Method for extracting tetrandrine in stephania tetrandra and application of stephania tetrandra extract

Abstract

The invention discloses a method for extracting tetrandrine in stephania tetrandra, which comprises the following steps: (1) mixing stephania tetrandra and ethanol, extracting, collecting filtrate, and removing ethanol to obtain stephania tetrandra extract; (2) mixing organic matters, inorganic salts and water to obtain an aqueous two-phase system; and (3) adding the stephania tetrandra extract into the aqueous two-phase system, and then carrying out ultrasonic treatment, shaking extraction and centrifugation to obtain an upper-phase system and a lower-phase system which contain tetrandrine. According to the method for extracting the tetrandrine in the stephania tetrandra, an aqueous two-phase extraction method and an ultrasonic technology are combined, the extraction rate of the tetrandrine in the stephania tetrandra is taken as a target, and an extraction and separation process of the tetrandrine in the stephania tetrandra extract is optimized by adopting the aqueous two-phase extraction method. The tetrandrine extracted by the ultrasonic-assisted aqueous two-phase method is high in yield and low in solvent residue. The extract obtained by the method has a good inhibition effect on pancreatic lipase.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine extraction, in particular to a method for extracting tetrandrine from Stephania tetrandra and application of Stephania tetrandra extract. BACKGROUND

[0002] Tetrandrine (Tet), also known as fangchinoline, is a kind of bisbenzylisoquinoline alkaloid extracted from the rhizome of traditional Chinese medicine Stephania tetrandra S. Moore, which is the main effective component of Stephania tetrandra. Tetrandrine has good biological activity in terms of antihypertensive, anti-inflammatory, anti-fibrosis, and anti-cancer. Tetrandrine tablets and tetrandrine injection, which take tetrandrine as the main component, have been widely used in clinical practice. They are used to relieve symptoms such as gastrointestinal reactions and limb numbness caused by chemotherapy drugs. However, there are records of adverse reactions such as nausea, abdominal discomfort, and abnormal defecation, indicating that Stephania tetrandra extract and Tet can affect digestive function. However, there is no detailed report on the relationship between Stephania tetrandra extract and digestive function.

[0003] The excellent efficacy of tetrandrine has led to a significant increase in the demand for tetrandrine. Zhang et al. (Improvement of extraction process of Stephania tetrandra) used acid-base sedimentation method to separate and refine tetrandrine. However, the purity of the target product obtained by this method is low, and the production of waste acid and waste alkali is large. Wei et al. (Study on extraction, separation and purification process of tetrandrine from Stephania tetrandra) used ethanol reflux method to extract Stephania tetrandra extract, and then extracted it with chloroform and other organic solvents. This method requires repeated extraction, has large amount of waste liquid, is complicated, and chloroform is harmful to the human body.

[0004] The information disclosed in this section of the background art is only intended to increase the understanding of the overall background of the present application, and should not be considered as acknowledging or implying in any form that the information constitutes prior art known to those skilled in the art. SUMMARY

[0005] The purpose of the present application is to provide a method for extracting tetrandrine from Stephania tetrandra, so as to overcome the low extraction rate of tetrandrine from Stephania tetrandra in the prior art.

[0006] Another purpose of the present application is to provide a use of Stephania tetrandra extract.

[0007] To achieve the above-mentioned purpose, the present application provides a method for extracting tetrandrine from Stephania tetrandra, comprising the following steps:

[0008] (1) mixing Stephania tetrandra with ethanol for extraction, collecting the filtrate, removing ethanol, and obtaining Stephania tetrandra extract;

[0009] (2) mixing organic matter, inorganic salt and water to obtain a two-water phase system;

[0010] (3) adding the powder of step (1) into the two-water phase system, then extracting by ultrasonic and shaking, centrifuging to obtain an upper and lower phase system, and the upper phase system contains tetrandrine.

[0011] Preferably, in the above technical solution, the liquid-solid ratio mL / g of the powder of step (1) mixed with ethanol is 1-15:1, and the extraction is carried out at 90-120℃ for 0.5-5h, and the extraction is repeated 1-3 times, and the filtrate is collected.

[0012] Preferably, in the above technical solution, the organic matter of step (2) includes a high polymer or an organic small molecule alcohol, the high polymer includes one or more of L-31, L-35, L-44, L-64 and L-65, and the organic small molecule alcohol includes one or more of methanol, ethanol, n-propanol, isopropanol and n-butanol.

[0013] Preferably, in the above technical solution, the inorganic salt of step (2) includes one or more of sodium carbonate, ammonium sulfate, potassium carbonate and dipotassium hydrogen phosphate.

[0014] Preferably, in the above technical solution, the mass ratio of the organic matter, the inorganic salt and water of step (2) is 1-5:1-5:1-8.

[0015] Preferably, in the above technical solution, the solid-liquid ratio g / g of the powder of step (3) mixed with the two-water phase system is 1-4:100.

[0016] Preferably, in the above technical solution, the temperature of step (3) is 30-70℃.

[0017] Preferably, in the above technical solution, the ultrasonic time of step (3) is 15-60min.

[0018] Preferably, in the above technical solution, the extraction time of step (3) is 20-100min.

[0019] The use of the powder of step (3) for preparing a medicine for inhibiting the activity of lipase.

[0020] The principle of the two-water phase extraction technology:

[0021] The two-water phase extraction technology is realized by the selective adaptation of the extract between two different phases. Because the two phases are different in nature, the surface properties and chemical bonds of the extracted substances entering the two-phase medium are different, resulting in different concentrations of the extracted substances in the two phases. By analyzing the concentration ratio of the extracted substances in the two phases, that is, the partition coefficient value, it can be determined that the extracted substances are mainly concentrated in one of the two phases, so as to achieve the process of separation and purification of substances.

[0022] Compared with the prior art, the present application has the following beneficial effects: the method for extracting tetrandrine from Stephania tetrandra S. Moore combines aqueous two-phase extraction with ultrasonic technology, optimizes the extraction and separation process of tetrandrine in Stephania tetrandra S. Moore extract by aqueous two-phase extraction, and takes the extraction rate of tetrandrine in Stephania tetrandra S. Moore as the target. The tetrandrine extracted by the method has high yield and low solvent residue. The extract of Stephania tetrandra S. Moore has good inhibitory effect on pancreatic lipase and can be used for preparing drugs for inhibiting lipase activity. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the HPLC chromatogram of the tetrandrine reference solution and the test sample solution;

[0024] wherein, Figure 1 A: standard chromatogram; B: upper phase chromatogram of the test sample; C: lower phase chromatogram of the test sample (1. tetrandrine 2. tetrandrine), which is a sample prepared by the experiment numbered 1 in Example 11.

[0025] Figure 2 is a column chart of the influence of high polymer species on the extraction rate of tetrandrine;

[0026] Figure 3 is a column chart of the influence of small molecule organic species on the extraction rate of tetrandrine;

[0027] Figure 4 is a column chart of the influence of inorganic salt species on the extraction rate of tetrandrine;

[0028] Figure 5 is a curve chart of the influence of inorganic salt content on the extraction rate of tetrandrine;

[0029] Figure 6 is a curve chart of the influence of organic matter content on the extraction rate of tetrandrine;

[0030] Figure 7 is a curve chart of the influence of solid-liquid ratio on the extraction rate of tetrandrine;

[0031] Figure 8 is a curve chart of the influence of extraction time on the extraction rate of tetrandrine;

[0032] Figure 9 is a curve chart of the influence of extraction temperature on the extraction rate of tetrandrine;

[0033] Figure 10 is a curve chart of the influence of ultrasonic time on the extraction rate of tetrandrine;

[0034] Figure 11 is a curve chart of the inhibitory effect of Stephania tetrandra S. Moore purified liquid on pancreatic lipase.

[0035] Figure 12 This is a Lineweaver-Burk curve of the purified solution of Stephania tetrandra on pancreatic lipase. Detailed Implementation

[0036] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0037] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0038] I. Experimental Section

[0039] 1. Materials and Instruments

[0040] Tetrandrine A (batch number: K902585) and Tetrandrine B (batch number: DST230819) standards, with a content greater than 99.9% as measured by liquid chromatography, were purchased from Shanghai Dibai Biotechnology Co., Ltd.; methanol, ethanol, n-propanol, isopropanol, and n-butanol, analytical grade, were purchased from Guangdong Guanghua Technology Co., Ltd.; potassium carbonate, sodium citrate, dipotassium hydrogen phosphate, ammonium sulfate, sodium carbonate, and potassium dihydrogen phosphate, analytical grade, were purchased from Sinopharm Chemical Reagent Co., Ltd.; L-31 (a copolymer of ethylene oxide (EO) and propylene oxide (PO), molecular weight = 1100), L-35 (a copolymer of EO and PO, molecular weight = 1900), L-44 (a copolymer of EO and P, molecular weight = 2200), L-64 (a copolymer of EO and PO, molecular weight = 3000), and L-65 (a copolymer of EO and PO, molecular weight = 3900) were purchased from Jiangsu Haian Petrochemical Plant.

[0041] 2. Main instruments and equipment

[0042] Electronic balance (Shanghai Yue Scientific Instruments (Suzhou) Manufacturing Co., Ltd.), ultrasonic cleaner (Shenzhen Jiemeng Cleaning Equipment Co., Ltd.), constant temperature water bath shaker (Shanghai Yichun Industrial Co., Ltd.), automatic balancing centrifuge (Jinan Instrument Manufacturing Co., Ltd., Jintan City, Jiangsu Province), and analytical high performance liquid chromatograph (Waters).

[0043] 3. Test methods

[0044] (1) Preparation of Stephania tetrandra extract

[0045] Take 20.0 g of dry powder of Stephania tetrandra root solids (50-80 mesh) in a 250 mL flask, liquid-solid ratio 6:1 (mL / g) to add 70% ethanol. Extract at 102℃ for 2h, repeat extraction 2 times, collect the filtrate, rotary evaporation to no alcohol, get Stephania tetrandra extract.

[0046] (2) HPLC analysis conditions

[0047] The HPLC (high performance liquid chromatography) analysis conditions are shown in Table 1.

[0048] Table 1 HPLC analysis conditions

[0049]

[0050] (3) Drawing of standard curve

[0051] A certain amount of tetrandrine standard was precisely weighed and dissolved in methanol to prepare a mixed control solution, and the concentration of tetrandrine standard was 0.606 mg / mL. 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL and 1.4 mL of the mixed standard were precisely taken and diluted to 5 mL volumetric flask to obtain the corresponding concentration of tetrandrine solution. Methanol was used as a blank solution, and high performance liquid chromatography was used to measure the standard curve with tetrandrine peak area as the abscissa. As shown in Table 2. Figure 1

[0052] (4) Preparation of aqueous two-phase system ATPS

[0053] The aqueous two-phase system used mainly consists of organic matter, inorganic salt and distilled water. A certain amount of inorganic salt and organic matter was precisely weighed in a 10 mL centrifuge tube, distilled water was added to make the total mass 10 g, and a certain amount of Stephania tetrandra extract was added to obtain the aqueous two-phase system. After ultrasonic, shake extraction, the sample was centrifuged at 3000 r / min for 20 min to obtain the upper and lower phase system, and the volume of the upper and lower phase was accurately measured. The concentration of tetrandrine was measured by high phase liquid phase method, and the extraction rate of tetrandrine was used as the evaluation index.

[0054]

[0055] In the formula, c is the mass concentration of tetrandrine (mg / mL), N is the dilution multiple, V is the volume of the upper phase (mL), and m is the mass of the added Stephania tetrandra extract (g).

[0056] ​The organic matter includes one or more of L-31, L-35, L-44, L-64, L-65, and the organic small molecule alcohol includes one or more of methanol, ethanol, n-propanol, isopropanol, and n-butanol. The inorganic salt includes one or more of sodium carbonate, ammonium sulfate, potassium carbonate, and dipotassium hydrogen phosphate. The mass ratio of the mixture of the organic matter, the inorganic salt, and water is 1-5:1-5:1-8. The solid-liquid ratio g / g of the powder aconite extract and the aqueous two-phase system is 1-4:100. The extraction temperature is 30-70°C; the ultrasonic time is 15-60 min; and the extraction time is 20-100 min.

[0057] II. Results and analysis

[0058] Extraction of tetrandrine under different conditions

[0059] Example 1

[0060] The difference lies in the type of polymer

[0061] The aqueous two-phase system used mainly consists of organic matter, inorganic salt, and distilled water. A certain amount of inorganic salt and organic matter are precisely weighed in a 10 mL centrifuge tube, distilled water is added to make the total mass 10 g, and a certain amount of powder aconite extract is added to obtain the aqueous two-phase system. After ultrasonic treatment, the sample is shaken and extracted, and the sample is centrifuged at 3000 r / min for 20 min to obtain the upper and lower phase systems.

[0062] The extract mass is 0.2 g, the potassium carbonate mass is 2.0 g, and the organic matter mass is 2.0 g. The solid-liquid ratio g / g of the powder aconite extract and the aqueous two-phase system is 2:100. The extraction temperature is 30°C; the ultrasonic time is 50 min; and the extraction time is 60 min.

[0063] The difference lies in the type of organic small molecule alcohol

[0064] As shown in Figure 2 , with the increase of the molecular weight of the polymer, the extraction rate of tetrandrine in the extract first increases and then decreases. When the polymer L-44 is selected, the extraction rate of tetrandrine is the highest, which is 5.39%.

[0065] Example 2

[0066] The difference lies in the type of organic small molecule alcohol

[0067] The used aqueous two-phase system is mainly composed of organic matter, inorganic salt and distilled water. A certain amount of inorganic salt and organic matter is precisely weighed in a 10 mL centrifuge tube, distilled water is added to make the total mass 10 g, and then a certain amount of powder aconite extract is added to obtain the aqueous two-phase system. After ultrasonic treatment, the sample is extracted by shaking, and the sample is centrifuged at 3000 r / min for 20 min to obtain the upper and lower phase systems.

[0068] In the formula, the mass of the extract is 0.2 g, the mass of potassium carbonate is 2.0 g, and the mass of the organic matter is 2.0 g. The solid-liquid ratio g / g of the powder aconite extract mixed with the aqueous two-phase system is 2:100. The extraction temperature is 30°C; the ultrasonic time is 50 min. The extraction time is 60 min.

[0069] The difference lies in that the organic matter is selected from methanol, ethanol, n-propanol, isopropanol and n-butanol.

[0070] As shown in Figure 3 , with the increase of the number of carbon atoms of the small molecule alcohol, the content of tetraphylline in the extract first increases and then decreases (methanol as the organic phase does not separate), and the extraction rate of tetraphylline is the highest when n-propanol is selected, which is 6.24%. Tetraphylline has poor water solubility and is easily soluble in organic solvents such as alcohols, but with the increase of the molecular weight, the volume of the upper phase of the aqueous two-phase system is smaller, resulting in a low extraction rate.

[0071] Example 3

[0072] The difference lies in that the inorganic salt is different

[0073] The used aqueous two-phase system is mainly composed of organic matter, inorganic salt and distilled water. A certain amount of inorganic salt and organic matter is precisely weighed in a 10 mL centrifuge tube, distilled water is added to make the total mass 10 g, and then a certain amount of powder aconite extract is added to obtain the aqueous two-phase system. After ultrasonic treatment, the sample is extracted by shaking, and the sample is centrifuged at 3000 r / min for 20 min to obtain the upper and lower phase systems.

[0074] In the formula, the mass of the extract is 0.2 g, the mass of potassium carbonate is 2.0 g, and the mass of the organic matter is 2.0 g. The solid-liquid ratio g / g of the powder aconite extract mixed with the aqueous two-phase system is 2:100. The extraction temperature is 30°C; the ultrasonic time is 50 min. The extraction time is 60 min.

[0075] The difference lies in that the inorganic salt is selected from sodium citrate, potassium dihydrogen phosphate, sodium carbonate, ammonium sulfate, potassium carbonate and dipotassium hydrogen phosphate.

[0076] As shown in Figure 4 , when the inorganic salt is potassium carbonate (the inorganic salt is potassium dihydrogen phosphate or sodium citrate, which does not separate), the extraction rate of tetraphylline is the highest, which is 6.32%, which may be related to its strong alkalinity.

[0077] Example 4

[0078] The difference lies in the different content of inorganic salt

[0079] The used aqueous two-phase system mainly consists of organic matter, inorganic salt and distilled water. A certain amount of inorganic salt and organic matter are precisely weighed in a 10 mL centrifuge tube, distilled water is added to make the total mass 10 g, and a certain amount of powder aconite extract is added to obtain the aqueous two-phase system. After ultrasonic, shaking extraction is performed, and the sample is centrifuged at 3000 r / min for 20 min to obtain the upper and lower phase systems.

[0080] Among them, the mass of the extract is 0.2 g, the mass of n-propanol is 2 g; the content of inorganic salt potassium carbonate is 1.0 g, 2.0 g, 3.0 g, 4.0 g and 5.0 g. The solid-liquid ratio g / g of the mixture of powder aconite extract and aqueous two-phase system is 2:100. The extraction temperature is 30℃; the ultrasonic time is 50 min. The extraction time is 60 min.

[0081] As shown in Figure 5 The content of inorganic salt is 1.0-3.0 g, the extraction rate of tetrandrine shows an upward trend, and the content of inorganic salt is 3.0-4.0, which shows a downward trend. The extraction rate reaches a peak at 3.0 g, which may be because with the increase of the content of inorganic salt, the binding ability of inorganic salt to water in the aqueous two-phase system is enhanced, which makes the volume of organic phase in the upper phase increase, which promotes the dissolution of tetrandrine in the upper phase. When the inorganic salt is saturated, the increase of the mass has little effect on the extraction rate.

[0082] Example 5

[0083] The difference lies in the different content of organic matter

[0084] The used aqueous two-phase system mainly consists of organic matter, inorganic salt and distilled water. A certain amount of inorganic salt and organic matter are precisely weighed in a 10 mL centrifuge tube, distilled water is added to make the total mass 10 g, and a certain amount of powder aconite extract is added to obtain the aqueous two-phase system. After ultrasonic, shaking extraction is performed, and the sample is centrifuged at 3000 r / min for 20 min to obtain the upper and lower phase systems.

[0085] Among them, the mass of the extract is 0.2 g, the mass of n-propanol is 2 g; the content of inorganic salt potassium carbonate is 1.0 g, 2.0 g, 3.0 g, 4.0 g and 5.0 g. The solid-liquid ratio g / g of the mixture of powder aconite extract and aqueous two-phase system is 2:100. The extraction temperature is 30℃; the ultrasonic time is 50 min. The extraction time is 60 min.

[0086] As shown in Figure 6As shown in the table, the extraction rate of tetrandrine showed an upward trend at 1.0-2.0 g, reached a peak at 2.0 g, and decreased when the amount exceeded 2.0 g. This is because when the organic matter content is too low, the aqueous two-phase system is unstable, the solubility of tetrandrine in the upper phase is small, and when the organic matter content is too large, the stability of the aqueous two-phase system decreases, and inorganic salt crystals are easily precipitated, affecting the dissolution of tetrandrine.

[0087] Example 6

[0088] The difference lies in the solid-liquid ratio

[0089] The aqueous two-phase system used mainly consists of organic matter, inorganic salt and distilled water. A certain amount of inorganic salt and organic matter is precisely weighed in a 10 mL centrifuge tube, distilled water is added to make the total mass 10 g, and a certain amount of powder aconite extract is added to obtain the aqueous two-phase system. After ultrasonic treatment, the sample is extracted by shaking, and the sample is centrifuged at 3000 r / min for 20 min to obtain the upper and lower phase systems.

[0090] The mass of the extract is 0.2 g, the mass of potassium carbonate is 2 g, and the mass of n-propanol is 2 g. The solid-liquid ratio g / g of the powder aconite extract mixed with the aqueous two-phase system is 2:100. The extraction temperature is 30°C, and the ultrasonic time is 50 min.

[0091] As shown in the table, the extraction rate of tetrandrine showed an upward trend at 1.0-2.0 g, reached a peak at 2.0 g, and decreased when the amount exceeded 2.0 g. This is because when the organic matter content is too low, the aqueous two-phase system is unstable, the solubility of tetrandrine in the upper phase is small, and when the organic matter content is too large, the stability of the aqueous two-phase system decreases, and inorganic salt crystals are easily precipitated, affecting the dissolution of tetrandrine. Figure 7 Example 7

[0092] The difference lies in the extraction time

[0093] The aqueous two-phase system used mainly consists of organic matter, inorganic salt and distilled water. A certain amount of inorganic salt and organic matter is precisely weighed in a 10 mL centrifuge tube, distilled water is added to make the total mass 10 g, and a certain amount of powder aconite extract is added to obtain the aqueous two-phase system. After ultrasonic treatment, the sample is extracted by shaking, and the sample is centrifuged at 3000 r / min for 20 min to obtain the upper and lower phase systems.

[0094] The mass of the extract is 0.2 g, the mass of potassium carbonate is 2 g, and the mass of n-propanol is 2 g. The solid-liquid ratio g / g of the powder aconite extract mixed with the aqueous two-phase system is 2:100. The extraction temperature is 30°C, and the ultrasonic time is 50 min.

[0095] The mass of the extract is 0.2 g, the mass of potassium carbonate is 2 g, and the mass of n-propanol is 2 g. The solid-liquid ratio g / g of the powder aconite extract mixed with the aqueous two-phase system is 2:100. The extraction temperature is 30°C, and the ultrasonic time is 50 min.

[0096] The difference is that the extraction time is 20 min, 40 min, 60 min, 80 min, 100 min.

[0097] As shown in Figure 8 With the increase of extraction time, the extraction rate of tetrandrine first increases and then decreases, and reaches the peak at 60 min, and the extraction rate of tetrandrine is 6.35%.

[0098] Example 8

[0099] The difference is that the extraction temperature is different

[0100] The aqueous two-phase system used mainly consists of organic matter, inorganic salt and distilled water, and a certain amount of inorganic salt and organic matter is accurately weighed in a 10 mL centrifuge tube, distilled water is added to make the total mass 10 g, and a certain amount of powder aconite extract is added to obtain the aqueous two-phase system. After ultrasonic, shake extraction, the sample is centrifuged at 3000 r / min for 20 min to obtain the upper and lower phase system.

[0101] Among them, the mass of the extract is 0.2 g, the mass of potassium carbonate is 2 g, and the mass of n-propanol is 2 g; the solid-liquid ratio g / g of the mixture of powder aconite extract and aqueous two-phase system is 2:100. The ultrasonic time is 50 min. The extraction time is 60 min.

[0102] The difference is that the extraction temperature is 30℃, 40℃, 50℃, 60℃, 70℃.

[0103] As shown in Figure 9 The extraction rate of tetrandrine gradually increases at 25-30℃, and decreases when the temperature exceeds 30℃, and reaches the peak at 30℃, and the extraction rate of tetrandrine is 6.34%.

[0104] Example 9

[0105] The difference is that the ultrasonic time is different

[0106] The aqueous two-phase system used mainly consists of organic matter, inorganic salt and distilled water, and a certain amount of inorganic salt and organic matter is accurately weighed in a 10 mL centrifuge tube, distilled water is added to make the total mass 10 g, and a certain amount of powder aconite extract is added to obtain the aqueous two-phase system. After ultrasonic, shake extraction, the sample is centrifuged at 3000 r / min for 20 min to obtain the upper and lower phase system.

[0107] Among them, the mass of the extract is 0.2 g, the mass of potassium carbonate is 2 g, and the mass of n-propanol is 2 g; the solid-liquid ratio g / g of the mixture of powder aconite extract and aqueous two-phase system is 2:100. The extraction temperature is 30℃. The extraction time is 60 min.

[0108] The difference is that the ultrasonic time is 15 min, 30 min, 40 min, 50 min, and 60 min.

[0109] As shown in Figure 10 Figure 2, the extraction rate of tetrandrine gradually increased from 15 min to 50 min, reached a peak at 50 min, and decreased after 50 min.

[0110] Example 10

[0111] Extraction rates of tetrandrine prepared under several different reaction conditions

[0112] The aqueous two-phase system used mainly consists of organic matter, inorganic salt, and distilled water. A certain amount of inorganic salt and organic matter were precisely weighed into a 10 mL centrifuge tube, distilled water was added to make the total mass 10 g, and a certain amount of powdered Stephania root extract was added to obtain the aqueous two-phase system. After ultrasonic treatment, the sample was shaken and extracted, and the upper and lower phase systems were obtained by centrifugation at 3000 r / min for 20 min.

[0113] The organic matter is n-propanol, the inorganic salt is potassium carbonate, the extraction time is 60 min, and the ultrasonic time is 50 min.

[0114] The difference is that the inorganic salt content (A), the organic matter content (B), the solid-liquid ratio (C), and the extraction temperature (D) are different. As shown in Table 2.

[0115] Table 2 Extraction rates of tetrandrine prepared under different reaction conditions

[0116]

[0117]

[0118] A quadratic polynomial regression model equation was obtained by multiple regression fitting of the results in Table 2: Y = 6.26 - 0.4364A + 0.1219B + 0.0386C - 0.1503D - 0.2056AB - 0.0672AC + 0.0018AD - 0.0018BC + 0.2037BD + 0.0385CD - 0.4906A 2 - 0.2064B 2 + 0.1036C 2 - 0.1373D 2 The regression model was subjected to variance analysis and significance analysis. The Design-Expert 12.0.1.0 software was used to deeply analyze the regression simulation equation, and the optimal extraction process of the aqueous two-phase extraction method for tetrandrine in powdered Stephania root extract was obtained.

[0119] Example 11 Verification test results

[0120] Other reaction conditions remain unchanged, organic matter is n-propanol, inorganic salt is potassium carbonate, extraction time is 60 min, ultrasonic time is 50 min. Select inorganic salt mass is 2.40 g, organic matter mass is 2.70 g, solid-liquid ratio is 2:100 g / g, extraction temperature is 36.0℃. Extract Hanfengjie A in Hanfengjie extract. According to the optimized best process conditions, 3 parallel experiments were carried out, and the results are shown in Table 3, and the average value of 3 extraction rates is 6.46%. As shown in Table 3.

[0121] Table 3

[0122]

[0123] Three, Hanfengjie A inhibits lipase activity test

[0124] 1. Inhibition of lipase activity test

[0125] Pig pancreatic lipase (Type II) was dissolved in ultrapure water and different concentrations were prepared and stored at 4℃. 0.04 g of lauric acid 4-nitrophenyl ester was dissolved in 50 mL of 5 mol / L sodium acetate solution (containing 1% Trition X-100), heated in boiling water for 1 min to help dissolve, and the concentration of lauric acid 4-nitrophenyl ester solution was 0.8 mg / L. Cool to room temperature and reserve. Different volumes of powder fuzi extract solution (powder fuzi extract prepared under the conditions of Example 11), 80 μL of pancreatic lipase, 20 μL of PBS phosphate buffer (pH = 8) were added to a 96-well plate, incubated at 37℃ for 10 min, then 40 μL of lauric acid 4-nitrophenyl ester solution was added, gently shaken and mixed, incubated at 37℃ for 20 min in the dark, and the absorbance value was measured at 405 nm with an enzyme marker and recorded. Repeat three times and take the average value.

[0126] Inhibition rate formula: pancreatic lipase inhibition rate (%) = [1-(A3-A4) / (A1-A2)] x 100%

[0127] In the formula: A1, A2, A3, A4 are the absorbance of the blank group (distilled water instead of sample), the blank background group (distilled water instead of sample, PBS instead of pancreatic lipase), the sample group, and the sample background group (PBS instead of pancreatic lipase).

[0128] 2. Effect of powder fuzi extract on inhibition of pancreatic lipase activity

[0129] (1) Inhibition rate of powder fuzi purified liquid on pancreatic lipase

[0130] As Figure 11As shown, the inhibition rate of the purified Stephania tetrandra extract on pancreatic lipase gradually increases with increasing volume. The inhibition curve of Stephania tetrandra extract volume on pancreatic lipase was obtained by multiple linear regression fitting, yielding y = -0.0011x. 2 +0.0479x+0.3782, R 2 =0.9970; Tet purification buffer half-inhibitory volume (IC50) 50 The value was 2.41 μL.

[0131] (2) Enzyme inhibition kinetics

[0132] To determine the type of inhibition of pancreatic lipase by the purified *Stephania tetrandra* extract, a graph was plotted with the reciprocal of the purified extract volume (1 / V) on the x-axis and the reciprocal of the reaction rate (1 / V) on the y-axis. The results are as follows: Figure 12 As shown in Table 4, the Lineweaver-Burk curves of the purified Stephania tetrandra solution all intersected in the first quadrant, and K increased with increasing sample concentration (Table 4). m A decrease in V indicates an increase in the enzyme's affinity for its substrate, suggesting competition between the enzyme, substrate, and inhibitor. max The decrease is typical of anti-competitive inhibition, indicating that the sample can only bind to the enzyme-substrate complex to inhibit product release, thereby reducing enzyme activity. This suggests that the sample exhibits anti-competitive inhibition.

[0133] Table 4. Kinetic parameters of pancreatic lipase

[0134]

[0135] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for extracting tetrandrine from powdered Stephania tetrandra S. Moore, characterized in that, The method comprises the following steps: (1) mixing powder of Stephania tetrandra S. Moore with ethanol to extract, collecting the filtrate, removing ethanol to obtain powder of Stephania tetrandra S. Moore extract; (2) mixing organic matter, inorganic salt and water to obtain a two-water-phase system; (3) adding the powder of Stephania tetrandra S. Moore extract of step (1) into the two-water-phase system, then ultrasonic extraction, shaking extraction, centrifugation to obtain an upper phase and a lower phase, and the upper phase contains tetrandrine.

2. The method of claim 1, wherein the extraction powder is prepared by mixing the roots of Stephania tetrandra S. Moore with water, and then drying the mixture. In step (1), the liquid-solid ratio mL / g of powder of Stephania tetrandra S. Moore mixed with ethanol is 1-15:1, extraction is carried out at 90-120℃ for 0.5-5h, and the extraction is repeated for 1-3 times, and the filtrate is collected.

3. The method of claim 1, wherein the extraction powder is prepared by mixing the roots of Stephania tetrandra S. Moore with water, and then drying the mixture. In step (2), the organic matter includes high polymer or organic small molecule alcohol, the high polymer includes one or more of L-31, L-35, L-44, L-64 and L-65, and the organic small molecule alcohol includes one or more of methanol, ethanol, n-propanol, isopropanol and n-butanol.

4. The method of claim 1, wherein the extraction powder is prepared by mixing the roots of Stephania tetrandra S. Moore with water, and then drying the mixture. In step (2), the inorganic salt includes one or more of sodium carbonate, ammonium sulfate, potassium carbonate and dipotassium hydrogen phosphate.

5. The method of claim 1, wherein the extraction powder is prepared by mixing the roots of Stephania tetrandra S. Moore with water, and then drying the mixture. In step (2), the mass ratio of the organic matter, inorganic salt and water is 1-5:1-5:1-8.

6. The method of claim 1, wherein the extraction powder is prepared by mixing the roots of Stephania tetrandra S. Moore with water, and then drying the mixture. In step (3), the solid-liquid ratio g / g of powder of Stephania tetrandra S. Moore extract mixed with the two-water-phase system is 1-4:

100.

7. The method of claim 1, wherein the extraction powder is Stephania tetrandra S. Moore, and the tetrandrine is tetrandrine. In step (3), the extraction temperature is 30-70℃.

8. The method of claim 1, wherein the extraction powder is prepared by mixing the roots of Stephania tetrandra S. Moore with water, and then drying the mixture. In step (3), the ultrasonic time is 15-60min.

9. The method of claim 1, wherein the extraction powder is Stephania tetrandra S. Moore, and the tetrandrine is tetrandrine. In step (3), the extraction time is 20-100min.

10. Use of powder of Stephania tetrandra S. Moore extract obtained by the method of any one of claims 1-9 for preparing a medicine for inhibiting lipase activity.

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