Preparation process, detection method and application of total glucosides of paeony

Abstract

The invention belongs to the field of plant extracts, and relates to a preparation process of a total glucosides of paeony extract as well as a content detection method and application of the total glucosides of paeony extract. The radix paeoniae alba total glycoside extract is prepared through the steps of superfine grinding, bionic extraction, ultrasonic-assisted extraction, macroporous adsorption resin column chromatography separation, membrane separation and purification and the like. The total glucosides of paeony extract prepared by the method is high in content of active ingredients. The invention also provides a method for detecting the content of main active components in the total glucosides of paeony root extract by adopting a high performance liquid chromatography. The invention also provides application of the total glucosides of paeony in preparation of anti-premature ovarian failure drugs.

Description

Technical Field

[0001] The invention belongs to the field of plant extracts, and particularly relates to a total glycosides of white peony extract and a preparation method and a content detection method thereof. Technical Background

[0002] White peony root is the dried root of the dicotyledonous plant Ranunculaceae peony (Paeonialactiflora pall), which is commonly used in traditional Chinese medicine compound, and has multiple effects such as anti-inflammatory and analgesic, immunomodulatory, anti-liver damage and immune liver fibrosis. The effective parts of white peony are mainly a series of glycosides, including paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycosides, benzoylpeoniflorin, etc., collectively known as total glucosides of peony (TGP).

[0003] In clinical practice, total glucosides of white peony (TGP) are widely used to treat autoimmune diseases. Experiments by Liu Xingyan and others from Ningbo Vocational and Technical College showed that in response to the issue of the effect of TGP on the intestinal microenvironment of Sjögren's syndrome, by observing the effects of TGP on intestinal motility, intestinal barrier function, intestinal flora structure and metabolites in SS model mice, the effects of TGP on the intestinal microenvironment, intestinal flora and its metabolites (short-chain fatty acids) of SS model mice were clarified, and the possible regulatory mechanism of total glucosides of white peony in the occurrence and development of SS disease was explored from the perspective of the intestinal environment. TGP improves the symptoms of constipation in SS mice by increasing the water content of feces and improving intestinal motility; TGP regulates the structure of intestinal flora, increases the content of acetate and butyrate, increases the expression of tight junction proteins, reduces the permeability of the intestinal barrier of SS mice, and promotes the recovery of damaged intestinal barrier function. The research results formed the paper "Regulatory Effects of Total Glucosides of White Peony on the Intestinal Microenvironment of Mice with Sjögren's Syndrome", which was published in the "Chinese Journal of Experimental Traditional Chinese Medicine" in July 2021.

[0004] In the article "Research Progress on the Pharmacological Actions and Mechanisms of Total Glucosides of White Peony", Yang Shanjing and others from Shandong University of Traditional Chinese Medicine summarized the research on the pharmacological actions and mechanisms of total glycosides of white paeony, pointing out that total glycosides of white paeony are the effective parts of the extracts of the traditional Chinese medicine white peony, mainly including paeoniflorin, paeoniflorin lactone glycosides, oxidized paeoniflorin and other ingredients. Modern pharmacological studies have found that total glycosides of white paeony not only have immunomodulatory, anti-inflammatory, and liver protection effects, but also have multiple effects such as brain and nerve protection, cardiovascular protection, kidney protection, and inhibition of cell proliferation. The article was published in "Chinese Modern Applied Pharmacy" in July 2021.

[0005] At present, the main methods for extracting total glucosides of white peony include water immersion extraction, microwave extraction, ultrasonic extraction, flash extraction, and thermal reflux extraction.

[0006] Li Shan et al. from Qingdao University conducted an experimental study on the extraction and purification process of total glycosides of white paeony. The results showed that the extraction of total glycosides of white paeony was 9 (3 4 ) was used in an orthogonal experimental design, and water extraction was used. The amount of water added, extraction time, and extraction times were taken as the main considerations. The optimal extraction process of total glycosides of white paeony was determined as follows: adding 12 times the amount of water and boiling three times, each time for 3 hours. On this basis, D 101 The macroporous adsorption resin separation and purification can finally achieve a transfer rate of 80.5% of paeoniflorin in white peony. The research results were published in the journal Food Science in 2007.

[0007] Su Ting and others from Changchun University of Chinese Medicine conducted an experimental study on the extraction process of total glycosides of white paeony. The results showed that the optimal extraction process of total glycosides of white paeony was: 12 times the volume of 50% ethanol reflux extraction twice, 1h / time. This optimal process was applied to pilot production, and the total glycosides of white paeony paste yield was 17.03%, the content of total glycosides of white paeony was 34.75mg / g, and the content of paeoniflorin was 26.53mg / g. The paper "Research on Industrial Extraction Process of Total Glucosides of White Peony" based on the research results was published in the Journal of Clinical Medical Literature in April 2015.

[0008] The inventors of the present invention have conducted research on white peony root for many years, and accidentally discovered a unique method for extracting and separating total glycosides of white peony root from white peony root, which effectively improves the extraction rate of total glycosides of white peony root extract.

[0009] The white peony root medicinal material used in the present invention was purchased from the Qingyutang chain store of Hangzhou Hu Qingyutang Guoyaohao Co., Ltd. Summary of the invention

[0010] The purpose of the invention is to provide a total glucosides of white peony root extract.

[0011] Another object of the present invention is to provide a method for preparing the total glucosides of white peony root extract.

[0012] Another object of the present invention is to provide a method for detecting the content of the total glucosides of white peony extract.

[0013] The invention also provides a pharmaceutical dosage form of the total glucosides of white peony extract and a preparation method thereof.

[0014] The invention also provides application of the total glucosides of white peony extract in preparing a drug for preventing premature ovarian failure.

[0015] The object of the present invention is achieved in the following ways:

[0016] A total glycosides extract of white peony root is prepared by the following method:

[0017] (1) ultrafine grinding white peony root material by using a jet mill to obtain ultrafine white peony root powder A;

[0018] (2) taking the white peony superfine powder A obtained in step (1), adding it to the mixed enzyme solution, stirring and extracting in a water bath, centrifuging, and collecting the supernatant B and the precipitate C respectively; concentrating the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside;

[0019] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction, adding ethanol as an extract, filtering the extract, recovering ethanol from the filtrate, and obtaining an extract E for later use;

[0020] (4) taking the extract D obtained in step (2) and the extract E obtained in step (3) and combining them, mixing them evenly, performing column chromatography separation, collecting the eluate, and filtering the solution after column chromatography to obtain a filtrate F;

[0021] (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, and the solution obtained by membrane separation is freeze-dried to obtain the total glucosides of white peony extract.

[0022] The above-mentioned total glucosides of white peony extract is preferably prepared by the following method:

[0023] (1) placing the white peony root in an oven to dry, taking out the dried white peony root, and ultrafine grinding it with a jet mill to obtain ultrafine white peony root powder A;

[0024] (2) taking the white peony superfine powder A obtained in step (1), adding it to the mixed enzyme solution, stirring and extracting in a water bath, centrifuging, and collecting the supernatant B and the precipitate C respectively; concentrating the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution is prepared into a solution by cellulase, pectinase, xylanase and citric acid;

[0025] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction, adding ethanol as an extract, performing extraction three times in total, combining the extracts, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use;

[0026] (4) taking the extract D obtained in step (2) and the extract E obtained in step (3) and combining them, mixing them evenly, and performing column chromatography separation, wherein the filler is magnesium silicate, collecting the eluate, wherein the eluate is a mixed solvent of ethanol-methanol, filtering the solution after the column chromatography, and obtaining a filtrate F;

[0027] (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, a polyamide composite membrane is selected, and the solution obtained by membrane separation is freeze-dried to obtain the total glucosides of white peony extract.

[0028] The above-mentioned total glucosides of white peony extract is more preferably prepared by the following method:

[0029] (1) After placing the white peony root in an oven at 30-40° C. and drying for 5-7 hours, the dried white peony root is taken out and ultrafinely pulverized by an air flow mill. The air flow pressure of the air flow mill is 1200-1400 kPa, and the feed speed is 30-50 r·min -1 , the classification frequency is 20-30 Hz, the crushing time is 30-50 min, and the white peony superfine powder A is obtained;

[0030] (2) Take the white peony superfine powder A obtained in step (1), add it to 5-15 times the amount of the mixed enzyme solution, stir and extract in a water bath at 50-60° C. for 1-3 hours, centrifuge for 15-25 minutes at a centrifugal speed of 2000-3000 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution comprises 0.5-1.5% cellulase, 0.2-0.6% pectinase, 0.3-0.7% xylanase and 0.02-0.04 mol·L -1 Citric acid is prepared into a solution;

[0031] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction, the extraction temperature is 30-40° C., the ultrasonic frequency is 25-35 kHz, and 65-75% ethanol is added as the extracting solution at a solid-liquid ratio of 1 g:7-11 mL. Each extraction is performed for 25-45 min, and the extraction is performed three times in total. The extracts are combined, filtered, and ethanol is recovered from the filtrate to obtain an extract E for later use;

[0032] (4) taking the extract D obtained in step (2) and the extract E obtained in step (3) and combining them, mixing them, and performing column chromatography separation, the chromatography column used is filled with magnesium silicate, the eluent is a mixed solvent of ethanol-methanol in a ratio of 60-80:40-20, the liquid-to-solid ratio is 1:8-12, the flow rate is 5-15 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain a filtrate F;

[0033] (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, using a polyamide composite membrane with a pore size of 0.1 μm, a membrane thickness of 20 to 40 μm, and a membrane area of ​​150 to 250 m 2 , the operating pressure is 1-3 MPa, and the solution obtained by membrane separation is freeze-dried to obtain the total glucosides of white peony extract.

[0034] The above-mentioned total glucosides of white peony extract is most preferably prepared by the following method:

[0035] (1) After the white peony root was placed in an oven and dried at 35°C for 6 hours, the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r / min.-1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0036] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution;

[0037] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz, adding 70% ethanol as an extracting solution at a solid-liquid ratio of 1 g:9 mL, extracting for 35 min each time, extracting three times in total, combining the extracts, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use;

[0038] (4) taking the extract D obtained in step (2) and the extract E obtained in step (3) and combining them, mixing them evenly, and performing column chromatography separation, the chromatography column used is 20 cm × 2 cm, the filler is magnesium silicate, the eluent is a mixed solvent of ethanol and methanol in a ratio of 70:30, the liquid-to-solid ratio is 1:10, the flow rate is 10 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain filtrate F;

[0039] (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, using a polyamide composite membrane with a pore size of 0.1 μm, a membrane thickness of 30 μm, and a membrane area of ​​200 m 2 , the operating pressure is 2 MPa, and the solution obtained by membrane separation is freeze-dried to obtain the total glucosides of white peony extract.

[0040] A method for detecting the content of total glycosides of white peony root extract is as follows:

[0041] (1) Preparation of reference solution: accurately weigh the reference substances of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycoside and benzoylpeoniflorin, and add methanol to prepare a mixed reference solution containing paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycoside and benzoylpeoniflorin;

[0042] (2) Preparation of test solution: Take the powder of total glucosides of white peony extract, weigh it accurately, put it into a stoppered conical flask, add methanol accurately, stopper it tightly, weigh it accurately, treat it with ultrasound, let it cool, weigh it accurately again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution;

[0043] (3) Chromatographic conditions: Chromatographic column: Waters chromatographic column, specification: 4.6×250 mm, 5 μm, mobile phase: acetonitrile-0.1% phosphoric acid solution; gradient elution; column temperature: 20-50°C; flow rate: 0.5-2.0 mL / min; detection wavelength: 210-240 nm; injection volume: 5-20 μL;

[0044] (4) Sample injection test: Take 5-20 μL of the reference solution and the test solution and inject them into the high performance liquid chromatography instrument for detection.

[0045] The above-mentioned method for detecting the content of the total glucosides of white peony extract is preferably carried out by high performance liquid chromatography as follows:

[0046] (1) Preparation of reference solution: accurately weigh the reference substances of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycoside and benzoylpeoniflorin, and add 60-80% methanol to prepare a mixed reference solution containing 270-290 μg / mL, 30-50 μg / mL, 20-40 μg / mL, 30-50 μg / mL and 40-60 μg / mL of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycoside and benzoylpeoniflorin, respectively;

[0047] (2) Preparation of test solution: Take 0.4-0.6 g of total glycosides of white peony extract powder, weigh accurately, place in a stoppered conical flask, add 40-60 ml of 60-80% methanol accurately, stopper tightly, weigh, and treat with ultrasound at a power of 240-260 W and a frequency of 40-60 kHz for 20-40 minutes. Let cool, weigh again, make up the lost weight with 60-80% methanol, shake well, filter, and take the filtrate to obtain the solution.

[0048] (3) Chromatographic conditions: Chromatographic column: Waters Atlantis chromatographic column, specification: 4.6×250 mm, 5 μm, mobile phase: acetonitrile-0.1% phosphoric acid solution; gradient elution, 0 min to 5 min: acetonitrile ratio is maintained at 5%, 0.1% phosphoric acid solution ratio is maintained at 95%; 6 min to 25 min: acetonitrile ratio is linearly increased from 5% to 25%, 0.1% phosphoric acid solution ratio is linearly decreased from 95% to 75%; 26 min to 40 min: acetonitrile ratio is linearly increased from 25% to 32%, 0.1% phosphoric acid solution ratio is linearly decreased from 75% to 68%; column temperature: 30-40°C; flow rate: 0.5-1.5 mL / min; detection wavelength: 220-230 nm; injection volume is 5-15 μL;

[0049] (4) Sample injection test: Take 5-15 μL of the reference solution and the test solution and inject them into the high performance liquid chromatograph for detection.

[0050] The above-mentioned method for detecting the content of the total glucosides of white peony extract is most preferably carried out by high performance liquid chromatography as follows:

[0051] (1) Preparation of reference solution: Paeoniflorin, hydroxypaeoniflorin, paeoniflorin, paeoniflorin lactone glycosides and benzoylpaeoniflorin reference substances were accurately weighed and added with 70% methanol to prepare a mixed reference solution containing 840 μg / mL, 120 μg / mL, 90 μg / mL, 120 μg / mL and 150 μg / mL of paeoniflorin, hydroxypaeoniflorin, paeoniflorin, paeoniflorin lactone glycosides and benzoylpaeoniflorin, respectively;

[0052] (2) Preparation of test solution: Take 0.5 g of total glycosides of white peony extract powder, weigh accurately, place in a stoppered conical flask, add 50 ml of 70% methanol accurately, stopper tightly, weigh, and treat with ultrasound at a power of 250 W and a frequency of 50 kHz for 30 minutes. Let cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the solution;

[0053] (3) Chromatographic conditions: Chromatographic column: Waters Atlantis T3 chromatographic column, specification: 4.6×250 mm, 5 μm, mobile phase: acetonitrile-0.1% phosphoric acid solution; gradient elution, 0 min to 5 min: acetonitrile ratio maintained at 5%, 0.1% phosphoric acid solution ratio maintained at 95%; 6 min to 25 min: acetonitrile ratio linearly increased from 5% to 25%, 0.1% phosphoric acid solution ratio linearly decreased from 95% to 75%; 26 min to 40 min: acetonitrile ratio linearly increased from 25% to 32%, 0.1% phosphoric acid solution ratio linearly decreased from 75% to 68%; column temperature: 35°C; flow rate: 1.0 mL / min; detection wavelength: 225 nm; injection volume: 10 μL;

[0054] (4) Sample injection test: Take 10 μL of the reference solution and the test solution and inject them into the high performance liquid chromatograph for detection.

[0055] The above-mentioned total glucosides of white peony extract can be prepared into oral preparations such as tablets, pills, soft capsules, hard capsules, oral liquids, and granules by conventional pharmaceutical methods.

[0056] A use of a total glucosides of white peony root extract in the preparation of an anti-premature ovarian failure drug, wherein the total glucosides of white peony root extract is prepared by the following method:

[0057] (1) After the white peony root was placed in an oven and dried at 35°C for 6 hours, the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r / min. -1, the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0058] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution;

[0059] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz, adding 70% ethanol as an extracting solution at a solid-liquid ratio of 1 g:9 mL, extracting for 35 min each time, extracting three times in total, combining the extracts, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use;

[0060] (4) taking the extract D obtained in step (2) and the extract E obtained in step (3) and combining them, mixing them evenly, and performing column chromatography separation, the chromatography column used is 20 cm × 2 cm, the filler is magnesium silicate, the eluent is a mixed solvent of ethanol and methanol in a ratio of 70:30, the liquid-to-solid ratio is 1:10, the flow rate is 10 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain filtrate F;

[0061] (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, using a polyamide composite membrane with a pore size of 0.1 μm, a membrane thickness of 30 μm, and a membrane area of ​​200 m 2 , the operating pressure is 2 MPa, and the solution obtained by membrane separation is freeze-dried to obtain the total glucosides of white peony extract.

[0062] The technical effect of the present invention is verified by the following experimental studies:

[0063] Experiment 1: Drug screening experiment of different white peony root extracts against premature ovarian failure

[0064] 1 Materials and methods

[0065] 1.1 Experimental animals

[0066] Female ICR mice, SPF grade, weighing 18-22g, 6-8 weeks old, were purchased from the Comparative Medicine Center of Yangzhou University, animal qualification certificate number: NO.202206983. The breeding environment is a barrier system, use license number: SYXK (Su) 20180026, room temperature 20-22℃, relative humidity 40-70%, light 12h / 12h. Experimental mouse maintenance feed was purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., in accordance with GB 14924.1 "General Quality Standard for Experimental Animal Formula Feed", production license number: Su Feed Certificate (2019) 01003.

[0067] 1.2 Main instruments and drugs used in the experiment

[0068] 1.2.1 Experimental instruments

[0069] Electronic balance, Beijing Sartorius Instrument System Co., Ltd., model: BS224S; Shanghai Youke Instrument Co., Ltd., model: FA2004B

[0070] Animal weighing scale, Shanghai Xiangchuan Electronic Scale Co., Ltd., model: ACX-SC-DA;

[0071] High-speed refrigerated centrifuge, Eppendorf, model: Centrifuge 5810R; Dalong Xingchuang Experimental Instrument (Beijing) Co., Ltd., model: D3024R

[0072] Purified water instrument, Millipore, Germany, model: Milli-Q Plus;

[0073] Multifunctional microplate reader, MD Company, USA, model: Flexstation 3; Thermo Fisher (Shanghai) Instrument Co., Ltd., model: Multiskan FC

[0074] Biological microscope, Leica Instruments, Germany, model: DM4000B. Ningbo Sunny Instruments Co., Ltd., model: ES

[0075] 1.2.2 Main reagents and drugs

[0076] 1.2.2.1 Test drug S1: Prescription: White peony root 500g;

[0077] Preparation method: (1) 500 g of white peony root was placed in an oven at 35°C and dried for 6 hours, then the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r·min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0078] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D; mix the precipitate C with the extract D, and freeze-dry to obtain the test drug S1; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid was prepared as a solution.

[0079] 1.2.2.2 Test drug S2: Prescription: White peony root 500g;

[0080] Preparation method: (1) 500 g of white peony root was placed in an oven at 35°C and dried for 6 hours, then the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r·min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0081] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution;

[0082] (3) The precipitate C obtained in step (2) was subjected to ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz. Ethanol with a concentration of 70% was added as the extract at a solid-liquid ratio of 1 g:9 mL. Each extraction lasted for 35 min, and the extractions were performed three times in total. The extracts were combined, filtered, and ethanol was recovered from the filtrate to obtain extract E. Extract D was mixed with extract E, and freeze-dried to obtain the test drug S2.

[0083] 1.2.2.3 Test drug S3: Prescription: White Peony Root 500g;

[0084] Preparation method: (1) 500 g of white peony root was placed in an oven at 35°C and dried for 6 hours, then the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r·min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0085] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution;

[0086] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz, adding 70% ethanol as an extracting solution at a solid-liquid ratio of 1 g:9 mL, extracting for 35 min each time, extracting three times in total, combining the extracts, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use;

[0087] (4) Take the extract D obtained in step (2) and the extract E obtained in step (3) and combine them, mix them evenly, and separate them by column chromatography. The chromatography column used is 20 cm×2 cm, the filler is magnesium silicate, the eluent is a mixed solvent of ethanol and methanol in a ratio of 70:30, the liquid-to-solid ratio is 1:10, the flow rate is 10 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain filtrate F. The filtrate F is freeze-dried to obtain the test drug S3.

[0088] 1.2.2.4 Test drug S4: Prescription: White Peony Root 500g;

[0089] Preparation method: (1) 500 g of white peony root was placed in an oven at 35°C and dried for 6 hours, then the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r·min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0090] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution;

[0091] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz, adding 70% ethanol as an extracting solution at a solid-liquid ratio of 1 g:9 mL, extracting for 35 min each time, extracting three times in total, combining the extracts, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use;

[0092] (4) taking the extract D obtained in step (2) and the extract E obtained in step (3) and combining them, mixing them evenly, and performing column chromatography separation, the chromatography column used is 20 cm × 2 cm, the filler is magnesium silicate, the eluent is a mixed solvent of ethanol and methanol in a ratio of 70:30, the liquid-to-solid ratio is 1:10, the flow rate is 10 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain filtrate F;

[0093] (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, using a polyamide composite membrane with a pore size of 0.1 μm, a membrane thickness of 30 μm, and a membrane area of ​​200 m 2 , the operating pressure is 2 MPa, and the solution obtained by membrane separation is freeze-dried to obtain the test drug S4.

[0094] 1.2.2.5 Progynova (estradiol valerate tablets): Guangzhou Branch of Bayer Healthcare Co., Ltd., batch number 060823, specification: 1 mg / tablet, 21 tablets / bottle, batch number: 600A20201112.

[0095] 1.2.2.6 Tripterygium wilfordii glycosides tablets: Zhejiang Deende Pharmaceutical Co., Ltd., specifications: 10 mg / tablet, 50 tablets / bottle, batch number: 2107115B, 2111131B0.

[0096] 1.2.2.7 Estradiol (E2) ELISA kit, Shanghai Bioengineering Co., Ltd., batch number: LOT: l528AD0334.

[0097] 1.2.2.8 Testosterone (T) ELISA kit, Shanghai Bioengineering Co., Ltd., batch number: LOT: l518AD0346.

[0098] 1.3 Experimental methods

[0099] 1.3.1 Grouping, modeling and drug administration

[0100] 70 healthy female ICR mice were randomly divided into 7 groups, namely normal group, model group, positive group, test drug S1 group, test drug S2 group, test drug S3 group, and test drug S4 group, with 10 mice in each group. Except for the normal group, the other groups were given Tripterygium wilfordii polyglycosides tablets by gavage at a dose of 40 mg / kg every day, and the gavage volume was 10 mL / kg mouse, and the administration was continued for 10 weeks. The normal control group was gavaged with distilled water every day in the same way. Starting from the 5th week, each group was given Tripterygium wilfordii polyglycosides tablets and gavage at the above dose for 6 consecutive weeks.

[0101] 1.3.2 Observation of estrus in mice

[0102] Ten days before the last administration, vaginal cells were observed regularly every afternoon to determine the changes in the estrous cycle. Specific operation method: Fix the mouse in the back position of the left hand, hold a pipette in the right hand, which is pre-absorbed with 0.1-0.2 mL of saline, gently insert it into the mouse vagina 0.05-1 cm, aspirate 2-3 times, drop the aspirated liquid into a 96-well culture plate, and observe the morphology of vaginal cells under an inverted biological microscope. Judgment criteria: Proestrus: All are nucleated epithelial cells, occasionally a small number of keratinized (anucleated) epithelial cells, and no white blood cells; estrus: All are anucleated keratinized cells, large and irregular in shape, with a small number of epithelial cells in between; metestrus: There are white blood cells, keratinized cells, and nucleated epithelial cells; diestrus: A large number of white blood cells and a small number of epithelial cells and mucus are seen; mouse estrus cycle: 5-6 days.

[0103] 1.3.3 Serum estradiol (E2) and testosterone (T) level detection

[0104] One hour after the last administration, the eyeballs of mice in each group were removed to collect blood, which was centrifuged at 3000 rpm for 10 min to collect serum. The levels of E2 and T in serum were detected by ELISA according to the operating instructions of the kit.

[0105] 1.3.4 Organ Index

[0106] After the mice were killed by cervical dislocation, the uterus and ovaries on both sides were removed, the wet weight was measured after fat was removed, and the uterine index and ovarian index were calculated.

[0107] 1.3.5 Pathological testing

[0108] After weighing, the ovaries were divided into groups and fixed in 10% formaldehyde solution, routinely sampled, dehydrated, paraffin-embedded, sliced, HE-stained, and sealed. The slides were read under an optical microscope, and the number of various follicles and corpora lutea in the ovaries was counted.

[0109] 1.4 Statistical analysis

[0110] Statistical data processing The data were expressed and analyzed using EXCEL, and the t test was used for comparison among the groups.

[0111] 2 Results

[0112] 2.1 Effects on the estrous cycle of mice

[0113] The experimental results showed that the estrous cycle of mice in the normal group was normal, with an average of about 5.3 days. Compared with the normal group, the estrous cycle of mice in the model group was significantly prolonged, and the estrous period and the proportion of the estrous period were significantly shortened. The results showed that the model of premature ovarian failure caused by Tripterygium wilfordii in mice was successfully established.

[0114] Compared with the model group, the estrous cycle of the test drug S3, test drug S4 and Progynova groups was significantly shortened, the estrous period was significantly prolonged, and the proportion of the estrous period was significantly prolonged. This indicates that the test drug S3 and the test drug S4 both have estrogen-like effects, which can prolong the estrous period of mice after premature ovarian failure caused by Tripterygium wilfordii polyglycosides and shorten the estrous cycle. Among them, the test drug S4 has the best effect. The results are shown in Table 1.

[0115] Table 1 Effect of Tripterygium wilfordii polyglycosides on the composition ratio of estrus in mice after administration of Tripterygium wilfordii polyglycosides induced premature ovarian failure

[0116]

[0117] Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group, *P<0.05, **P<0.01. 2.2 Effects on serum E2 and T levels

[0118] The experimental results showed that compared with the normal group, after Tripterygium wilfordii polyglycosides induced premature ovarian failure in mice, the serum estradiol E2 level of the model group mice decreased significantly, and the testosterone T level increased significantly.

[0119] Compared with the model group, the serum estradiol level of mice in the test drug S3, test drug S4 and Progynova groups was significantly increased, and the serum testosterone level was significantly decreased. This indicates that the test drug S3 and the test drug S4 can increase the serum estradiol (E2) level of mice and reduce the testosterone T level after premature ovarian failure induced by Tripterygium wilfordii polyglycosides, and have estrogen-like effects. Among them, the test drug S4 had the best effect. The results are shown in Table 2.

[0120] Table 2 Effect of E on premature ovarian failure in mice induced by Tripterygium wilfordii polyglycosides 2 , the influence of T level

[0121]

[0122] Compared with the normal group, # P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0123] 2.3 Effects on mouse ovary and uterine index

[0124] The experimental results showed that compared with the normal group, the uterine weight, uterine index, ovarian weight and ovarian index of mice in the model group were significantly reduced.

[0125] Compared with the model group, the uterine weight and uterine index of the mice in the test drug S3, test drug S4 and Progynova groups were significantly increased; the ovarian weight and ovarian index of the mice in the test drug S3, test drug S4 and Progynova groups were significantly increased. This indicates that the test drugs S2, test drug S3 and test drug S4 have a certain promoting effect on the growth of the ovaries and uterus of mice with premature ovarian failure caused by Tripterygium wilfordii polyglycosides, among which the test drug S4 has the best effect. The results are shown in Table 3.

[0126] Table 3 Effects of Tripterygium wilfordii polyglycosides on ovarian and uterine indexes in mice with premature ovarian failure ( n=10)

[0127]

[0128]

[0129] Compared with the normal group, # P<0.05Compared with the model group, *P<0.05, **P<0.01.

[0130] 2.4 Pathological Detection

[0131] The experimental results showed that the ovarian tissue of mice in the model group was atrophied, the cortical and medullary structures were disordered, there were more atretic follicles than in the normal group, and fewer developing follicles than in the normal group. The ratio of atretic follicles to developing follicles was significantly increased, and the number of corpora lutea was significantly decreased, indicating that the model was successful.

[0132] Compared with the model group, the number of atretic follicles in each drug group decreased, and the number of developing follicles and corpora lutea increased. The number of corpora lutea in the ovaries of the mice in the test drug S3 group, the test drug S4 group and the Progynova group increased significantly, and the ratio of atretic follicles to developing follicles in the test drug S3 group and the test drug S4 group decreased significantly. The S4 group was slightly better than the S3 group, but there was no significant difference. The results are shown in Table 4, as well as the ovarian pathological pictures (×100) of the mice with premature ovarian failure caused by Tripterygium wilfordii polyglycosides, which are attached to the instructions. Figure 1 -Attached Figure 7 .

[0133] Table 4 Statistical results of the number of follicles and corpus luteum in each group

[0134]

[0135] Compared with the normal group, # P<0.05Compared with the model group, *P<0.05, **P<0.01.

[0136] Figure 1 Display: Ovarian pathology images of the normal group (×100): The ovaries are of medium size, with clear cortical and medullary structures, a large number of corpora lutea in the ovaries, good development, and large size. Follicles of all levels can be seen (primordial follicles, developing follicles, mature follicles, etc.), with a large number of interstitial blood vessels and no obvious congestion.

[0137] Figure 2 Display: Ovarian pathological images of the model group (×100): The number of follicles at all stages in the ovarian cortex was significantly reduced, especially the number of developing follicles. The total number of follicles was reduced compared with the normal group, and the number of atretic follicles increased.

[0138] Figure 3 Display: Ovarian pathology images of Progynova (×100): The total number of follicles and the number of developing follicles increased significantly compared with the model group, the number of atretic follicles was less than that of developing follicles, and the number of developing follicles at all levels of the cortex increased compared with the model group.

[0139] Figure 4 Display: Ovarian pathological images of the test drug S1 group (×100): The total number of follicles and the number of developing follicles increased compared with the model group, but the number of atretic follicles was higher, and the number of developing follicles at all levels of the cortex increased slightly compared with the model group.

[0140] Figure 5 Shown: Ovarian pathological images of the test drug S2 group (×100): the total number of follicles and the number of developing follicles increased compared with the model group, but the number of atretic follicles was larger, and the number of developing follicles at all levels of the cortex increased compared with the model group.

[0141] Figure 6 Display: Ovarian pathological images of the test drug S3 group (×100): the total number of follicles and the number of developing follicles increased significantly compared with the model group, and the number of atretic follicles decreased.

[0142] Figure 7 Shown: Ovarian pathological images of the test drug S4 group (×100): the total number of follicles and the number of developing follicles were significantly increased compared with the model group, the number of atretic follicles was reduced, and the number of developing follicles at all levels of the cortex was increased compared with the model group.

[0143] 3 Conclusion

[0144] This study showed that 10 weeks after modeling with Tripterygium wilfordii polysaccharide, the estrous cycle of mice in the model group was prolonged, the estrus period was shortened, the serum E2 level was decreased, the testosterone T level was increased, the weight and index of the uterus and ovary were significantly reduced, the ovarian tissue was atrophied, the cortical medulla structure was chaotic, the number of atretic follicles increased, and the number of developing follicles and corpus luteum was significantly reduced, indicating that the premature ovarian failure model was successfully prepared.

[0145] After 6 weeks of administration, the test drugs S3 and S4 were able to prolong the estrus period of mice after premature ovarian failure induced by Tripterygium wilfordii tablets, shorten the estrus cycle, promote the growth of ovaries and uterus, increase the serum E2 level of mice, reduce the T level, etc., and also reduce the number of atretic follicles in the ovaries and increase the number of developing follicles and corpora lutea. This suggests that the test drugs S3 and S4 have therapeutic effects on premature ovarian failure induced by Tripterygium wilfordii tablets in mice, and the therapeutic effect of the test drug S4 is relatively the best.

[0146] Experiment 2: HPLC method to detect the contents of five components in the total glycosides of white peony extract

[0147] Combined with the chemical composition and pharmacological effects of the total glucosides of white peony extract, which are also introduced in the background technology of this application, this study uses paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin, and benzoylpeoniflorin as indicator components to establish a method for simultaneously detecting the contents of these five components in the total glucosides of white peony extract, in order to provide a basis for the content detection of the total glucosides of white peony extract.

[0148] 1. Instruments and reagents

[0149] 1.1 Instrument

[0150] Agilent 1260 high performance liquid chromatograph, DAD detector; Mettler XSE104 electronic analytical balance; Mettler MS205DU electronic analytical balance; Deyisheng 040S ultrasonic cleaning machine.

[0151] 1.2 Drug testing

[0152] Paeoniflorin (batch number 202202, purity ≥ 98%), Wuhan Tianzhi Biotechnology Co., Ltd.; hydroxypaeoniflorin (batch number 202101, purity ≥ 98%), Chengdu Zhibiao Huachun Biotechnology Co., Ltd.; peony flower glycosides (batch number 202204, purity ≥ 98%), Baike Biotechnology (Shanghai) Co., Ltd.; paeoniflorin (batch number 111702-202107, content 98.4%), China Food and Drug Inspection Institutes; benzoylpaeoniflorin (batch number 202301, purity ≥ 98%), Sichuan Weikeqi Biotechnology Co., Ltd. Acetonitrile and phosphoric acid were chromatographically pure, water was homemade purified water, and methanol was analytically pure.

[0153] 2 Methods and Results

[0154] 2.1 Solution preparation

[0155] 2.1.1 Reference solution

[0156] Take paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycoside and benzoylpeoniflorin reference substances, accurately weigh them, add 70% methanol to prepare a mixed reference substance solution containing 840 μg / mL, 120 μg / mL, 90 μg / mL, 120 μg / mL and 150 μg / mL of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycoside and benzoylpeoniflorin, respectively.

[0157] 2.1.2 Test solution

[0158] Preparation of total glycosides of white peony extract: (1) 500 g of white peony root was placed in an oven and dried at 35°C for 6 hours, then the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r·min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0159] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution;

[0160] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz, adding 70% ethanol as an extracting solution at a solid-liquid ratio of 1 g:9 mL, extracting for 35 min each time, extracting three times in total, combining the extracts, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use;

[0161] (4) taking the extract D obtained in step (2) and the extract E obtained in step (3) and combining them, mixing them evenly, and performing column chromatography separation, the chromatography column used is 20 cm × 2 cm, the filler is magnesium silicate, the eluent is a mixed solvent of ethanol and methanol in a ratio of 70:30, the liquid-to-solid ratio is 1:10, the flow rate is 10 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain filtrate F;

[0162] (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, using a polyamide composite membrane with a pore size of 0.1 μm, a membrane thickness of 30 μm, and a membrane area of ​​200 m 2, the operating pressure is 2 MPa, and the solution obtained by membrane separation is freeze-dried to obtain the total glycosides extract of white peony to be tested.

[0163] Take about 0.5 g of the above-mentioned total glycosides of white peony extract powder, weigh it accurately, put it in a stoppered conical flask, accurately add 50 ml of 70% methanol, stopper it, weigh it, ultrasonically treat it (power 250 W, frequency 50 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0164] 2.2 Chromatographic conditions

[0165] Chromatographic column: Waters Atlantis T3 (4.6×250mm, 5μm); mobile phase: acetonitrile-0.1% phosphoric acid solution; gradient elution, 0-5min: 5% acetonitrile; 5-25min: 5%→25% acetonitrile; 25-40min: 25%→32% acetonitrile; column temperature: 35℃; flow rate: 1.0mL / min; detection wavelength: 225nm; injection volume: 10μL. See attached Figure 8 , i.e. HPLC chromatogram, wherein: Figure A is the mixed reference; Figure B is the test sample of white peony total glucosides extract, Peak 1 is: paeoniflorin, Peak 2 is: hydroxypeoniflorin, Peak 3 is: paeoniflorin, Peak 4 is: paeoniflorin lactone glycoside, Peak 5 is: benzoylpeoniflorin.

[0166] 2.3 Precision test

[0167] The mixed reference solution in item 2.1.1 was sampled and analyzed continuously for 6 injections. The RSDs of the peak areas of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone and benzoylpeoniflorin measured after 6 consecutive injections were 1.45%, 1.06%, 1.79%, 0.98% and 1.42%, respectively, indicating good injection precision.

[0168] 2.4 Investigation of linear relationship

[0169] Take paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycoside and benzoylpeoniflorin reference substances, add 70% methanol to prepare mixed reference substance stock solutions with concentrations of 2383.50μg / mL, 183.15μg / mL, 166.20μg / mL, 235.05μg / mL and 244.80μg / mL for paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycoside and benzoylpeoniflorin, respectively, take the mixed reference substance stock solution and dilute it with 70% methanol to form a series of linear reference substance solutions, and inject the sample for detection. Use concentration (μg / mL) as the abscissa and peak area as the ordinate to perform regression and obtain a linear equation. The results are shown in Table 5.

[0170] Table 5 Linear regression results of the five components

[0171]

[0172] 2.5 Stability test

[0173] Take the powder of total glucosides of white peony extract numbered S1 to prepare a test solution, place it at room temperature for 0, 4, 8, 12, 18 and 24 hours, and then inject it for detection. The peak area RSDs of the five components, paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycosides and benzoylpeoniflorin, were calculated, and the results were 1.92%, 2.01%, 1.28%, 1.77% and 1.09%, respectively, indicating that the test solution was stable at room temperature for 24 hours.

[0174] 2.6 Repeatability test

[0175] Six portions of total glucosides of white peony extract powder numbered S1 were taken, and the test solution was prepared according to the method in 2.1.2. The samples were injected and analyzed, and the contents of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycosides and benzoylpeoniflorin in each portion of the test sample were calculated. Results The average contents of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycosides and benzoylpeoniflorin in the six test samples were 84.39 mg / g (RSD=1.84%), 10.86 mg / g (RSD=1.34%), 8.94 mg / g (RSD=2.03%), 15.18 mg / g (RSD=1.99%) and 12.87 mg / g (RSD=2.17%), respectively, indicating that the method had good repeatability.

[0176] 2.7 Recovery test

[0177] Take 6 portions of total glycosides of white peony extract powder numbered S1, take about 0.25g of each portion, accurately weigh, place in a stoppered conical flask, and add 1.5mL of mixed reference solution (the concentrations of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycosides and benzoylpeoniflorin in the mixed reference solution are 14.286mg / mL,

[0178] 1.824mg / mL, 1.515mg / mL, 2.469mg / mL and 2.106mg / mL), prepare the test solution according to the method under 2.1.2, inject and analyze, and calculate the results. The results are shown in Table 6.

[0179] Table 6 Sample recovery results (n = 6)

[0180]

[0181] 2.8 Sample testing

[0182] The total glucosides of white peony extract were sampled 5 times and the contents of the 5 components were tested. The results are shown in Table 7.

[0183] Table 7 Contents of five components in the total glycosides of white peony extract (mg / g)

[0184]

[0185] 3 Discussions

[0186] The full wavelength scanning method was used to collect the absorption spectrum in the range of 200-400nm. The results showed that paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin and benzoylpeoniflorin all had strong absorption at 225nm, so the wavelength was selected as 225nm. In this study, the chromatographic column was optimized, and the Waters Atlantis T3 column was finally selected to make the highly polar paeoniflorin and hydroxypeoniflorin have better retention, and the separation of the five index components in the chromatogram of the test sample can meet the quantitative requirements.

[0187] The extraction method of the test solution was investigated, and the results showed that 70% methanol ultrasonic treatment could completely extract paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin and benzoylpeoniflorin from the total glycosides of white peony. This experiment simultaneously detected the five components in the total glycosides of white peony extract. The method is simple and accurate, and can provide a basis for the content detection of the total glycosides of white peony extract.

[0188] Experiment 3: Comparative study on the purity and content of total glycosides of white peony extract prepared by different extraction and purification methods

[0189] 1 Preparation of white peony root extracts by different extraction and purification methods

[0190] 1.1 Drug to be tested S1:

[0191] Prescription: White Peony Root 500g;

[0192] Preparation method: (1) 500 g of white peony root was placed in an oven at 35°C and dried for 6 hours, then the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r·min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0193] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D; mix the precipitate C with the extract D, and freeze-dry to obtain the test drug S1; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid was prepared as a solution.

[0194] 1.2 Drug to be tested S2:

[0195] Prescription: White Peony Root 500g;

[0196] Preparation method: (1) 500 g of white peony root was placed in an oven at 35°C and dried for 6 hours, then the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r·min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0197] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution;

[0198] (3) Take the precipitate C obtained in step (2) and perform ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz. Add 70% ethanol as the extracting solution at a solid-liquid ratio of 1 g:9 mL. Each extraction lasts 35 min, and a total of three extractions are performed. The extracts are combined, filtered, and ethanol is recovered from the filtrate to obtain extract E. Extract D is mixed with extract E, and freeze-dried to obtain the test drug S2.

[0199] 1.3 Drug to be tested S3:

[0200] Prescription: White Peony Root 500g;

[0201] Preparation method: (1) 500 g of white peony root was placed in an oven at 35°C and dried for 6 hours, then the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r·min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0202] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution;

[0203] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz, adding 70% ethanol as an extracting solution at a solid-liquid ratio of 1 g:9 mL, extracting for 35 min each time, extracting three times in total, combining the extracts, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use;

[0204] (4) Combine the extract D obtained in step (2) and the extract E obtained in step (3), mix well, and separate by column chromatography. The chromatography column used is 20 cm × 2 cm, the filler is magnesium silicate, the eluent is a mixed solvent of ethanol and methanol in a ratio of 70:30, the liquid-to-solid ratio is 1:10, the flow rate is 10 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain a filtrate F. The filtrate F is freeze-dried to obtain the test drug S3.

[0205] 1.4 Drug to be tested S4:

[0206] Prescription: White Peony Root 500g;

[0207] Preparation method: (1) 500 g of white peony root was placed in an oven at 35°C and dried for 6 hours, then the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r·min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0208] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution;

[0209] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz, adding 70% ethanol as an extracting solution at a solid-liquid ratio of 1 g:9 mL, extracting for 35 min each time, extracting three times in total, combining the extracts, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use;

[0210] (4) taking the extract D obtained in step (2) and the extract E obtained in step (3) and combining them, mixing them evenly, and performing column chromatography separation, the chromatography column used is 20 cm × 2 cm, the filler is magnesium silicate, the eluent is a mixed solvent of ethanol and methanol in a ratio of 70:30, the liquid-to-solid ratio is 1:10, the flow rate is 10 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain filtrate F;

[0211] (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, using a polyamide composite membrane with a pore size of 0.1 μm, a membrane thickness of 30 μm, and a membrane area of ​​200 m 2 , the operating pressure is 2 MPa, and the solution obtained by membrane separation is freeze-dried to obtain the drug S4 to be tested.

[0212] 2. Detection Method

[0213] The HPLC method provided in the above Experiment 2 of the present invention was used to detect the contents of the five components in the above white peony extract (ie, test drug S1, test drug S2, test drug S, test drug S3).

[0214] 3. Test results

[0215] The test results are shown in Table 8.

[0216] Table 8 Contents of five components in white peony root extracts obtained by different extraction and purification methods (mg / g)

[0217]

[0218] 4 Conclusion

[0219] As can be seen from the experimental results in Table 1, the total glucosides of white peony extract (i.e., test drug S4) obtained by extraction and purification using the preparation method provided by the present invention not only extracts more total glucosides of white peony, but also has a high purity of the extract. This basically explains why the test drugs S3 and S4 provided in this study have a therapeutic effect on premature ovarian failure, among which the therapeutic effect of the test drug S4 is relatively the best, while the test drugs S1 and S2 have basically no therapeutic effect on premature ovarian failure, because the contents of the main active ingredients of the drugs, such as paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycosides, and benzoylpeoniflorin, contained in the test drugs S1 and S2 are too low. BRIEF DESCRIPTION OF THE DRAWINGS

[0220] Figure 1 : Ovarian pathology images of the normal group (×100): The ovaries are of medium size, with clear cortical and medullary structures, a large number of corpora lutea in the ovaries, good development, and large size. Follicles of all levels can be seen (primordial follicles, developing follicles, mature follicles, etc.), a large number of interstitial blood vessels, and no obvious congestion.

[0221] Figure 2 : Ovarian pathological images of the model group (×100): The number of follicles at all stages in the ovarian cortex was significantly reduced, especially the number of developing follicles. The total number of follicles was reduced compared with the normal group, and the number of atretic follicles increased.

[0222] Figure 3 : Ovarian pathological images of Progynova (×100): The total number of follicles and the number of developing follicles were significantly increased compared with the model group, the number of atretic follicles was less than that of developing follicles, and the number of developing follicles at all levels of the cortex increased compared with the model group.

[0223] Figure 4 : Ovarian pathological images of the test drug S1 group (×100): The total number of follicles and the number of developing follicles increased compared with the model group, but the number of atretic follicles was higher, and the number of developing follicles at all levels of the cortex increased slightly compared with the model group.

[0224] Figure 5 : Ovarian pathological images of the test drug S2 group (×100): The total number of follicles and the number of developing follicles increased compared with the model group, but the number of atretic follicles was larger, and the number of developing follicles at all levels of the cortex increased compared with the model.

[0225] Figure 6 : Ovarian pathological images of the test drug S3 group (×100): The total number of follicles and the number of developing follicles increased significantly compared with the model group, and the number of atretic follicles decreased.

[0226] Figure 7 : Ovarian pathological images of the test drug S4 group (×100): The total number of follicles and the number of developing follicles were significantly increased compared with the model group, the number of atretic follicles was reduced, and the number of developing follicles at all levels of the cortex was increased compared with the model group.

[0227] Figure 8 : HPLC chromatogram, where: Figure A is the mixed reference; Figure B is the test sample of the total glycosides of white peony extract, peak 1 is: paeoniflorin, peak 2 is: hydroxypeoniflorin, peak 3 is: paeoniflorin, peak 4 is: paeoniflorin lactone glycosides, peak 5 is: benzoylpeoniflorin DETAILED DESCRIPTION

[0228] Embodiment 1:

[0229] 1. Preparation of total glycosides of white peony root extract:

[0230] (1) 1000 g of white peony root was placed in an oven and dried at 35°C for 6 hours, then the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r / min. -1, the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained;

[0231] (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 hours, centrifuge for 20 minutes at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution;

[0232] (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz, adding 70% ethanol as an extracting solution at a solid-liquid ratio of 1 g:9 mL, extracting for 35 min each time, extracting three times in total, combining the extracts, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use;

[0233] (4) taking the extract D obtained in step (2) and the extract E obtained in step (3) and combining them, mixing them evenly, and performing column chromatography separation, the chromatography column used is 20 cm × 2 cm, the filler is magnesium silicate, the eluent is a mixed solvent of ethanol and methanol in a ratio of 70:30, the liquid-to-solid ratio is 1:10, the flow rate is 10 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain filtrate F;

[0234] (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, using a polyamide composite membrane with a pore size of 0.1 μm, a membrane thickness of 30 μm, and a membrane area of ​​200 m 2 , the operating pressure is 2 MPa, and the solution obtained by membrane separation is freeze-dried to obtain the total glucosides of white peony extract.

[0235] 2. Content detection of total glycosides of white peony extract:

[0236] The above-mentioned total glucosides of white peony extract is detected by the following high performance liquid chromatography method:

[0237] (1) Preparation of reference solution: Paeoniflorin, hydroxypaeoniflorin, paeoniflorin, paeoniflorin lactone glycosides and benzoylpaeoniflorin reference substances were accurately weighed and added with 70% methanol to prepare a mixed reference solution containing 840 μg / mL, 120 μg / mL, 90 μg / mL, 120 μg / mL and 150 μg / mL of paeoniflorin, hydroxypaeoniflorin, paeoniflorin, paeoniflorin lactone glycosides and benzoylpaeoniflorin, respectively;

[0238] (2) Preparation of test solution: Take 0.5 g of total glycosides of white peony extract powder, weigh accurately, place in a stoppered conical flask, add 50 ml of 70% methanol accurately, stopper tightly, weigh, and treat with ultrasound at a power of 250 W and a frequency of 50 kHz for 30 minutes. Let cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the solution;

[0239] (3) Chromatographic conditions: Chromatographic column: Waters Atlantis T3 chromatographic column, specification: 4.6×250 mm, 5 μm, mobile phase: acetonitrile-0.1% phosphoric acid solution; gradient elution, 0 min to 5 min: acetonitrile ratio maintained at 5%, 0.1% phosphoric acid solution ratio maintained at 95%; 6 min to 25 min: acetonitrile ratio linearly increased from 5% to 25%, 0.1% phosphoric acid solution ratio linearly decreased from 95% to 75%; 26 min to 40 min: acetonitrile ratio linearly increased from 25% to 32%, 0.1% phosphoric acid solution ratio linearly decreased from 75% to 68%; column temperature: 35°C; flow rate: 1.0 mL / min; detection wavelength: 225 nm; injection volume: 10 μL;

[0240] (4) Sample injection test: Take 10 μL of the reference solution and the test solution and inject them into the high performance liquid chromatography instrument for detection;

[0241] (5) Test results: The contents of the five main active ingredients in the total glucosides of white peony extract were: paeoniflorin 85.43 mg / g, hydroxypeoniflorin 11.54 mg / g, paeoniflorin 8.86 mg / g, paeoniflorin lactone 15.64 mg / g, and benzoylpeoniflorin 12.96 mg / g.

[0242] 3. Preparation of drug preparations

[0243] Take 2g of the above-mentioned total glucosides of white peony extract, crush it through a 100-mesh sieve, add 20g of starch that has been sieved through a 100-mesh sieve, 50g of dextrin, mix well, sieve through a 100-mesh sieve again, add appropriate amount of 50% ethanol, mix well, granulate through a 16-mesh iron sieve, dry the whole granules below 60°C, add appropriate amount of magnesium stearate, mix well, and tablet to make 1000 tablets.

[0244] 4. Indications: Treatment of premature ovarian failure.

[0245] 5. Dosage and Administration: 2 to 4 tablets at a time, three times a day, after meals.

Claims

1. A total glycosides of white peony extract, characterized in that The total glycosides of white peony root extract is prepared by the following method: (1) Ultrafine grinding of white peony root is performed by using a jet mill to obtain ultrafine white peony root powder A; (2) Take the white peony superfine powder A obtained in step (1), add it to the mixed enzyme solution, stir and extract in a water bath, separate and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction, adding ethanol as an extract, filtering the extract, recovering ethanol from the filtrate, and obtaining an extract E for later use; (4) combining the extract D obtained in step (2) and the extract E obtained in step (3), mixing them evenly, performing column chromatography separation, collecting the eluate, and filtering the solution after column chromatography to obtain a filtrate F; (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, and the solution obtained by membrane separation is freeze-dried to obtain the total glucosides of white peony extract.

2. The total glucosides of white peony extract according to claim 1, characterized in that The total glycosides of white peony root extract is prepared by the following method: (1) After placing the white peony root in an oven for drying, the dried white peony root is taken out and ultrafinely ground using a jet mill to obtain ultrafine white peony root powder A; (2) taking the white peony superfine powder A obtained in step (1), adding it to the mixed enzyme solution, stirring and extracting in a water bath, centrifuging, and collecting the supernatant B and the precipitate C respectively; concentrating the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution is prepared into a solution by cellulase, pectinase, xylanase and citric acid; (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction, adding ethanol as the extracting solution, performing extraction three times in total, combining the extracting solutions, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use; (4) taking the extract D obtained in step (2) and the extract E obtained in step (3) and combining them, mixing them evenly, and separating them by column chromatography, wherein the filler is magnesium silicate and the eluent is a mixed solvent of ethanol-methanol, collecting the eluent, and filtering the solution after column chromatography to obtain a filtrate F; (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, a polyamide composite membrane is selected, and the solution obtained by membrane separation is freeze-dried to obtain the total glucosides of white peony extract.

3. The total glucosides of white peony extract according to claim 2, characterized in that The total glycosides of white peony root extract is prepared by the following method: (1) Place the white peony root in an oven at 30-40°C and dry for 5-7 hours. Take out the dried white peony root and use an air flow mill to perform ultrafine grinding. The air flow pressure of the air flow mill is 1200-1400 kPa and the feed speed is 30-50 r / min. -1 , the classification frequency is 20-30 Hz, the crushing time is 30-50 min, and the white peony superfine powder A is obtained; (2) Take the white peony superfine powder A obtained in step (1), add it to 5-15 times the amount of the mixed enzyme solution, stir and extract in a water bath at 50-60°C for 1-3 hours, centrifuge for 15-25 minutes at a centrifugal speed of 2000-3000 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, and set it aside; the precipitate C is set aside; the mixed enzyme solution comprises 0.5-1.5% cellulase, 0.2-0.6% pectinase, 0.3-0.7% xylanase and 0.02-0.04 mol·L -1 Citric acid is prepared into a solution; (3) taking the precipitate C obtained in step (2), performing ultrasonic-assisted extraction at an extraction temperature of 30 to 40° C. and an ultrasonic frequency of 25 to 35 kHz, adding 65 to 75% ethanol as an extracting solution at a solid-liquid ratio of 1 g:7 to 11 mL, extracting for 25 to 45 min each time, extracting three times in total, combining the extracts, filtering, and recovering ethanol from the filtrate to obtain an extract E for later use; (4) Combine the extract D obtained in step (2) and the extract E obtained in step (3), mix them evenly, and separate them by column chromatography. The chromatographic column used is filled with magnesium silicate, and the eluent is a mixed solvent of ethanol and methanol in a ratio of 60 to 80:40 to 20, with a liquid-to-solid ratio of 1:8 to 12 and a flow rate of 5 to 15 ml / min. Collect the eluent, and filter the solution after column chromatography to obtain a filtrate F. (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, using a polyamide composite membrane with a pore size of 0.1 μm, a membrane thickness of 20 to 40 μm, a membrane area of ​​150 to 250 m2, and an operating pressure of 1 to 3 MPa. The solution obtained by membrane separation is freeze-dried to obtain a total glycosides extract of white peony.

4. The total glucosides of white peony extract according to claim 3, characterized in that The total glycosides of white peony root extract is prepared by the following method: (1) After the white peony root was dried in an oven at 35°C for 6 hours, the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r / min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained; (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 h, centrifuge for 20 min at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution; (3) The precipitate C obtained in step (2) was subjected to ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz. Ethanol with a concentration of 70% was added as an extracting solution at a solid-liquid ratio of 1 g:9 mL. Each extraction lasted for 35 min, and the extraction was performed three times in total. The extracts were combined and filtered, and ethanol was recovered from the filtrate to obtain an extract E for later use. (4) Combine the extract D obtained in step (2) and the extract E obtained in step (3), mix well, and separate by column chromatography. The chromatography column used is 20 cm × 2 cm, the filler is magnesium silicate, the eluent is a mixed solvent of ethanol and methanol in a ratio of 70:30, the liquid-to-solid ratio is 1:10, the flow rate is 10 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain a filtrate F; (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, using a polyamide composite membrane with a pore size of 0.1 μm, a membrane thickness of 30 μm, a membrane area of ​​200 m2, and an operating pressure of 2 MPa. The solution obtained by membrane separation is freeze-dried to obtain the total glycosides of white peony extract.

5. A method for detecting the content of total glucosides of white peony extract, characterized in that: The total glucosides of white peony extract is tested for content by the following method: (1) Preparation of reference solution: accurately weigh the reference substances of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycoside and benzoylpeoniflorin, and add methanol to prepare a mixed reference solution containing paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycoside and benzoylpeoniflorin; (2) Preparation of test solution: Take the powder of total glucosides of white peony extract, weigh it accurately, put it into a stoppered conical flask, add methanol accurately, stopper it tightly, weigh it accurately, treat it with ultrasound, cool it, weigh it accurately again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution; (3) Chromatographic conditions: Column: Waters column, specification: 4.6×250 mm, 5 μm, mobile phase: acetonitrile-0.1% phosphoric acid solution; gradient elution; column temperature: 20-50°C; flow rate: 0.5-2.0 mL / min; detection wavelength: 210-240 nm; injection volume: 5-20 μL; (4) Sample injection test: Take 5-20 μL of the reference solution and the test solution and inject them into the high performance liquid chromatography for detection.

6. The method for detecting the content of the total glucosides of white peony extract according to claim 5, wherein the total glucosides of white peony extract is detected by high performance liquid chromatography as follows: (1) Preparation of reference solution: accurately weigh the reference substances of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone and benzoylpeoniflorin, and add 60-80% methanol to prepare a mixed reference solution containing 820-860 µg / mL, 100-140 µg / mL, 70-110 µg / mL, 100-140 µg / mL and 130-170 µg / mL of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone and benzoylpeoniflorin, respectively; (2) Preparation of test solution: Take 0.4-0.6 g of total glycosides of white peony extract powder, weigh accurately, place in a stoppered conical flask, add 40-60 ml of 60-80% methanol accurately, stopper tightly, weigh, and treat with ultrasound at a power of 240-260 W and a frequency of 40-60 kHz for 20-40 minutes. Let cool, weigh again, make up the lost weight with 60-80% methanol, shake well, filter, and take the filtrate to obtain the test solution. (3) Chromatographic conditions: Chromatographic column: Waters Atlantis chromatographic column, specification: 4.6×250 mm, 5μm, mobile phase: acetonitrile-0.1% phosphoric acid solution; gradient elution, 0min to 5min: acetonitrile ratio is maintained at 5%, 0.1% phosphoric acid solution ratio is maintained at 95%; 6min to 25min: acetonitrile ratio is linearly increased from 5% to 25%, 0.1% phosphoric acid solution ratio is linearly decreased from 95% to 75%; 26min to 40min: acetonitrile ratio is linearly increased from 25% to 32%, 0.1% phosphoric acid solution ratio is linearly decreased from 75% to 68%; column temperature: 30-40℃; flow rate: 0.5-1.5mL / min; detection wavelength: 220-230nm; injection volume is 5-15μL; (4) Sample injection test: Take 5-15 μL of the reference solution and the test solution and inject them into the high performance liquid chromatography for detection.

7. The method for detecting the content of the total glucosides of white peony extract according to claim 6, wherein the total glucosides of white peony extract is detected by high performance liquid chromatography as follows: (1) Preparation of reference solution: accurately weigh the reference substances of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone and benzoylpeoniflorin, and add 70% methanol to prepare a mixed reference solution containing 840 µg / mL, 120 µg / mL, 90 µg / mL, 120 µg / mL and 150 µg / mL of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone and benzoylpeoniflorin, respectively; (2) Preparation of test solution: Take 0.5 g of total glycosides of white peony extract powder, weigh accurately, place in a stoppered conical flask, add 50 ml of 70% methanol accurately, stopper tightly, weigh, and treat with ultrasound at 250 W and 50 kHz for 30 min. Let cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the solution. (3) Chromatographic conditions: Chromatographic column: Waters Atlantis T3 chromatographic column, specification: 4.6×250mm, 5μm, mobile phase: acetonitrile-0.1% phosphoric acid solution; gradient elution, 0min to 5min: acetonitrile ratio is maintained at 5%, 0.1% phosphoric acid solution ratio is maintained at 95%; 6min to 25min: acetonitrile ratio is linearly increased from 5% to 25%, 0.1% phosphoric acid solution ratio is linearly decreased from 95% to 75%; 26min to 40min: acetonitrile ratio is linearly increased from 25% to 32%, 0.1% phosphoric acid solution ratio is linearly decreased from 75% to 68%; column temperature: 35℃; flow rate: 1.0mL / min; detection wavelength: 225nm; injection volume is 10μL; (4) Sample injection test: Take 10 μL of the reference solution and the test solution and inject them into the high performance liquid chromatography for detection.

8. The total glucosides extract of white peony according to any one of claims 1 to 4, characterized in that The total glucosides of white peony extract is prepared into an oral preparation by a conventional pharmaceutical method in pharmacy.

9. The total glucosides of white peony extract according to claim 7, characterized in that The total glucosides of white peony extract is prepared into tablets, pills, soft capsules, hard capsules, oral liquids and granules by conventional pharmaceutical methods.

10. An application of total glucosides of white peony extract in the preparation of an anti-premature ovarian failure drug, characterized in that: The total glycosides of white peony root extract is prepared by the following method: (1) After the white peony root was dried in an oven at 35°C for 6 hours, the dried white peony root was taken out and ultrafinely pulverized using an air flow mill. The air flow pressure of the air flow mill was 1300 kPa and the feed speed was 40 r / min. -1 , the classification frequency is 25Hz, the crushing time is 40min, and the white peony superfine powder A is obtained; (2) Take the white peony superfine powder A obtained in step (1), add it to 10 times the amount of the mixed enzyme solution, stir and extract in a 55°C water bath for 2 h, centrifuge for 20 min at a centrifugal speed of 2500 r / min, and collect the supernatant B and the precipitate C respectively; concentrate the supernatant B to obtain the extract D, which is set aside, and the precipitate C is set aside; the above-mentioned mixed enzyme solution consists of 1.0% cellulase, 0.4% pectinase, 0.5% xylanase and 0.03 mol·L -1 Citric acid is prepared into a solution; (3) The precipitate C obtained in step (2) was subjected to ultrasonic-assisted extraction at an extraction temperature of 35° C. and an ultrasonic frequency of 30 kHz. Ethanol with a concentration of 70% was added as an extracting solution at a solid-liquid ratio of 1 g:9 mL. Each extraction lasted for 35 min, and the extraction was performed three times in total. The extracts were combined and filtered, and ethanol was recovered from the filtrate to obtain an extract E for later use. (4) Combine the extract D obtained in step (2) and the extract E obtained in step (3), mix well, and separate by column chromatography. The chromatography column used is 20 cm × 2 cm, the filler is magnesium silicate, the eluent is a mixed solvent of ethanol and methanol in a ratio of 70:30, the liquid-to-solid ratio is 1:10, the flow rate is 10 ml / min, the eluent is collected, and the solution after column chromatography is filtered to obtain a filtrate F; (5) The filtrate F obtained in step (4) is separated and enriched by a membrane separation system, using a polyamide composite membrane with a pore size of 0.1 μm, a membrane thickness of 30 μm, a membrane area of ​​200 m2, and an operating pressure of 2 MPa. The solution obtained by membrane separation is freeze-dried to obtain the total glycosides of white peony extract.

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