 0.0764mg/mL, tetrahydropalmatine 0.1254mg/mL and palmatine 0.0446 mg/mL. HPLC (high performance liquid chromatography) chromatogram of each standard substance in rhizoma corydalis Decumbentis total alkaloids is shown in FIG. 4; the HPLC (high performance liquid chromatography) spectrum of each alkaloid in supercritical extract of rhizoma corydalis Decumbentis is shown in FIG. 5.

The chromatographic conditions were as follows: a chromatographic column: discovery (5 μm. times.250 mm). The detection conditions are phase A: 8mL of triethylamine and 30mL of glacial acetic acid are added into water to reach the constant volume of 1000 mL; phase B: methanol: acetonitrile 1: 4; the elution mobile phase ratio is shown in table 1. Flow rate: 1 mL/min. Detection wavelength: 282 nm.

Table 1 flow phase ratio example

Time (min)	A(％)	B(％)
			0	88	12
50	88	12
			75	75	25

Example 1

The supercritical total alkaloid extract of corydalis amabilis is prepared into absolute ethyl alcohol solutions with the concentrations of 0.05g/mL, 0.02g/mL, 0.01g/mL and 0.002g/mL respectively. 0.05g/mL supercritical extract solution of corydalis amabilis was found to contain a lot of oily insoluble substances, and could not be dissolved completely under the condition of heat and ultrasonic sound. Taking 20mL of each concentration solution, respectively adding 5-10 mL of 0.5mol/LNaOH aqueous solution into 0.05g/mL, 0.02g/mL, 0.01g/mL and 0.002g/mL of decumbent corydalis tuber supercritical extract absolute ethyl alcohol solution, and stirring and standing for overnight color. The next day, drying under reduced pressure, adding 25mL of double distilled water respectively, and ultrasonically mixing uniformly. The mixture was transferred to a separatory funnel at room temperature, and extracted 3 times with 25mL of ethyl acetate until colorless. After spin-drying, 0.05g of the suspension was dissolved in a mobile phase and the volume was adjusted to 25mL, and the solution was injected into a chromatograph to analyze the results.

At a concentration of 0.05g/mL, the removal rate of biconazole was lower than the remaining concentrations, probably due to incomplete dissolution. The removal rates of the remaining concentrations were not significantly different.

Example 2

3 parts of 0.5g supercritical total alkaloid extract of corydalis decumbens are respectively dissolved in 25mL absolute ethyl alcohol, and after complete dissolution, 0.1mol/L NaOH, 0.5mol/L NaOH and 0.8mol/L NaOH are respectively added to 5 mL-10 mL. Standing overnight, drying under reduced pressure, adding double distilled water, and ultrasonically mixing. The mixture was transferred to a separatory funnel at room temperature and extracted 3 times with an equal amount of ethyl acetate until colorless. After spin-drying, the mixture was injected into a chromatograph for analysis. When NaOH is about 0.5 mol/L-0.8 mol/L, the reaction can be completely carried out, and the specific data are shown in Table 2.

TABLE 2 influence of alkali concentration on bicolor removal

Different alkali concentrations (mol/L)	Sample content	0.10	0.50	0.80	1.5	2.0
							Bixiaokouling (%)	7.81	5.21	0.78	0.81	0.83	0.86

Example 3

Taking 3 parts of 0.5g decumbent corydalis tuber supercritical total alkaloid extract, respectively dissolving in 25mL of absolute ethyl alcohol, respectively adding 5 mL-10 mL of 0.5mol/NaOH after complete dissolution, reacting at normal temperature for 0.5h, 1h, 2h, carrying out rotary evaporation drying, adding double-distilled water, and carrying out ultrasonic mixing. The mixture was transferred to a separatory funnel at room temperature and extracted 3 times with an equal amount of ethyl acetate until colorless. After spin-drying, the mixture was injected into a chromatograph for analysis. When the reaction time is longer than 1h, the content of the bicuculline is lower, the reaction is more complete, and the specific data are shown in Table 3.

TABLE 3 influence of hydrolysis reaction time on bicolor removal

Different reaction times	Sample content	0.5h	1h	2h
					Bixiaokouling (%)	7.81	4.33	0.75	0.67

Example 4

3 parts of 0.5g supercritical total alkaloid extract of corydalis decumbens is taken and respectively dissolved in 25mL absolute ethyl alcohol, after complete dissolution, 5 mL-10 mL0.5mol/NaOH is respectively added, and the mixture is respectively placed in a water bath kettle at 20 ℃, 40 ℃ and 60 ℃ for reaction for 1 h. Rotary steaming and drying, adding double distilled water, and ultrasonically mixing. The mixture was transferred to a separatory funnel at room temperature and extracted 3 times with an equal amount of ethyl acetate until colorless. After spin-drying, the mixture was injected into a chromatograph for analysis. The reaction can be completed by the Bifunoline at the normal temperature of 20 ℃, but the recovery rates of other alkalis are slightly different at different temperatures, and the specific data are shown in Table 4.

TABLE 4 influence of temperature of hydrolysis reaction on bicolor removal

Different reaction temperatures	Sample content		20℃	40℃	60℃
						Bixiaokouling (%)	7.81	0.77	0.63	0.65

Example 5

Respectively dissolving 3 parts of 0.5g decumbent corydalis tuber supercritical total alkaloid extract in 25mL of absolute ethyl alcohol, respectively adding 5 mL-10 mL of 0.5mol/NaOH after complete dissolution, standing at normal temperature for 2h, rotatably evaporating to dryness, adding 25mL of double distilled water, and ultrasonically mixing uniformly. The mixture was transferred to a separatory funnel at room temperature, and group A was extracted with 50mL of ethyl acetate for the first time and 25mL for the second and third times, respectively. Group B was extracted a first time with 37.5mL of ethyl acetate and a second and third time with 25mL each. Group C was extracted a first time with 25mL of ethyl acetate and a second and third time with 25mL each. Each group was separately weighed by spin drying, diluted to volume, injected into chromatograph, and analyzed by HPLC. The extract obtained by the first extraction has the highest alkaloid content, the more extraction times are, and the salt in the water phase is reduced back to the buckling-restrained form probably due to reversible reaction. Taking the content of the Bifunoline, the content of other alkalis and the requirement of industrial production into consideration, extracting twice with an organic solvent with the volume 2 times of that of water and an organic solvent with the volume 1 time of that of water respectively, and combining the organic solvents.

Example 6

Since the esterification reaction is a reversible reaction and there may be residual NaOH in the ethyl acetate, the effect of the addition of the water-washing extract was investigated.

Weighing 0.5g of sample, respectively dissolving in 25mL of absolute ethyl alcohol, respectively adding 5 mL-10 mL of 0.5mol/NaOH after complete dissolution, standing at normal temperature for 2h, evaporating to dryness, adding 25mL of double distilled water, and ultrasonically mixing uniformly. The mixture was transferred to a separatory funnel at room temperature and extracted a first time with 50mL of ethyl acetate and a second time with 25mL of ethyl acetate. Combining the two extraction solutions, dividing into two parts with equal volume, and directly evaporating one part to dryness under reduced pressure to obtain a sample. Adding the other part into a separating funnel, adding isogenic double distilled water, uniformly mixing, standing for layering, taking an organic layer, and evaporating to dryness under reduced pressure to obtain a sample, wherein the specific data are shown in Table 5.

TABLE 5 influence of extraction on bicolor removal

Content (wt.)	No water washing	With water washing	Recovery rate	No water washing	With water washing
						Bixiaokouling (%)	0.71	0.47	Bixiaokouling (%)	7.62	3.19

Example 7

0.5g of a sample was weighed, dissolved in 25mL of absolute ethanol, and added with 8mL of 0.5mol/L aqueous sodium hydroxide solution, followed by reaction at room temperature for 1 hour. After completion of the reaction, the reaction mixture was evaporated to dryness under reduced pressure, and the residue was mixed with 25mL of water. The mixture was transferred to a separatory funnel at room temperature and extracted a first time with 37.5mL of ethyl acetate and a second time with 25 mL. Mixing the two extractive solutions, transferring to a separating funnel, adding water with equal volume, mixing, standing for layering, collecting organic layer, and evaporating to dryness under reduced pressure to obtain rhizoma corydalis Decumbentis total alkaloids with buckling eliminated. The removal rate of the Bifunoline is 99.8%, the recovery rate of other alkalis is more than 92.5%, the content of the total alkaloid reaches 99%, wherein, the xiawuning alkali is 34.2%, the protopine is 20.1%, the tetrahydropalmatine is 43.5%, and the palmatine hydrochloride is 1.5%. The extract is dried to be powder, so that the water solubility is increased, and the bioavailability is greatly improved.

Example 8

0.5g of a sample was weighed, dissolved in 25mL of absolute ethanol, and 4mL of a 0.5mol/L aqueous solution of sodium hydroxide was added, followed by reaction at 30 ℃ for two hours. After the reaction is completed, the reaction solution is evaporated to dryness under reduced pressure, and the residue is mixed with water in the volume of the anhydrous ethanol solution of the supercritical extract of corydalis amabilis. The mixture was transferred to a separatory funnel at room temperature and extracted a first time with 50mL of ethyl acetate and a second time with 25 mL. Mixing the two extractive solutions, transferring to a separating funnel, adding water with equal volume, mixing, standing for layering, collecting organic layer, and evaporating to dryness under reduced pressure to obtain rhizoma corydalis Decumbentis total alkaloids with buckling eliminated. The removal rate of the Bifunoline is 99.2 percent, the recovery rate of other alkalis is more than 92.8 percent, the content of the total alkaloid reaches 99 percent, wherein, the xiawuning alkali is 33.8 percent, the protopine is 20.5 percent, the tetrahydropalmatine is 43.7 percent, and the palmatine hydrochloride is 1.4 percent. The extract is dried to be powder, so that the water solubility is increased, and the bioavailability is greatly improved.

Example 9

0.5g of a sample was weighed, dissolved in 25mL of absolute ethanol, and 4mL of a 0.5mol/L aqueous solution of sodium hydroxide was added, followed by reaction at room temperature for two hours. After the reaction is completed, the reaction solution is evaporated to dryness under reduced pressure, and the residue is mixed with water in the volume of the anhydrous ethanol solution of the supercritical extract of corydalis amabilis. The mixture was transferred to a separatory funnel at room temperature and extracted a first time with 50mL of ethyl acetate and a second time with 25 mL. Mixing the two extractive solutions, transferring to a separating funnel, adding water with equal volume, mixing, standing for layering, collecting organic layer, and evaporating to dryness under reduced pressure to obtain rhizoma corydalis Decumbentis total alkaloids with buckling eliminated. The removal rate of the Bifunoling is 98.9%, the recovery rate of the rest alkalis is more than 93.5%, the content of the total alkaloid reaches 99%, wherein, the xiawuning alkali is 33.7%, the protopine is 20.9%, the tetrahydropalmatine is 43.3%, and the palmatine hydrochloride is 1.6%. The extract is dried to be powder, so that the water solubility is increased, and the bioavailability is greatly improved.

Example 10

Taking 100g of supercritical extract of corydalis amabilis, adding 5L of absolute ethanol to prepare 0.02g/mL of absolute ethanol solution, adding 1.5L of 0.8mol/L of sodium hydroxide aqueous solution, and reacting for two hours at 30 ℃. After completion of the reaction, the reaction mixture was evaporated to dryness under reduced pressure, and the residue was mixed with 5L of water. The mixture was transferred to a separatory funnel at room temperature and extracted a first time with 7.5L of ethyl acetate and a second time with 5L of ethanol. Mixing the two extractive solutions, transferring to a separating funnel, adding water with equal volume, mixing, standing for layering, collecting organic layer, and evaporating to dryness under reduced pressure to obtain rhizoma corydalis Decumbentis total alkaloids with buckling eliminated. The removal rate of the bicolor is 99.3 percent, and the recovery rate of other alkalis is more than 94.5 percent. The experiment is an amplification experiment, and proves that under the amplification condition, the experimental result of the method is not influenced by the amplification condition, the removal rate is still high, and the experimental effect is good. The HPLC chart is shown in FIG. 6.

Comparative example 1

The method disclosed in patent CN102475743A is adopted to prepare 4g of the same batch of samples by the method, the total alkaloid extract of rhizoma corydalis Decumbentis is dissolved in 10mL of DMSO, then is mixed with 1mL of NaOH solution of 4g/mL, and the mixed solution is clarified. The mixture was magnetically stirred at 35 ℃ for 30 minutes and transferred to a 250mL separatory funnel to which was added 80mL of HCl₃Adding 80mL of water, shaking, standing for layering, and separating out lower CHCl layer₃. Add 80mL of CHCl again to the separatory funnel₃Shaking, standing, and separating lower layer CHCl₃This operation is repeated once. Combine 3 CHCl₃Extracting, recovering solvent under reduced pressure to obtain extract 2.0g, and collecting CHCl in the process₃The emulsion is easy to generate, the standing time is long, the emulsion breaking is not easy, the sample loss caused by an emulsion layer is large, the recovery rate is high, and the removal rate of the biconazole in the extract is found to be 85 percent through HPLC analysis and is lower than the removal rate of the biconazole in the extract which can be more than 98 percent. In addition, the experiment can only be carried out in a small test, the emulsification phenomenon is more serious in a large-scale pilot test, the recovery rate of the total alkaloids of corydalis amabilis is lower than 40 percent, and the requirement of industrialization on removing the bicuculline sample cannot be met.

For the following examples 11 to 14 preparation of supercritical extract of corydalis amabilis:

taking dried tubers of corydalis amabilis, and crushing into medicinal material powder of 20-100 meshes; taking a certain amount of medicinal powder, adding ammonia water, and stirring until the pH is 8-10; putting the medicinal materials into an extraction kettle, extracting at the extraction temperature of 40-75 ℃, the temperature of a resolving kettle I of 30-70 ℃, the temperature of a resolving kettle II of 30-70 ℃ and the extraction pressure of 30-50 MPa, continuously adding ethanol as an entrainer in the extraction process, and adding the ethanol at the flow rate of 260 mL/h; and stopping extracting after 2-3 hours, and taking the rhizoma corydalis decumbentis total alkaloid extract obtained from the analysis kettle I as a sample for further research. The total alkaloids of corydalis amabilis used in all the following examples are the same batch of samples, and the content of each alkali is as follows: 9.26 percent of Bifunoline, 14.79 percent of Xiajonine, 21.49 percent of procatropin, 23.18 percent of tetrahydropalmatine and 0.09 percent of palmatine.

Sample pretreatment

Adding 1mol/L sodium hydroxide solution into supercritical extract of summer substance for reaction, and removing solvent from the reaction solution under-0.1 MPa and 50 deg.C under reduced pressure to obtain sample. HPLC (high performance liquid chromatography) chromatogram of five alkaloids (Bifujiaoling, xiawuning, Pritopin, tetrahydropalmatine and palmatine) in rhizoma corydalis total alkaloids is shown in figure 2; the supercritical fluid extract HPLC (high performance liquid chromatography) of rhizoma corydalis Decumbentis is shown in figure 3.

Detection method

Each alkaloid control was quantitatively weighed and compared with mobile phase a (%): b (%) ═ 88: 12 (the% indicates volume percentage) and preparing control solutions with the following concentrations of bicolor 0.0532mg/mL, xiaolingnin 0.1202mg/mL, procaine 0.0764mg/mL, tetrahydropalmatine 0.1254mg/mL and palmatine 0.0446 mg/mL.

The chromatographic conditions were as follows: a chromatographic column: discovery (5 μm. times.250 mm). The detection conditions are phase A: 8mL of triethylamine and 30mL of glacial acetic acid are added into water to reach the constant volume of 1000 mL; phase B: methanol: acetonitrile 1: 4; the elution ratios are shown in Table 1. Flow rate: 1 mL/min. Detection wavelength: 282 nm.

Table 1 flow phase ratio example

Time (min)	A(％)	B(％)
			0	88	12
50	88	12
			75	75	25

FCPC operation method

Adding the solvents into a separating funnel according to the proportion, violently shaking to fully mix the solvents, after balancing, respectively taking an upper phase and a lower phase, and degassing for 30 minutes by ultrasound before use. In the Ascending mode, the FCPC column was filled with the stationary phase (lower phase) at a low rpm and high flow rate (8 ml/min, 600 rpm). After the stationary phase is filled, pumping at a flow rate of 8ml/min and a rotation speed of 600rpm for 2min to remove bubbles in a desending mode and an centering mode respectively, adjusting parameters to the required flow rate and rotation speed to start pumping the mobile phase until the two phases are balanced, and then injecting the sample.

Ascending mode: the lower phase with higher density is used as stationary phase, and the upper phase with lower density is used as mobile phase.

Desending mode: the upper phase with lower density is used as the stationary phase, and the upper phase with higher density is used as the mobile phase.

Example 15

Selecting three selected factors of 20mg/mL, 50mg/mL, 80mg/mL, 1200r/min, 1600r/min, 2000r/min of rotation speed, 1mL/min, 2mL/min and 3mL/min of flow rate to carry out orthogonal L by taking the biculine removal rate as an evaluation index on the alkalized supercritical corydalis amabilis alkaloid extract₉3³And (5) carrying out experiments. The influence on the bicolor removal rate is as follows: rotational speed>Flow rate of flow>And (5) sample injection concentration.

Example 16

Selecting chloroform: the water volume ratio is 2: the system 1 is a separation system, various solvents are respectively added into a separating funnel according to the proportion, the solvents are violently shaken and fully mixed, and after balance, an upper phase and a lower phase are respectively taken. In the Ascending mode, the FCPC column was filled with the lower phase (stationary phase, 64mL) at a flow rate of 8.0mL/min at 600 rpm; the mode was changed and 64mL of the lower phase was charged at 600rpm with a flow rate of 8.0mL/min in Despending mode. Dissolving the alkalized supercritical rhizoma corydalis Decumbentis alkaloid extract with upper phase or lower phase to obtain sample solution with concentration of 50mg/mL, injecting sample, and separating Bishaoling at 1600r/min and 3mL/min under instrument parameters. Under the condition, the removal rate of the bicolor reaches 98.42 percent, the recovery rate of other alkalis reaches more than 90 percent, and the content of total alkaloids reaches 99 percent. The extract is dried to be powder, so that the water solubility is increased, and the bioavailability is greatly improved.

Example 17

Selecting chloroform: the volume ratio of water is 1: the system 1 is a separation system, various solvents are respectively added into a separating funnel according to the proportion, the solvents are violently shaken and fully mixed, and after balance, an upper phase and a lower phase are respectively taken. In the Ascending mode, the FCPC column was filled with the lower phase (stationary phase, 64mL) at a flow rate of 8.0mL/min at 600 rpm; the mode was changed and 64mL of the lower phase was charged at 600rpm with a flow rate of 8.0mL/min in Despending mode. Dissolving the alkalized supercritical rhizoma corydalis Decumbentis alkaloid extract with upper phase or lower phase to obtain sample solution with concentration of 80mg/mL, injecting sample, and separating Bishaoling at 1200r/min and 2mL/min under instrument parameters. Under the condition, the removal rate of the bicolor reaches 98.29 percent, the recovery rate of other alkalis reaches more than 90 percent, and the content of total alkaloids reaches 99 percent. The extract is dried to be powder, so that the water solubility is increased, and the bioavailability is greatly improved.

Example 18

Selecting ethyl acetate: water 3: the system 1 is a separation system, various solvents are respectively added into a separating funnel according to the proportion, the solvents are violently shaken and fully mixed, and after balance, an upper phase and a lower phase are respectively taken. In the Ascending mode, the FCPC column was filled with the lower phase (stationary phase, 64mL) at a flow rate of 8.0mL/min at 600 rpm; the mode was changed and 64mL of the lower phase was charged at 600rpm with a flow rate of 8.0mL/min in Despending mode. Dissolving the alkalized supercritical rhizoma corydalis Decumbentis alkaloid extract with upper phase or lower phase to obtain sample solution with concentration of 30mg/mL, injecting sample, and separating Bishaoling at 1600r/min and 4mL/min under instrument parameters. Under the condition, the removal rate of the bicolor reaches 98.94 percent, the recovery rate of other alkalis reaches more than 90 percent, and the content of total alkaloids reaches 99 percent. The extract is dried to be powder, so that the water solubility is increased, and the bioavailability is greatly improved. The FCPC is shown in FIG. 1, and the HPLC is shown in FIG. 4.

Example 19

For the optimal procedure, the loading was scaled up linearly according to the FCPC drum volume (50ml vs. 200 ml). Three replicates were performed at 320mg, a flow rate of 8mL/min, and a rotation rate of 600 mL/min. After FCPC separation, about 260mg of total alkaloid of corydalis amabilis with BI can be obtained, and the removal rate of bicolor is more than 98% in three repeated experiments. The recovery rate of each of the other alkalis is more than 90 percent.

Effect example 1 half Lethal Dose (LD)₅₀) Test of

Respectively dissolving the bicuculline, the supercritical extract (XTW-SFE) of the decumbent corydalis tuber and the supercritical extract (XTW-SFE-RB) of the decumbent corydalis tuber without the bicuculline with absolute ethanol, mixing with absolute ethanol solution of hydroxypropyl-beta-cyclodextrin (HP- β -CD), stirring, and finally decompressing and recovering the solvent to obtain powdery water-soluble alkaloid inclusion compound, cleaning grade Kunming mouse, wherein each dose of 10 mice is half female and half male, the weight is 18-22 g, (administration) is carried outThe food was fasted for 16h before, and water was freely drunk. Dissolving a sample in water with equal volume (10mL/kg) and performing disposable gavage at different concentrations. Symptoms of intoxication and death were observed and recorded immediately after dosing for 7 days and LD was calculated₅₀、LD₅₀95% mean confidence limit (SLD)₅₀). Obtaining the LD of XTW-SFE, bicuculine and XTW-SFE-RB₅₀100.5mg/kg, 18.6mg/kg and 422.1mg/kg, SLD, respectively₅₀Respectively 86.9 mg/kg-116.5 mg/kg, 17.1 mg/kg-20.3 mg/kg and 369.9 mg/kg-480.9 mg/kg, as shown in Table 2.

TABLE 2 test results of supercritical extract of corydalis amabilis (XTW-SFE), Bifunoline and LD50 and SLD50 from which the supercritical extract of corydalis amabilis (XTW-SFE-RB) was removed

The results prove that the bicuculline is the main component causing convulsion, and the toxicity of the supercritical corydalis amabilis extract without the bicuculline is greatly reduced, thereby ensuring the safety of the corydalis amabilis as clinical medication.

Effect example 2

With H₂O₂Induced cell injury of rat adrenal pheochromocytoma cell (PC12) is a model, and XTW-SFE-RB samples are observed for H by measuring cell Lactate Dehydrogenase (LDH) release rate, MTT and apoptosis rate₂O₂Causing protection of PC12 cells from injury.

The cells were digested, counted and formulated to a concentration of 1 × 10⁵Cell suspension/mL, 2mL cell suspension per well of cell culture plate (1 × 10 per well)⁵Individual cells); the cell culture plate was placed at 37 ℃ in 5% CO₂Culturing in an incubator for 24 hours; diluting the test sample with complete medium to desired concentration, adding 500 μ L of corresponding drug-containing medium to each well, 37 deg.C, 5% CO₂Incubating for 1h in the incubator; dilution of H with complete Medium₂O₂And when the concentration is 25 mu mol/L, 500 mu L of corresponding drug-containing culture medium is added into each hole, and a negative control group and a model control group are simultaneously established. LDH (layered double hydroxide) based on Nanjing Kai-based biotechnologyAnd (3) measuring by using a company Limited LDH kit method. The measurement wavelength was 440 nm. The results of the experiment are shown in figure 5 (P < 0.05, P < 0.01 compared to model group). FIG. 5 analysis of results: as can be seen from FIG. 5, H is added₂O₂And then, compared with a negative control group, the LDH leakage amount in the cell culture solution of the model group is obviously increased, which indicates that the cell membrane is seriously damaged, a large amount of LDH is leaked, and the model building is successful. The XTW-SFE3.125 and 12.5mg/mL solubility groups can obviously reduce the LDH release rate of the cell PC12, and have obvious difference compared with the model control group, P<0.01. Compared with the model control group, the three concentration groups of XTW-SFE-RB have the following advantages that the LDH release rate is obviously reduced: 0.78125mg/mL (P)<0.05)；3.125、12.5mg/mL(P<0.01). The LDH release rate of PC12 in the XTW-SFE-RB12.5mg/mL concentration group is close to that of the negative control group, which shows that the cell membrane damage is light and the function of protecting the cell membrane is excellent.

Culturing the cells in a 37 deg.C incubator for 24 hr, digesting with 0.25% pancreatin (without EDTA) to collect the cells after the drug is used up, washing the cells with PBS buffer solution twice (centrifuging at 2000rpm for 5min), and collecting 5 × 10⁵A cell; adding 500. mu.L Binding Buffer to suspend the cells; adding 5 μ L Annexin V-FITC (Annexin V-fluorescein isothiocyanate), mixing, adding 5 μ L Propidium Iodide, and mixing; reacting for 5-15 min at room temperature in a dark place; apoptosis was detected by flow cytometry (Ex 488 nm; Em 530 nm). The results of the experiment are shown in fig. 6 (P < 0.05, P < 0.01 compared to model group). Fig. 6 analysis of experimental results: comparison of model control group with negative control group, H₂O₂The apoptosis rate of the cell PC12 is obvious, indicating that H₂O₂The induction of the apoptosis of the PC12 cells is successful in modeling. The solubility groups of XTW-SFE0.78125, 3.125 and 12.5mg/mL can obviously reduce the cell apoptosis rate of cell PC12, and the apoptosis rate of the XTW-SFE12.5mg/mL concentration group PC12 is only one third of that of the model control group. Three concentrations of XTW-SFE-RB all can cause the apoptosis rate of PC12 cells to be obviously reduced, and the difference is statistically significant along with the increase of the concentration of XTW-SFE-RB. These results show that XTW-SFE, XTW-SFE-RB can significantly reduce H₂O₂Induced apoptosis rate of PC12 cells. The pH of the PBS buffer solution is 7.4, and the PBS buffer solution is 1LPBS bufferThe preparation method of the solution comprises the following steps: disodium hydrogen phosphate (Na)₂HPO₄)1.44g, potassium dihydrogen phosphate (KH)₂PO₄)0.24g, 8g of sodium chloride (NaCl), 0.2g of potassium chloride (KCl), dissolved in 800mL of distilled water, the pH of the solution adjusted to 7.4 with HCl, and water added to 1000 mL.

Effect example 3MTT method for determining PC12 cell viability

PC12 cells were experimentally grouped, with 6 wells per group and 3-well blanks in parallel. After adding the drugs, the culture was continued for 24h, the supernatant was discarded, 5mg/mL MTT10L was added and the mixture was put into an incubator for 4h, after the culture was terminated, the supernatant was carefully discarded, DMSO100L was added to each well and shaken in a shaker for 10min to terminate the reaction and sufficiently dissolve formazan particles, and then colorimetric was performed with a full-automatic enzyme calibrator at a wavelength of 490nm and each group of OD values was recorded (OD measured as OD real-OD empty). The cell viability of the negative group is recorded as 100%, and the cell viability of the model group and the administration group is the ratio of the OD value to the OD value of the negative group. The experiment was repeated 3 times. The results are shown in figure 7 (P < 0.05, P < 0.01 compared to model group; compared to negative group,^#P＜0.05，^##p is less than 0.01). Fig. 7 analysis of results: three dose groups of XTW-SFE all increased H significantly₂O₂The viability of PC12 cells was impaired (three dose groups P were all less than 0.01). Only two of the three dose groups of XTW-SFE-RB significantly increased H₂O₂The viability of PC12 cells was impaired (3.13g/mL, P < 0.05; 12.50g/mL, P < 0.01).

Supercritical CO used in the following examples₂The extraction and membrane coupled extractor was assembled according to the schematic shown in fig. 8. Examples 20 to 24 are the preparation of an extract of total alkaloids from corydalis decumbens.

Example 20: preparation of total alkaloid extract of corydalis

Crushing dried tubers of corydalis amabilis to obtain medicinal material powder with the granularity of 10-40 meshes; taking 300g of medicine powder, and adding 6% ammonia water in volume ratio as an alkalizer (the volume ratio refers to the percentage of the volume of an ammonia water reagent in the total volume of the ammonia water solution, the mass concentration of the ammonia water reagent is 25% -28%, and the mass concentration refers to the mass of ammonia gas in the total mass of the ammonia water reagentThe amount of the medicinal material is 50 percent of the weight of the corydalis amabilis, and the medicinal material is soaked for 2 hours; adding the medicinal materials into supercritical CO₂Extracting with methanol as entrainer for 30 min in an extraction instrument with membrane coupling (ultrafiltration membrane with pore diameter of 0.8 μm), stopping extraction to obtain total alkaloid extract, and drying. The content of total alkaloids measured by high performance liquid chromatography is 98.47%. The weight percentage composition of the composite material determined by HPLC comprises: protopine: 34.89%, tetrahydropalmatine: 24.56%, buckling: 3.51%, palmatine hydrochloride: 1.84%, xiawuning alkali: 19.77%, violmine: 9.58 percent and the content of other alkalis is 4.32 percent.

Example 21: preparation of total alkaloid extract of corydalis

Taking dried tubers of corydalis amabilis, and crushing to obtain medicinal material powder with the granularity of 20-40 meshes; taking 300g of medicinal material powder, adding 10% by volume of ammonia water as an alkalizer (the volume ratio refers to the percentage of the volume of an ammonia water reagent in the total volume of the ammonia water solution, the mass concentration of the ammonia water reagent is 25-28%, the mass concentration refers to the percentage of the mass of ammonia gas in the total mass of the ammonia water reagent), soaking for 1h, wherein the dosage is 60% of the weight of the corydalis amabilis medicinal material; adding the medicinal materials into supercritical CO₂In an extraction instrument of extraction and membrane coupling (adopting a nanofiltration membrane with the aperture of 2 nanometers), 95% by mass of ethanol is taken as an entrainer (the mass percentage refers to the mass of the ethanol accounting for the total mass of the ethanol water solution), the extraction time is 1.5 hours, and the content of the total alkaloids measured by the high performance liquid chromatography is 98.95%. The weight percentage composition of the composite material determined by HPLC comprises: protopine: 30.52%, tetrahydropalmatine: 28.93%, buckling: 0.01%, palmatine hydrochloride: 0.09%, xiawuning alkali: 25.64%, corydalmine: 9.73 percent and the content of other alkalis is 4.03 percent.

Example 22: method for preparing total alkaloid extract of rhizoma corydalis Decumbentis

Taking dried tubers of corydalis amabilis, and pulverizing to obtain medicinal material powder with the granularity of 10-20 meshes; 200g of the medicine powder is taken, and an ammonia water solution with the volume ratio of 7 percent is added as an alkalizer (the volume ratio refers to the percentage of the volume of the ammonia water reagent to the total volume of the ammonia water solution, and the ammonia waterThe mass concentration of the reagent is 25-28%, the mass concentration refers to the mass percentage of ammonia gas in the total mass of the ammonia water reagent), the dosage is 50% of the weight of the corydalis amabilis medicinal material, and the soaking is carried out for 3 h; adding the medicinal materials into supercritical CO₂In an extraction instrument of extraction and membrane coupling (adopting a hollow fiber membrane with the aperture of 0.1 micrometer), 95% by mass of ethanol is taken as an entrainer (the mass percentage refers to the mass percentage of the ethanol to the total mass of the ethanol water solution), the extraction time is 40 minutes, the obtained total alkaloid is 300mL of 1% hydrochloric acid aqueous solution (the mass percentage refers to the mass percentage of the hydrogen chloride to the total mass of the hydrochloric acid aqueous solution), and ultrasound (the ultrasonic frequency is 80H)_Z) Extracting for 2h, filtering with a 1.0 μm filter membrane, adding 8% by mass of potassium hydroxide solution to adjust pH to 10-11 (the mass percentage refers to the mass percentage of potassium hydroxide in the total mass of potassium hydroxide), filtering to obtain precipitate, and measuring the total alkaloid content to be 98.88% by high performance liquid chromatography. The weight percentage composition of the composite material determined by HPLC comprises: protopine: 35.68%, tetrahydropalmatine: 19.36%, bitloline: 4.64%, palmatine hydrochloride: 0.61%, xiawuning alkali: 28.71%, violamine: 8.75 percent and other alkali content is 1.13 percent.

Example 23: method for preparing total alkaloid extract of rhizoma corydalis Decumbentis

Taking dried tubers of corydalis amabilis, and pulverizing to obtain corydalis amabilis medicinal material powder with the granularity of 20-40 meshes; taking 300g of corydalis amabilis medicinal material powder, adding ammonia water with the volume ratio of 10% as an alkalizer (the volume ratio refers to the percentage of the volume of an ammonia water reagent in the total volume of an ammonia water solution, the mass concentration of the ammonia water reagent is 25% -28%, the mass concentration refers to the percentage of the mass of ammonia gas in the total mass of the ammonia water reagent), soaking for 2.5h, wherein the dosage is 50% of the weight of the corydalis amabilis medicinal material; adding the medicinal materials into supercritical CO₂Extracting for 1 hour by using 95% ethanol as entrainer (the mass percentage refers to the mass percentage of the ethanol in the total mass of the ethanol water solution) in an extractor of extraction and membrane coupling (adopting a composite nanofiltration membrane with the aperture of 1 nanometer). Using 500ml of 1 percent hydrochloric acid aqueous solution by mass percent and carrying out ultrasonic treatment (ultrasonic frequency 60H)_Z) Extracting for 1h, filtering with a 0.45-micrometer filter membrane, adding 10% by mass of sodium hydroxide solution to adjust pH to 9-10 (the mass percentage refers to the mass percentage of potassium hydroxide in the total mass of potassium hydroxide), filtering to obtain precipitate, and measuring the total alkaloid content by high performance liquid chromatography to be 98.68%. The weight percentage composition of the composite material determined by HPLC comprises: protopine: 20.68%, tetrahydropalmatine: 32.16%, buckling: 8.84%, palmatine hydrochloride: 0.50%, xiawuning alkali: 28.71%, violamine: 6.75 percent and other alkali 1.04 percent.

Example 24: method for preparing total alkaloid extract of rhizoma corydalis Decumbentis

Taking dried tubers of corydalis amabilis, and pulverizing to obtain corydalis amabilis medicinal material powder with the granularity of 30-40 meshes; taking 8000g of corydalis amabilis medicinal material powder, adding ammonia water with the volume ratio of 10% as an alkalizer (the volume ratio refers to the percentage of the volume of an ammonia water reagent in the total volume of an ammonia water solution, the mass concentration of the ammonia water reagent is 25-28%, the mass concentration refers to the percentage of the mass of ammonia gas in the total mass of the ammonia water reagent), soaking for 24h, wherein the dosage is 70% of the weight of the corydalis amabilis medicinal material; adding the medicinal materials into supercritical CO₂Extracting with 95% ethanol (the mass percentage is the percentage of the mass of the ethanol in the total mass of the ethanol water solution) in an extractor of extraction and membrane coupling (adopting a reverse osmosis membrane with the aperture of 15 nanometers), extracting for 1h, and drying to obtain the product. Then 300mL of hydrochloric acid aqueous solution with the mass percent of 2% (the mass percent refers to the mass percent of the hydrogen chloride in the total mass of the hydrochloric acid aqueous solution), and ultrasonic treatment (the ultrasonic frequency is 80H)_Z) Extracting for 0.5h, filtering with a filter membrane with a pore diameter of 0.1 micrometer, adding 10% by mass of sodium hydroxide solution to adjust pH to 9-10 (the mass percentage is the mass percentage of sodium hydroxide in the total mass of sodium hydroxide), filtering to obtain precipitate, recrystallizing with methanol, and measuring total alkaloid content to be 99.88% by high performance liquid chromatography. The weight percentage composition of the composite material determined by HPLC comprises: protopine: 29.28%, tetrahydropalmatine: 23.83%, Bifibula: 8.52%, palmatine hydrochloride: 1.90%, xiawuning alkali: 29.71%, corydalmine: 4.62 percent and other alkali 2.02 percent.

Claims

1. A method for removing bicolor from total alkaloids of corydalis amabilis is characterized by comprising the following steps: taking dried tubers of corydalis amabilis, and crushing into medicinal material powder of 20-100 meshes; taking medicine powder, adding ammonia water, and stirring until the pH is 8-10; putting the medicinal materials into an extraction kettle, extracting at the extraction temperature of 40-75 ℃, the temperature of a resolving kettle I of 30-70 ℃, the temperature of a resolving kettle II of 30-70 ℃ and the extraction pressure of 30-50 MPa, continuously adding ethanol as an entrainer in the extraction process, and adding the ethanol at the flow rate of 260 mL/h; stopping extracting after 2-3 hours, and obtaining a rhizoma corydalis decumbentis total alkaloid extract from the resolution kettle I; the total alkaloids of rhizoma corydalis Decumbentis contain the following alkalis: 9.26 percent of Bifenuling, 14.79 percent of Xiajonine, 21.49 percent of procatropin, 23.18 percent of tetrahydropalmatine and 0.09 percent of palmatine;

sample pretreatment: adding 1mol/L sodium hydroxide solution into supercritical extract of rhizoma corydalis Decumbentis for reaction, and removing solvent from the reaction solution under-0.1 MPa at 50 deg.C under reduced pressure to obtain sample;

selecting chloroform: the water volume ratio is 2: the system 1 is a separation system, various solvents are respectively added into a separating funnel according to the proportion, the solvents are violently shaken and fully mixed, and after balance, an upper phase and a lower phase are respectively taken; in the Ascending mode, the FCPC column is filled with a lower phase of 64mL of stationary phase at a flow rate of 8.0mL/min and a rotation speed of 600 rpm; changing the mode, and filling 64mL of lower phase at a flow rate of 8.0mL/min and 600rpm in a Despending mode; dissolving the alkalized supercritical rhizoma corydalis Decumbentis alkaloid extract with upper phase or lower phase to obtain 50mg/mL sample solution, injecting sample, and separating Bishaoling at 1600r/min and 3mL/min under instrument parameters;

wherein, FCPC is a high-speed centrifugal distribution chromatograph; the Ascending mode is as follows: taking a lower phase with higher density as a stationary phase and an upper phase with lower density as a mobile phase; the desending mode is as follows: the upper phase with lower density is used as stationary phase, and the lower phase with higher density is used as mobile phase.

2. A method for removing bicolor from total alkaloids of corydalis amabilis is characterized by comprising the following steps:

taking dried tubers of corydalis amabilis, and crushing into medicinal material powder of 20-100 meshes; taking medicine powder, adding ammonia water, and stirring until the pH is 8-10; putting the medicinal materials into an extraction kettle, extracting at the extraction temperature of 40-75 ℃, the temperature of a resolving kettle I of 30-70 ℃, the temperature of a resolving kettle II of 30-70 ℃ and the extraction pressure of 30-50 MPa, continuously adding ethanol as an entrainer in the extraction process, and adding the ethanol at the flow rate of 260 mL/h; stopping extracting after 2-3 hours, and obtaining a rhizoma corydalis decumbentis total alkaloid extract from the resolution kettle I; the total alkaloids of rhizoma corydalis Decumbentis contain the following alkalis: 9.26 percent of Bifenuling, 14.79 percent of Xiajonine, 21.49 percent of procatropin, 23.18 percent of tetrahydropalmatine and 0.09 percent of palmatine;

selecting chloroform: the volume ratio of water is 1: the system 1 is a separation system, various solvents are respectively added into a separating funnel according to the proportion, the solvents are violently shaken and fully mixed, and after balance, an upper phase and a lower phase are respectively taken; in the Ascending mode, the FCPC column is filled with a lower phase of 64mL of stationary phase at a flow rate of 8.0mL/min and a rotation speed of 600 rpm; changing the mode, and filling 64mL of lower phase at a flow rate of 8.0mL/min and 600rpm in a Despending mode; dissolving the alkalized supercritical rhizoma corydalis Decumbentis alkaloid extract with upper phase or lower phase to obtain sample solution with concentration of 80mg/mL, introducing sample, and separating Bishaoling at 1200r/min and 2mL/min under instrument parameters;

3. A method for removing bicolor from total alkaloids of corydalis amabilis is characterized by comprising the following steps:

selecting ethyl acetate: water 3: the system 1 is a separation system, various solvents are respectively added into a separating funnel according to the proportion, the solvents are violently shaken and fully mixed, and after balance, an upper phase and a lower phase are respectively taken; in the Ascending mode, the FCPC column is filled with a lower phase of 64mL of stationary phase at a flow rate of 8.0mL/min and a rotation speed of 600 rpm; changing the mode, and filling 64mL of lower phase at a flow rate of 8.0mL/min and 600rpm in a Despending mode; dissolving the alkalized supercritical rhizoma corydalis Decumbentis alkaloid extract with upper phase or lower phase to obtain sample solution with concentration of 30mg/mL, injecting sample, and separating Bifenuling at 1600r/min and 4mL/min under instrument parameters;