Method for identifying dayuan liquid preparation

Abstract

The invention relates to the field of modernization of traditional Chinese medicines, in particular to a method for identifying a Dayuan liquid preparation. The invention respectively provides thin-layer chromatography identification methods for pogostemon cablin, bupleurum chinense and glabrous sarcandra herb in the daoyuan liquid preparation, the thin-layer chromatography identification methods for the three components are good in specificity, repeatability and stability, and effective quality control of the daoyuan liquid preparation can be realized.

Description

[0001] This application is a divisional application of the Chinese patent application with application date of November 23, 2022 and application number 202211476780.8, entitled "A Dayuan liquid preparation and its preparation and identification method". Technical Field

[0002] The invention relates to the field of modernization of traditional Chinese medicine, and in particular to an identification method of a Dayuan liquid preparation. Background Art

[0003] In recent years, the new coronavirus has ravaged the world. In the thousands of years of precipitation, Chinese medicine for warm disease has accumulated valuable experience in the treatment of infectious diseases. It is believed that when warm disease first occurs, the evil is hidden in the membrane. Patients infected with the new coronavirus often show symptoms and signs of "dampness and turbidity", which is consistent with the characteristics of warm disease. According to the membrane theory, the treatment of such diseases should use drugs that eliminate filth and turbidity to attack the pathogenic evil hidden in the membrane, which is to open the membrane. In the Chinese medicine classic "Discussion on Plague", the Chinese medicine decoction Dayuan Drink is recorded. It is made of seven Chinese medicines: betel nut, magnolia bark, grass fruit, anemarrhena, peony, scutellaria, and licorice. It can open the membrane, eliminate filth and turbidity, and treat the initial stage of warm disease. With the development of traditional Chinese medicine to date, people have developed various types of Dayuan prescriptions such as Chaihu Dayuan Drink and Dayuan Baidu Powder for different clinical needs.

[0004] The dosage forms of traditional Chinese medicine are mainly decoctions, pills, powders, etc. With the development of science and technology, Chinese medicine preparations such as granules and tablets have emerged. The dosage forms of Chinese medicine preparations are aimed at meeting clinical medical needs, and are selected based on factors such as the composition of the medicinal flavor, clinical needs, the target of the medication, the nature of the drug, the prescription dose, and the safety of the drug. For example, for emergency medication, the drug effect should be fast, so injections, sublingual tablets (pills), aerosols and other dosage forms are used. For chronic medication, the drug effect should be long-lasting, and honey pills, pastes, sustained-release tablets, etc. are often used; drug ingredients that are easily decomposed and ineffective by gastric acid should be made into enteric-coated capsules or enteric-coated tablets; when some drugs are unstable when made into liquid preparations, they can be made into powders, tablets, powder injections or oil solutions.

[0005] Traditional Dayuan medicines are generally prepared in decoction form. Decoctions are taken into the human body in liquid form, which is easy to absorb and quick to take effect. However, decoctions are not easy to carry, not easy to store for a long time, and the concentration of effective drug ingredients is low. When it is made into capsules, the volume of the drug extract is too large. To achieve the desired therapeutic effect, a large number of capsules need to be taken, and the drug enters the human body in a solid state, so the effect is relatively slow. When preparing granules, the drug extract is too viscous and the granules are not easy to form. Combining modern extraction and preparation processes, people have developed Dayuan Yinwei Floating Tablets, which improve the problem of inconvenience in carrying, but the release rate of drug ingredients in tablets is lower than that of liquid preparations, and the effect is slower, which is not conducive to rapid control of the disease at the beginning of the epidemic. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for identifying a Dayuan liquid preparation. The Dayuan decoction piece extract is obtained by extracting a Dayuan medicinal material decoction piece composition.

[0007] The Dayuan medicinal material decoction piece composition comprises, by weight, 26.75-27.25 parts of Bupleurum chinense, 26.75-27.25 parts of Patchouli, 17.67-18.5 parts of Perilla frutescens, 19.75-20.50 parts of Platycodon grandiflorum, 12.50-14.00 parts of Honey Ephedra, 26.75-27.25 parts of Polygonatum odoratum, 12.50-14.00 parts of White Wild Marsh, 12.50-14.00 parts of Rhizoma Atractylodis Macrocephalae, 26.75-27.25 parts of Cortex Magnoliae Officinalis, 19.75-20.50 parts of Areca Nut, 26.75-27.25 parts of Rhizoma Anemarrhenae, and 26.75-27. 25 parts, 32.50-34.00 parts of red peony root, 32.50-34.00 parts of scutellaria, 32.50-34.00 parts of thunbergii, 12.50-14.00 parts of cirrhosa, 8.5-9.5 parts of tsaoko, 32.50-34.00 parts of lotus leaf, 19.75-20.50 parts of stir-fried green peel with bran, 32.50-34.00 parts of stir-fried malt, 26.75-27.25 parts of stewed bitter almond, 19.75-20.50 parts of jianqu, 32.50-34.00 parts of swollen joint herb and 21.50-23.00 parts of notopterygium.

[0008] The honey ephedra is prepared by processing ephedra according to the honey-roasting method of honey ephedra described in the Chinese Pharmacopoeia.

[0009] The ginger magnolia bark, bran-fried atractylodes, salt-fried anemarrhena, bran-fried green peel and stewed bitter almonds are prepared by processing magnolia bark, atractylodes, anemarrhena, green peel and bitter almonds according to the ginger-fried, bran-fried, salt-fried and stewed methods described in the general processing rules 0213 of the Chinese Pharmacopoeia.

[0010] The roasted malt is prepared according to the single roasting method described in the general rules for processing in 0213 of the Chinese Pharmacopoeia.

[0011] The Dayuan decoction piece extract needs to be concentrated and then mixed with flavoring agents and preservatives.

[0012] The flavoring agent is xylitol.

[0013] The preservative is potassium sorbate or ethylparaben.

[0014] Preferably, the Dayuan medicinal material decoction piece composition comprises, by weight, 27.00-27.25 parts of northern bupleurum, 27.00-27.25 parts of patchouli, 18.00-18.50 parts of perilla leaves, 20.00-20.25 parts of platycodon, 13.00-13.50 parts of honey ephedra, 27.00-27.25 parts of polygonatum, 13.00-13.50 parts of white mulberry, 13.00-13.50 parts of bran-fried atractylodes, 27.00-27.25 parts of ginger magnolia bark, 20.00-20.25 parts of betel nut, 27.00-20.25 parts of salt anemarrhena, and 13.00-13.50 parts of honey ephedra. 27.25 parts, 33.00-33.75 parts of red peony root, 33.00-33.75 parts of scutellaria, 33.00-33.75 parts of thunbergii bulbs, 13.00-13.50 parts of cirrhosa, 9-9.5 parts of tsaoko, 33.00-33.75 parts of lotus leaf, 20.00-20.25 parts of stir-fried green peel with bran, 33.00-33.75 parts of stir-fried malt, 27.00-27.25 parts of stewed bitter almonds, 20.00-20.25 parts of jianqu, 33.00-33.75 parts of swollen joint wind and 22.00-22.50 parts of notopterygium.

[0015] Preferably, the amount of xylitol added is 6-10% by weight of the original extract concentrate.

[0016] Preferably, the preservative is ethylparaben.

[0017] Furthermore, the amount of xylitol added is 8% by weight of the original extract concentrate.

[0018] Furthermore, the amount of ethylparaben added is up to 0.05% by weight of the original extract concentrate.

[0019] The indicative ingredient of the Dayuan liquid preparation is paeoniflorin (C 23 H 28 O 11 ), hesperidin (C 28 H 34 O 15 ) and baicalin (C 21 H 18 O 11 ).

[0020] The contents of the index components in the Dayuan liquid preparation are as follows: paeoniflorin (C 23 H 28 O 11 )≥0.30mg / mL, hesperidin (C 28 H 34 O 15 )≥0.23mg / mL, baicalin (C 21 H 18 O 11)≥1.4mg / mL.

[0021] The present invention provides a method for preparing the above-mentioned Dayuan liquid preparation, which specifically comprises the following steps:

[0022] The first step is to prepare the Dayuan extract: by weight, add 26.75-27.25 parts of Bupleurum chinense, 26.75-27.25 parts of Patchouli, 17.67-18.5 parts of Perilla frutescens, 19.75-20.50 parts of Platycodon grandiflorum, 12.50-14.00 parts of Ephedra sinica, 26.75-27.25 parts of Polygonatum odoratum, 12.50-14.00 parts of White Vitex, 12.50-14.00 parts of Atractylodes macrocephala with bran, 26.75-27.25 parts of Magnolia officinalis, 19.75-20.50 parts of Areca catechu, 26.75-27.25 parts of Anemarrhena asphodeloides, 32.50-3 4.00 parts, 32.50-34.00 parts of Scutellaria baicalensis, 32.50-34.00 parts of Fritillaria thunbergii, 12.50-14.00 parts of Fritillaria cirrhosa, 8.5-9.5 parts of Amomum villosum, 32.50-34.00 parts of Lotus leaf, 19.75-20.50 parts of Fried green peel with bran, 32.50-34.00 parts of Fried malt, 26.75-27.25 parts of Boiled bitter almond, 19.75-20.50 parts of Jianqu, 32.50-34.00 parts of Sargassum sarmentosum and 21.50-23.00 parts of Notopterygium wilfordii are soaked in 10 times amount of purified water for 1.5-7 hours, and the decoction is filtered out after decoction.

[0023] Step 2, preparing a concentrated Dayuan extract: concentrating the Dayuan extract obtained in the first step to 1.08-1.10 g / ml (60±5° C.);

[0024] The third step is to add flavoring agents and preservatives to the concentrated solution obtained in the second step, add purified water twice the weight of the slices by weight, stir evenly, centrifuge, filter, sterilize and then fill. The speed of the centrifugation step is 5000-10000r / min, and the centrifugation time is 20min.

[0025] Preferably, the soaking time of the medicinal material slices in the first step is 3-7 hours, and the decoction times are not less than 2 times.

[0026] Preferably, the concentration temperature of the Dayuan extract in the second step is 60-80°C.

[0027] Preferably, the rotation speed adopted in the centrifugation step in the third step is 8000r / min.

[0028] Furthermore, the medicinal material slices in the first step are soaked for 3 hours and decocted 3 times.

[0029] Furthermore, the concentration temperature of the Dayuan extract in the second step is 70-80°C.

[0030] The present invention provides a method for identifying the above-mentioned Dayuan liquid preparation, which specifically uses thin layer chromatography to identify Bupleurum chinense, Patchouli and Sarcandra chinensis in the liquid preparation, and comprises the following steps:

[0031] (1) Preparation of test sample solution: When identifying Pogostemon cablin and Bupleurum chinense, the test sample solution was prepared by column chromatography, and when identifying Sarcandra glabra, the test sample solution was prepared by shaking method;

[0032] (2) Preparation of control medicinal material solution: extracting the control medicinal material, and then preparing the extract into a control medicinal material solution;

[0033] (3) Control TLC: Spot the test sample solution and three control medicinal material solutions on the same silica gel plate, and compare the chromatograms of the test sample and control medicinal materials at the same position. The identification of Pogostemon cablin and Bupleurum chinense requires spraying of a color developer.

[0034] Preferably, for the identification method, when preparing the test sample solution for identifying Pogostemon cablin in step (1), the chromatographic column used is an ODS-C18 extraction column, and the column is rinsed with 5 mL of distilled water, 20% ethanol, 60% ethanol and 80% ethanol respectively, and the 60% ethanol layer eluate is collected, evaporated to dryness in a water bath, and the residue is dissolved in 2 mL of methanol; when preparing the test sample solution for identifying Bupleurum chinense, a macroporous adsorption resin is used to load the column (wet column loading), and the column is rinsed with 30 mL of distilled water, 20% ethanol, 60% ethanol and 80% ethanol respectively, and the 80% ethanol layer eluate is collected, evaporated to dryness in a water bath, and the residue is dissolved in 2 mL of methanol; when preparing the test sample solution for identifying Sarcandra sylvestris, the solvent used is ethyl acetate.

[0035] Preferably, for the identification method, when preparing the reference medicinal material extract in step (2), the coarse powders of Pogostemon cablin and Bupleurum chinense are respectively extracted by reflux extraction with distilled water, and the medicinal materials of Sarcandra scabra are extracted by ultrasonic extraction with water; when preparing the reference medicinal material solution from the medicinal material extract, the reference medicinal material extract is taken according to the treatment method of the test sample solution to prepare the reference drug solution.

[0036] Preferably, for the identification method, when spotting in step (3), the developing agent for identifying Pogostemon cablin is toluene-ethyl acetate-n-hexane-formic acid (2:4:8:0.1), the developing agent for identifying Bupleurum chinense is ethyl acetate-ethanol-water-formic acid (10:2:1:0.1), and the developing agent for identifying Sarcandra scabra is an upper layer solution of toluene-ethyl acetate-methanol-water (10:4:2:0.5); the color developer for identifying Pogostemon cablin is a 10% sulfuric acid ethanol solution, and the color developer for identifying Bupleurum chinense is a 2% p-dimethylaminobenzaldehyde 10% sulfuric acid solution.

[0037] Preferably, in the identification method, during the spotting in step (3), the development humidity is controlled to be above 66%, preferably 66%-88%.

[0038] Preferably, in the identification method, when identifying Sarcandra glabra in step (3), the operating temperature is controlled between 5-35°C, preferably at 25°C.

[0039] The present invention also provides a method for detecting the mass content and transfer rate of the active ingredient of the above-mentioned Dayuan liquid preparation, which comprises the following steps:

[0040] (1) Preparation of reference solution: Take appropriate amount of paeoniflorin, hesperidin, and baicalin reference substances, weigh accurately, and add methanol to prepare a mixed reference solution containing 102 μg paeoniflorin, 100 μg hesperidin, and 100 μg baicalin per 1 mL of solution;

[0041] (2) Preparation of test sample solution: extract the original liquid preparation with solvent and filter;

[0042] (3) High performance liquid chromatography analysis: octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B for gradient elution.

[0043] Preferably, for the detection method, the solvent in step (2) is selected from one of methanol, ethanol, 70% methanol, 50% methanol and 70% ethanol.

[0044] Preferably, for the detection method, the extraction in step (2) is performed by ultrasonic extraction or shaking extraction.

[0045] Preferably, for the detection method, the gradient elution in step (3) is divided into four stages, the first stage is the elution period of 0-10 minutes, the volume ratio of A to B is 10:90; the second stage is the elution period of 10-15 minutes, the volume ratio of A to B is 12:88; the third stage is the elution period of 15-35 minutes, the volume ratio of A to B is 20:80; the fourth stage is the elution period of 35-45 minutes, the volume ratio of A to B is 30:70.

[0046] Preferably, for the detection method, in step (3), 230 nm is selected as the detection wavelength for determining the content of paeoniflorin in the Dayuan liquid preparation, and 280 nm is selected as the detection wavelength for determining the content of hesperidin and baicalin in the Dayuan liquid preparation.

[0047] Furthermore, the solvent in step (2) is methanol, and the extraction method is a shaking method.

[0048] The present invention provides another set of technical solutions as follows:

[0049] The present invention provides a method for identifying a Dayuan liquid preparation, specifically, using thin layer chromatography to identify Bupleurum chinense, Pogostemon cablin and Sarcandra chinensis in the preparation;

[0050] The identification comprises the following steps: (1) preparing a test sample solution; (2) preparing a control medicinal material solution; and (3) a control chromatogram.

[0051] Preferably, the preparation method of the test sample solution is different according to the components to be identified: when identifying Pogostemon cablin and Bupleurum chinense, the test sample solution is prepared by column chromatography, and when identifying Sarcandra glabra, the test sample solution is prepared by dissolution.

[0052] Preferably, in the identification method, when preparing the test sample solution of Patchouli, the chromatographic column used is an ODS-C18 extraction column, and the column is rinsed with distilled water, 20% ethanol, 60% ethanol and 80% ethanol in sequence, the 60% ethanol layer eluate is collected, evaporated to dryness, and the residue is dissolved with methanol; when preparing the test sample solution for identification of Bupleurum chinense, a macroporous adsorption resin is used to load the column (wet column loading), the test sample and Bupleurum chinense negative reagent are added respectively, the column is rinsed with distilled water, 20% ethanol, 60% ethanol and 80% ethanol in sequence, the 80% ethanol layer eluate is collected, evaporated to dryness, and the residue is dissolved with methanol.

[0053] Preferably, in the identification method, when preparing the test sample solution of Sarcandra salsa, the solvent used in the dissolution method is ethyl acetate.

[0054] Preferably, the method for preparing the control drug solution comprises the following steps: (1) preparing a control medicinal material extract: taking coarse powders of Pogostemon cablin and Bupleurum chinense respectively and extracting them by reflux with distilled water, and taking medicinal materials of Sarcandra scabra with water and ultrasonic extraction; (2) preparing a control medicinal material solution: taking the control drug extract obtained in the previous step and preparing control drug solutions respectively according to the preparation method of the test sample solution.

[0055] Preferably, 2.0 g of Radix Sarcandrae control medicinal material is taken, added with 50 mL of water and ultrasonicated for 30 minutes, filtered, and the filtrate is extracted twice with ethyl acetate by shaking, each time with 20 mL, the ethyl acetate layer solution is combined, evaporated to dryness in a water bath, and the residue is dissolved with 2 mL of methanol as the control medicinal material solution.

[0056] Preferably, in the chromatography experiment, the developing solvent for identifying Pogostemon cablin is toluene-ethyl acetate-n-hexane-formic acid (2:4:8:0.1), the developing solvent for identifying Bupleurum chinense is ethyl acetate-ethanol-water-formic acid (10:2:1:0.1), and the developing solvent for identifying Sarcandra scabra is an upper layer solution of toluene-ethyl acetate-methanol-water (10:4:2:0.5); the color developer for identifying Pogostemon cablin is a 10% sulfuric acid ethanol solution, and the color developer for identifying Bupleurum chinense is a 2% p-dimethylaminobenzaldehyde 10% sulfuric acid solution.

[0057] Preferably, the development humidity is controlled above 66%.

[0058] Preferably, the humidity is controlled to be 66%-88%.

[0059] Preferably, when identifying Sarcandra glabra, the temperature is controlled between 5-35°C.

[0060] Preferably, when identifying Sarcandra glabra, the temperature is controlled at 25°C.

[0061] Preferably, the liquid preparation is prepared by concentrating, preparing, filtering and finally filling the Dayuan decoction slice extract, and the Dayuan decoction slice extract is prepared by decocting raw medicinal material decoction slices.

[0062] Preferably, the raw medicinal material slices include the following components by weight: 26.75-27.25 parts of northern bupleurum, 26.75-27.25 parts of patchouli, 17.67-18.5 parts of perilla leaves, 19.75-20.50 parts of platycodon, 12.50-14.00 parts of honey ephedra, 26.75-27.25 parts of polygonatum, 12.50-14.00 parts of white mulberry, 12.50-14.00 parts of bran-fried atractylodes, 26.75-27.25 parts of ginger magnolia bark, 19.75-20.50 parts of betel nut, 26.75-2 7.25 parts, 32.50-34.00 parts of red peony root, 32.50-34.00 parts of scutellaria, 32.50-34.00 parts of thunbergii, 12.50-14.00 parts of cirrhosa, 8.5-9.5 parts of tsaoko, 32.50-34.00 parts of lotus leaf, 19.75-20.50 parts of stir-fried green peel with bran, 32.50-34.00 parts of stir-fried malt, 26.75-27.25 parts of stewed bitter almond, 19.75-20.50 parts of jianqu, 32.50-34.00 parts of swollen joint wind and 21.50-23.00 parts of notopterygium.

[0063] Preferably, by weight, the Dayuan liquid preparation comprises 27.00-27.25 parts of Bupleurum chinense, 27.00-27.25 parts of Patchouli, 18.00-18.50 parts of Perilla frutescens, 20.00-20.25 parts of Platycodon grandiflorum, 13.00-13.50 parts of Honey Ephedra, 27.00-27.25 parts of Polygonatum odoratum, 13.00-13.50 parts of White Vitex, 13.00-13.50 parts of Fried Atractylodes lancea, 27.00-27.25 parts of Magnolia officinalis, 20.00-20.25 parts of Areca nut, 27.00-27.25 parts of Salt Anemarrhena. The invention discloses a novel ...

[0064] Preferably, the flavoring agent and preservative are added after the Dayuan decoction piece extract is concentrated.

[0065] Preferably, the flavoring agent is xylitol, and the added amount is 6-10% by weight of the concentrated Dayuan decoction piece extract, preferably 8-10%.

[0066] Preferably, the preservative is potassium sorbate or ethylparaben;

[0067] Preferably, the added amount is 0.04-0.05% by weight of the original decoction piece extract concentrate.

[0068] Beneficial effects achieved by the present invention:

[0069] 1. The Dayuan liquid preparation prepared by the present invention has been studied by many clinical and formulation experts, and the traditional Chinese medicine decoction has been developed into a Chinese patent medicine, which solves the problems of low effective ingredients of Dayuan decoction, inconvenience in carrying and easy deterioration, making it easier for it to play a role in epidemic prevention in patients with mild new coronavirus infection, people in epidemic areas and people in self-isolation at home.

[0070] 2. The Dayuan liquid preparation described in the present invention has a high content of active ingredients (peonidin, baicalin and hesperidin). After high temperature test, strong light irradiation test, accelerated stability test and long-term stability test, the content of peonidin, baicalin and hesperidin is stable, and the clarity and microbial content meet the requirements.

[0071] 3. The present invention provides a quality detection method for the above-mentioned Dayuan liquid preparation, which has strong specificity, good reproducibility, high precision and good stability, and can control the quality of its finished product in a more scientific, feasible, effective and reliable manner, effectively ensuring the clinical efficacy of the preparation and achieving the purpose of the invention of effectively controlling the quality of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The results of thin layer chromatography identification of Radix Scutellariae (from left to right, the spectra correspond to: Dayuan liquid preparation test sample, baicalin reference sample, baicalin reference sample, wogonin reference sample, negative test sample (lacking Radix Scutellariae));

[0073] Figure 2 The results of thin layer chromatography identification of betel nut (from left to right, the chromatograms correspond to: Dayuan liquid preparation test sample, betel nut control medicinal material, negative test sample (lacking betel nut);

[0074] Figure 3 The results of thin layer chromatography identification of patchouli (from left to right, the spectra correspond to: negative test sample (lacking patchouli), patchouli control medicinal material, and Dayuan liquid preparation test sample);

[0075] Figure 4The results of thin layer chromatography identification of Bupleurum chinense (from left to right, the spectra correspond to: negative test sample (lacking Bupleurum chinense), Dayuan liquid preparation test sample, and Bupleurum chinense reference medicinal material);

[0076] Figure 5 The results of thin layer chromatography identification of Sarcandra glabra (from left to right, the chromatograms correspond to: negative test sample (lacking Bupleurum chinense), Dayuan liquid preparation test sample, isoflurane reference substance, Sarcandra glabra reference medicinal material);

[0077] Figure 6 The results of thin layer chromatography identification of red peony root (from left to right, the corresponding spectra are: Dayuan liquid preparation test sample, paeoniflorin reference sample, negative test sample (lacking red peony root));

[0078] Figure 7 The results of thin layer chromatography identification of fried green peel of bran (from left to right, the corresponding spectra are: Dayuan liquid preparation test sample, hesperidin reference substance, negative test sample (fried green peel without bran));

[0079] Figure 8 To achieve the original liquid preparation identification method, the sample volume of the thin layer chromatography of Sarcandra sylvestris was investigated (from left to right, the graphs correspond to: 1, 2, 4, 6 μL of the test solution, and 2 μL of the Sarcandra sylvestris control drug);

[0080] Fig. 9 To investigate the amount of patchouli sampled in the thin layer chromatography method for the identification of the original liquid preparation (from left to right, the graphs correspond to: 3 μL of patchouli control drug sampled, 2, 3, 4, and 5 μL of the test solution sampled);

[0081] Fig.10 To investigate the amount of sample spotting for the thin layer chromatography of Radix Bupleuri in the original liquid preparation identification method (from left to right, the graphs correspond to: 1, 2, 4, 6 μL of sample spotting for the test solution, and 2 μL of sample spotting for Radix Bupleuri control drug);

[0082] Fig.11 To achieve the original liquid preparation identification method, the thin layer chromatograms of different brands of silica gel G plates were used to examine the sample of Scutellaria baicalensis (among which a is a thin layer chromatogram spotted using Qingdao Ocean Silica G Plate; b is a thin layer chromatogram spotted using Qingdao High-efficiency Silica G Plate; c is a thin layer chromatogram spotted using Sinopharm Silica G Plate; d is a thin layer chromatogram spotted using German Merck Plate);

[0083] Fig.12 To investigate the thin layer chromatograms of patchouli spotted on different brands of silica gel G plates in the identification method of the original liquid preparation (a is a thin layer chromatogram spotted on Qingdao Ocean Silica G plate, b is a thin layer chromatogram spotted on Qingdao High-efficiency Silica G plate, c is a thin layer chromatogram spotted on Sinopharm Silica G plate, and d is a thin layer chromatogram spotted on German Merck plate);

[0084] Fig.13 To investigate the thin layer chromatograms of different brands of silica gel G plates spotted with Bupleurum chinense in the identification method of the original liquid preparation (a is a thin layer chromatogram spotted with Qingdao Ocean Silica G Plate; b is a thin layer chromatogram spotted with Qingdao High-efficiency Silica G Plate; c is a thin layer chromatogram spotted with Sinopharm Silica G Plate; d is a thin layer chromatogram spotted with German Merck Plate);

[0085] Fig.14 To achieve the original liquid preparation identification method, the thin layer chromatograms of Radix Achyranthis Bidentatae at different temperatures were investigated (a is the thin layer chromatogram at 5°C, b is the thin layer chromatogram at 25°C, and c is the thin layer chromatogram at 35°C);

[0086] Fig.15 To investigate the thin layer chromatograms of Pogostemon cablin at different temperatures in the identification method of the original liquid preparation (a is the thin layer chromatogram at 5°C, b is the thin layer chromatogram at 25°C, and c is the thin layer chromatogram at 35°C);

[0087] Fig.16 To investigate the thin layer chromatograms of Radix Bupleuri at different temperatures in the identification method of the original liquid preparation (a is the thin layer chromatogram at 5°C, b is the thin layer chromatogram at 25°C, and c is the thin layer chromatogram at 35°C);

[0088] Fig.17 To achieve the original liquid preparation identification method, the thin layer chromatograms of Radix Achyranthis Bidentatae at different humidity levels were investigated (a is the thin layer chromatogram at a humidity of 33%, b is the thin layer chromatogram at a humidity of 66%, and c is the thin layer chromatogram at a humidity of 88%);

[0089] Fig.18 To investigate the thin layer chromatograms of patchouli at different humidity levels in the identification method of the original liquid preparation (a is the thin layer chromatogram at a humidity of 33%, b is the thin layer chromatogram at a humidity of 66%, and c is the thin layer chromatogram at a humidity of 88%);

[0090] Fig.19 To investigate the thin layer chromatograms of Radix Bupleuri at different humidity levels in the identification method of the original liquid preparation (a is the thin layer chromatogram at a humidity of 33%, b is the thin layer chromatogram at a humidity of 66%, and c is the thin layer chromatogram at a humidity of 88%);

[0091] Figure 20-1 The 190-400nm UV absorption diagram of the paeoniflorin control solution in the main component content detection method of the original liquid preparation;

[0092] Figure 20-2 The ultraviolet absorption diagram of the test solution sample at the absorption wavelength of paeoniflorin in the method for detecting the content of the main components in the Weidayuan liquid preparation;

[0093] Figure 20-3 The 190-400nm UV absorption diagram of the hesperidin control solution in the main component content detection method of the original liquid preparation;

[0094] Figure 20-4 The ultraviolet absorption diagram of the test solution sample at the hesperidin absorption wavelength in the method for detecting the content of the main components in the original liquid preparation;

[0095] Figure 20-5 The 190-400nm UV absorption diagram of the baicalin control solution in the main component content detection method of the original liquid preparation;

[0096] Figure 20-6 The ultraviolet absorption diagram of the test solution sample at the baicalin absorption wavelength in the method for detecting the content of the main components in the original liquid preparation;

[0097] Figure 21-1 The HPLC chromatogram of the methanol blank solvent in the method for detecting the content of the main components in the original liquid preparation;

[0098] Figure 21-2 The mixed standard HPLC chromatogram of the reference sample solution of paeoniflorin, baicalin and hesperidin in the main component content detection method of Weidayuan liquid preparation;

[0099] Figure 21-3 The HPLC chromatogram of the test solution in the method for detecting the content of the main components in the original liquid preparation;

[0100] Figure 21-4 The HPLC comparison chart of Scutellaria baicalensis negative sample solution and mixed standard control solution in the main component content detection method of Weidayuan liquid preparation;

[0101] Figure 21-5 The HPLC comparison chart of negative sample solution and mixed standard reference solution of fried green peel with bran in the main component content detection method of Weidayuan liquid preparation;

[0102] Figure 21-6 The HPLC comparison chart of red peony root negative sample solution and mixed standard control solution in the main component content detection method of Weidayuan liquid preparation;

[0103] Figure 22-1 To achieve a linear standard curve for examining the linear relationship between the concentration of paeoniflorin and the peak area in the concentration range in the main component content detection method of the original liquid preparation;

[0104] Figure 22-2 To achieve a linear standard curve for examining the linear relationship between hesperidin concentration and peak area within the concentration range in the main component content detection method of the original liquid preparation;

[0105] Figure 22-3 To achieve a linear standard curve for examining the linear relationship between the concentration of baicalin and the peak area in the concentration range in the main component content detection method of the original liquid preparation;

[0106] Figure 23-1 The HPLC chromatogram of the test sample liquid used to examine the durability of the No. 1 chromatographic column in the main component content detection method of the original liquid preparation;

[0107] Figure 23-2 The HPLC chromatogram of the test sample liquid used to examine the durability of the No. 2 chromatographic column in the main component content detection method of the original liquid preparation;

[0108] Figure 23-3 The HPLC chromatogram of the test sample liquid used to examine the durability of the No. 3 chromatographic column in the main component content detection method of the original liquid preparation;

[0109] Figure 23-4 This is the HPLC chromatogram of the test sample liquid used to investigate the durability of chromatographic column No. 4 in the detection method for the content of the main components in the original liquid preparation. DETAILED DESCRIPTION

[0110] 1. As mentioned above, the present invention provides a liquid preparation of Dayuan, the preparation method of which comprises the following steps:

[0111] The first step is to prepare the Dayuan extract: by weight, add 26.75-27.25 parts of Bupleurum chinense, 26.75-27.25 parts of Patchouli, 17.67-18.5 parts of Perilla frutescens, 19.75-20.50 parts of Platycodon grandiflorum, 12.50-14.00 parts of Honey Ephedra, 26.75-27.25 parts of Polygonatum odoratum, 12.50-14.00 parts of White Vitex, 12.50-14.00 parts of Fried Atractylodes lancea, 26.75-27.25 parts of Magnolia officinalis, 19.75-20.50 parts of Areca nut, 26.75-27.25 parts of Salt Anemarrhena, 32.50-34.00 parts of Red Peony Root, 32.50-34.00 parts of Scutellaria baicalensis, 32.50-34.00 parts of Fritillaria thunbergii, 12.50-14.00 parts of Fritillaria cirrhosa, 8.5-9.5 parts of Amomum villosum, 32.50-34.00 parts of Lotus leaf, 19.75-20.50 parts of stir-fried green peel with bran, 32.50-34.00 parts of stir-fried malt, 26.75-27.25 parts of stewed bitter almonds, 19.75-20.50 parts of Jianqu, 32.50-34.00 parts of Sargassum scutellariae and 21.50-23.00 parts of Notopterygium wilfordii are soaked in purified water 10 times the total weight of the medicinal material slices for 1.5-7 hours, and the decoction is filtered out after decoction;

[0112] Step 2, preparing a concentrated Dayuan extract: concentrating the Dayuan extract obtained in the first step to 1.08-1.10 g / ml (60±5° C.);

[0113] The third step is to add flavoring agents and preservatives to the concentrated solution obtained in the second step, add purified water twice the weight of the slices by weight, stir evenly, centrifuge, filter, sterilize and then fill. The speed of the centrifugation step is 5000-10000r / min, and the centrifugation time is 20min.

[0114] In a preferred embodiment of the present invention, the medicinal material slices are soaked for 3-7 hours in the first step, and the decoction times are not less than 2 times.

[0115] In a preferred embodiment of the present invention, the concentration temperature of the Dayuan extract in the second step is 60-80°C.

[0116] In a preferred embodiment of the present invention, the rotation speed adopted in the centrifugation step in the third step is 8000r / min.

[0117] Preferably, the medicinal material slices are soaked for 3 hours and decocted 3 times in the first step.

[0118] Preferably, the concentration temperature of the Dayuan extract in the second step is 70-80°C.

[0119] 2. The present invention provides a method for identifying the Dayuan liquid preparation, which is specifically identified by detecting Bupleurum chinense, Pogostemon cablin and Sarcandra chinensis by thin layer chromatography, and the identification steps are as follows:

[0120] (1) Preparation of test sample solution: When identifying Pogostemon cablin and Bupleurum chinense, the test sample solution was prepared by column chromatography, and when identifying Sarcandra glabra, the test sample solution was prepared by shaking method;

[0121] (2) Preparation of control medicinal material solution: extracting the control medicinal material, and then preparing the extract into a control medicinal material solution;

[0122] (3) Control TLC: Spot the test sample solution and three control medicinal material solutions on the same silica gel plate, and compare the chromatograms of the test sample and control medicinal materials at the same position. The identification of Pogostemon cablin and Bupleurum chinense requires spraying of a color developer.

[0123] In a preferred specific embodiment of the present invention, when preparing the test sample solution for identifying Pogostemon cablin in step (1), the chromatographic column used is an ODS-C18 extraction column, and the column is rinsed with 5 mL of distilled water, 20% ethanol, 60% ethanol and 80% ethanol respectively, and the 60% ethanol layer eluate is collected, evaporated to dryness in a water bath, and the residue is dissolved in 2 mL of methanol; when preparing the test sample solution for identifying Bupleurum chinense, a macroporous adsorption resin is used to load the column (wet column loading), and the column is rinsed with 30 mL of distilled water, 20% ethanol, 60% ethanol and 80% ethanol respectively, and the 80% ethanol layer eluate is collected, evaporated to dryness in a water bath, and the residue is dissolved in 2 mL of methanol; when preparing the test sample solution for identifying Sarcandra sylvestris, the solvent used is ethyl acetate.

[0124] In a preferred embodiment of the present invention, for the identification method, when preparing the reference medicinal material extract in step (2), the crude powders of Patchouli and Bupleurum chinense are respectively extracted with distilled water by reflux, and the medicinal material of Sarcandra scabra is extracted with water by ultrasonic extraction; when preparing the reference medicinal material solution from the medicinal material extract, the reference medicinal material extract is taken according to the treatment method of the test sample solution to prepare the reference drug solution.

[0125] In a preferred specific embodiment of the present invention, during the spotting in step (3), the developing agent for identifying Pogostemon cablin is toluene-ethyl acetate-n-hexane-formic acid (2:4:8:0.1), the developing agent for identifying Bupleurum chinense is ethyl acetate-ethanol-water-formic acid (10:2:1:0.1), and the developing agent for identifying Sarcandra scabra is an upper layer solution of toluene-ethyl acetate-methanol-water (10:4:2:0.5); the color developer for identifying Pogostemon cablin is a 10% sulfuric acid ethanol solution, and the color developer for identifying Bupleurum chinense is a 2% p-dimethylaminobenzaldehyde 10% sulfuric acid solution.

[0126] In a preferred embodiment of the present invention, during the spotting in step (3), the development humidity is controlled to be above 66%, preferably 66%-88%.

[0127] In a preferred embodiment of the present invention, during the step (3) of identifying Sarcandra glabra, the operating temperature is controlled between 5-35°C, preferably at 25°C.

[0128] 3. The present invention also provides a method for detecting the mass content and transfer rate of the active ingredient of the above-mentioned Dayuan liquid preparation, which comprises the following steps:

[0129] (1) Preparation of reference solution: Take appropriate amount of paeoniflorin, hesperidin, and baicalin reference substances, weigh accurately, and add methanol to prepare a mixed reference solution containing 102 μg paeoniflorin, 100 μg hesperidin, and 100 μg baicalin per 1 mL;

[0130] (2) Preparation of test sample solution: extract the original liquid preparation with solvent and filter;

[0131] (3) High performance liquid chromatography analysis: octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B for gradient elution.

[0132] In a preferred embodiment of the present invention, the solvent in step (2) is selected from one of methanol, ethanol, 70% methanol, 50% methanol and 70% ethanol, preferably methanol.

[0133] In a preferred embodiment of the present invention, the extraction in step (2) is carried out by ultrasonic extraction or shaking extraction, preferably shaking extraction.

[0134] In a preferred embodiment of the present invention, the gradient elution in step (3) is divided into four stages, the first stage is the 0-10 minutes of elution, the volume ratio of A to B is 10:90; the second stage is the 10-15 minutes of elution, the volume ratio of A to B is 12:88; the third stage is the 15-35 minutes of elution, the volume ratio of A to B is 20:80; the fourth stage is the 35-45 minutes of elution, the volume ratio of A to B is 30:70.

[0135] In a preferred embodiment of the present invention, in step (3), 230 nm is selected as the detection wavelength for determining the content of paeoniflorin in the Dayuan liquid preparation, and 280 nm is selected as the detection wavelength for determining the content of hesperidin and baicalin in the Dayuan liquid preparation.

[0136] The manufacturers of the raw materials used in this embodiment are described as follows. The information of the raw materials and the experimental equipment used in the examples are shown in Table 1 and Table 2, respectively.

[0137] Table 1 Information on the raw materials used in the examples

[0138]

[0139]

[0140] Table 2 Experimental equipment used in the examples

[0141]

[0142]

[0143] Example 1

[0144] 1. Preparation of Extract

[0145] Take 12g of Northern Bupleurum, 12g of Patchouli, 8g of Perilla Leaf, 9g of Platycodon, 6g of Honey Ephedra, 12g of Polygonatum, 6g of White Wei, 6g of Fried Atractylodes lancea with bran, 12g of Magnolia officinalis with ginger, 9g of Areca, 12g of Salt Anemarrhena, 15g of Red Peony Root, 15g of Scutellaria, 15g of Fritillaria thunbergii, 6g of Fritillaria cirrhosa, 4g of Amomum villosum, 15g of Lotus Leaf, 9g of Fried Citrus aurantium with bran, 15g of Fried malt, 12g of Stewed Bitter Almond, 9g of Jianqu, 15g of Sarcandra and 10g of Notopterygium wilfordii, and soak them in 8 times the amount of purified water for 3 hours.

[0146] After boiling for 0.5 hours, add 6 times the amount of water, continue boiling for 0.5 hours, add 6 times the amount of water again, boil for 0.5 hours, filter out, and combine the decoctions obtained from the three decoctions.

[0147] 2. Preparation of Concentrate

[0148] The decoction obtained in the previous step was concentrated by rotary evaporation at 70°C until the concentration of the concentrate reached about 1.08 g / ml at a temperature of 60±5°C.

[0149] 3. Liquid preparation, filtration and filling

[0150] Take 35g of the concentrate obtained in the previous step, add 6% xylitol and 0.05% potassium sorbate by weight of the concentrate, dissolve with 450mL purified water, stir evenly, centrifuge at 10000r / min for 10min, filter and bottle the obtained filtrate.

[0151] Example 2

[0152] 1. Preparation of Extract

[0153] Take 12g of Northern Bupleurum, 12g of Patchouli, 8g of Perilla Leaf, 9g of Platycodon, 6g of Honey Ephedra, 12g of Polygonatum, 6g of White Wei, 6g of Fried Atractylodes, 12g of Magnolia Bark, 9g of Areca, 12g of Salt Anemarrhena, 15g of Red Peony Root, 15g of Scutellaria, 15g of Fritillaria Thunbergii, 6g of Fritillaria Sichuan, 4g of Amomum villosum, 15g of Lotus Leaf, 9g of Fried Citrus aurantium, 15g of Fried Malt, 12g of Stewed Bitter Almond, 9g of Jianqu, 15g of Sarcandra and 10g of Notopterygium. Soak them in 10 times the amount of purified water for 1.5 hours.

[0154] After boiling for 2 hours, add 8 times the amount of water, continue boiling for 2 hours, add 8 times the amount of water again, boil for 2 hours, filter out, and combine the decoctions obtained from the three decoctions.

[0155] 2. Preparation of Concentrate

[0156] The decoction obtained in the previous step was concentrated by rotary evaporation at 80°C until the concentration of the concentrate reached about 1.08 g / ml.

[0157] 3. Liquid preparation, filtration and filling

[0158] Take 35g of the concentrate obtained in the previous step, add 8% xylitol and 0.05% ethylparaben (dissolved in a small amount of ethanol) of the concentrate, dissolve with 450mL of purified water, stir evenly, centrifuge at 8000r / min for 20min, filter and bottle the obtained filtrate.

[0159] Comparative Example 1

[0160] 1. Preparation of Extract

[0161] Take 12g of Northern Bupleurum, 12g of Patchouli, 8g of Perilla Leaf, 9g of Platycodon, 6g of Honey Ephedra, 12g of Polygonatum, 6g of White Wei, 6g of Fried Atractylodes, 12g of Magnolia Bark, 9g of Areca, 12g of Salt Anemarrhena, 15g of Red Peony Root, 15g of Scutellaria, 15g of Fritillaria Thunbergii, 6g of Fritillaria Sichuan, 4g of Amomum villosum, 15g of Lotus Leaf, 9g of Fried Green Peel, 15g of Fried Malt, 12g of Stewed Bitter Almond, 9g of Jianqu, 15g of Sarcandra and 10g of Notopterygium. Soak them in 8 times the amount of purified water for 0.5 hour.

[0162] After boiling for 2 hours, add 6 times the amount of water, continue boiling for 2 hours, filter out and combine the decoctions obtained from the two decoctions.

[0163] 2. Preparation of Concentrate

[0164] The decoction obtained in the previous step was concentrated by rotary evaporation at 60°C until the concentration of the concentrate reached about 1.08 g / ml.

[0165] 3. Liquid preparation, filtration and canning

[0166] Take 35g of the concentrate obtained in the previous step, add 4% xylitol and 0.04% potassium sorbate by weight of the concentrate, dissolve with 450mL of purified water, stir evenly, centrifuge at 8000r / min for 10min, filter and bottle the filtrate.

[0167] Comparative Example 2

[0168] 1. Preparation of Extract

[0169] Take 12g of Northern Bupleurum, 12g of Patchouli, 8g of Perilla Leaf, 9g of Platycodon, 6g of Honey Ephedra, 12g of Polygonatum, 6g of White Wei, 6g of Fried Atractylodes, 12g of Magnolia Bark, 9g of Areca, 12g of Salt Anemarrhena, 15g of Red Peony Root, 15g of Scutellaria, 15g of Fritillaria Thunbergii, 6g of Fritillaria Sichuan, 4g of Amomum villosum, 15g of Lotus Leaf, 9g of Fried Green Peel, 15g of Fried Malt, 12g of Stewed Bitter Almond, 9g of Jianqu, 15g of Sarcandra and 10g of Notopterygium. Soak them in 6 times the amount of purified water for 3 hours.

[0170] After boiling for 2 hours, filter out the decoction.

[0171] 2. Preparation of Concentrate

[0172] The decoction obtained in the previous step was concentrated by rotary evaporation at 70°C until the concentration of the concentrate reached about 1.08 g / ml.

[0173] 3. Liquid preparation, filtration and filling

[0174] Take 35g of the concentrate obtained in the previous step, add 8% xylitol and 0.04% ethylparaben (dissolved in a small amount of ethanol) of the concentrate, dissolve with 450mL of purified water, stir evenly, centrifuge at 5000r / min for 20min, filter and bottle the obtained filtrate.

[0175] Results Analysis

[0176] In a specific embodiment of the present invention, the evaluation indexes for the evaluation of the extraction process are as follows:

[0177] (1) Water absorption rate of decoction pieces:

[0178]

[0179] Where M2 is the mass (g) of the slice in the water-saturated state, and M1 is the mass (g) of the slice in the dry state.

[0180] (2) Active ingredient transfer rate:

[0181] Transfer rate % = (content of index component in drug solution * weight of drug solution) / (content of index component in decoction piece * weight of decoction piece)

[0182] The transfer rate of active ingredients indicates the content of active ingredients in the extract. The higher the transfer rate, the better the extraction effect.

[0183] (3) Extract / concentrate paste yield:

[0184] Paste yield % = (extract or concentrate volume × extract or concentrate solid content) / slice volume

[0185] The solid content calculation method of the extract / concentrate refers to the "hot immersion method" in the general rules (2201) of Part 4 of the 2020 edition of the "Chinese Pharmacopoeia" for the determination of extracts: accurately weigh 10 g of the extract / concentrate, place it in an evaporating dish with constant weight, evaporate it to dryness on a water bath, dry it at 105°C for 3 hours, cool it in a desiccator for 30 minutes, quickly and accurately weigh the weight, and calculate the solid content.

[0186] (4) Content of active ingredients in the product:

[0187] The content of the active ingredients paeoniflorin, baicalin and hesperidin in the product was determined by high performance liquid chromatography. The specific detection method is: using octadecylsilane bonded silica gel as a filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); using acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, eluting according to the following table; temperature is 30°C, flow rate is 0.3 mL / min; paeoniflorin detection wavelength is 230 nm, hesperidin and baicalin detection wavelength is 280 nm; injection volume is 1 μL. The theoretical plate number calculated based on the paeoniflorin peak should not be less than 13,000.

[0188] The experimental products of Examples 1, 2 and Comparative Examples 1, 2 are numbered 1, 2, 3, and 4. In the extract preparation experiment, the effect of the extraction process is investigated from three aspects: the water absorption rate of the slices, the transfer rate of paeoniflorin, baicalin and hesperidin in the extract, and the paste rate of the extract; in the concentrate preparation experiment, the effect of the concentration process is investigated from two aspects: the transfer rate of paeoniflorin, baicalin and hesperidin in the concentrate and the paste rate of the concentrate; in the liquid preparation and product molding experiment stage, the effect of the flavoring agent, preservative and centrifugation process on the product and the effect of the process method on the content of the active ingredient in the product are investigated from four aspects: the number of aerobic bacteria in the product, the taste of the product, the clarity of the product, and the content of the active ingredient in the product. The evaluation results are shown in Table 3 below.

[0189] Table 3 Summary of experimental results

[0190]

[0191]

[0192] Comparing the experimental results of Examples 1 and 2 and Comparative Examples 1 and 2, the soaking time of the slices in Example 1 and Comparative Example 4 is 3 hours, and the water absorption rate reaches about 170%. The soaking time in Example 2 is 1.5 hours, and the water absorption rate is about 160%. The slices in Comparative Example 1 are soaked for 0.5 hours, and the water absorption rate is 110%. It can be seen that when the soaking time is short (0.5-1.5h), the extension of the soaking time can effectively improve the water absorption rate of the slices, and the water absorption rate can be increased by 40% by increasing the soaking time by one hour; when the soaking time is long (1.5-3h), the extension of the soaking time has no significant effect on the water absorption rate of the slices. The water absorption rate of soaking for 3 hours is only about 10% higher than that of soaking for 1.5 hours. Therefore, extending the soaking time after the soaking time exceeds 3 hours does not significantly improve the water absorption rate of the slices, and the soaking time is preferably 3 hours.

[0193] In the process of extract preparation, although the water absorption rate of the slices of comparative example 2 is greater than that of the slices of embodiment 2 and comparative example 1, the paste yield (17.43%) and the transfer rate of effective ingredients (peony glycoside 64.54%, baicalin 33.00%, hesperidin 11.26%) of comparative example 2 are the lowest; the number of extractions of embodiments 1 and 2 is 3 times, the number of extractions of comparative example 1 is 2 times, the number of extractions of comparative example 2 is 1 time, the decoction time of embodiment 1 is 0.5 hours, and the decoction time of embodiment 2 and comparative example 1 is 2 hours. The obtained extract has an extract yield of 26.81% in Example 1, 32.39% in Example 2, and 21.44% in Comparative Example 1. The effective component transfer rates of Example 1 are 87.21% for paeoniflorin, 56.43% for baicalin, and 21.67% for hesperidin. The effective component transfer rates of Example 2 are 97.34% for paeoniflorin, 64.27% for baicalin, and 42.58% for hesperidin. The effective component transfer rates of Comparative Example 1 are 70.61% for paeoniflorin, 40.12% for baicalin, and 17.35% for hesperidin. The evaluation results show that the extraction scheme of Example 2 has the best extraction effect.

[0194] In the preparation process of the concentrated solution, the concentration temperature of Example 1 is 70°C, the concentration temperature of Example 2 is 80°C, the concentration temperature of Comparative Example 1 is 60°C, and the concentration temperature of Comparative Example 2 is 70°C. The paste yield of the obtained concentrated solution is about 18%; the transfer rate of paeoniflorin in the concentrated solution at 80°C is slightly higher than that at 70°C, while the transfer rates of baicalin and hesperidin are higher at 70°C; the concentration temperatures described in Examples 1 and 2 both have good extraction effects.

[0195] During the product preparation and filtration process, the preservative used in Example 1 is potassium sorbate, and the preservative used in Example 2 is ethyl hydroxybenzoate, and the added amount is 0.05% of the concentrate by weight. The preservative used in Comparative Example 1 is potassium sorbate, and the added amount is 0.04% of the concentrate, and the preservative used in Comparative Example 2 is ethyl hydroxybenzoate, and the added amount is also 0.04%. When the preservative addition amount is 0.05%, the total number of aerobic bacteria is less than 10 cfu / mL. When the preservative addition amount is 0.04%, the aerobic bacteria count is 145 cfu / mL with potassium sorbate as the preservative, and the aerobic bacteria count is 90 cfu / mL with ethyl hydroxybenzoate as the preservative. It can be seen that the preservative effect of ethyl hydroxybenzoate is better than that of potassium sorbate, and the amount of preservative added should be 0.05% of the weight of the concentrate.

[0196] The amount of xylitol added in Example 2 and Comparative Example 2 is 8% of the concentrated solution, the amount added in Example 1 is 6%, and the amount added in Comparative Example 2 is 4%. The products obtained in Example 2 and Comparative Example 2 have a sweet taste, while the taste of Example 1 is slightly sweet, and the taste of the product obtained in Comparative Example 1 is slightly bitter. Therefore, the amount of xylitol added should be no less than 6% of the concentrated extract, and 8% is better.

[0197] After the liquid preparation is completed, centrifugation is performed. The centrifugal process parameters of Example 1 are centrifugation at a rate of 10000 r / min for 10 minutes, Example 2 is centrifuged at a rate of 8000 r / min for 20 minutes, Comparative Example 1 is centrifuged at a rate of 8000 r / min for 10 minutes, and Comparative Example 2 is centrifuged at a rate of 5000 r / min for 20 minutes. The liquids of Example 1 and Comparative Example 1 are relatively clear, the liquid obtained in Comparative Example 2 is relatively turbid, and the liquid obtained in Example 2 is clear. It can be seen that the centrifugal effects of 10000 r / min and 8000 r / min are similar, which is better than 5000 r / min.

[0198] In summary, the method of the present invention is as follows: soak the Dayuan slices for 3 hours, extract them 3 times, concentrate the extract at 70°C to 80°C to a density of 1.08-1.10 g / ml (60±5°C), add more than 6% xylitol and 0.05% ethyl hydroxybenzoate by mass of the concentrate, add purified water twice the mass of the slices and stir evenly, stir the obtained concentrate and centrifuge at a rate of 8000 r / min for 20 minutes, filter and fill. The obtained Dayuan mixture has a transfer rate of 97.34% of paeoniflorin, 64.27% of baicalin and 42.58% of hesperidin in the extract.

[0199] The pilot test was carried out according to the above process method, and the identification experiment, main component detection experiment and stability experiment were carried out on the pilot test results.

[0200] 1. Identification experiment

[0201] The drugs used in the identification experiment of the Dayuan liquid preparation are shown in Table 4 below, and the materials and equipment used in the identification experiment are shown in Table 5 below.

[0202] Table 4 Drugs used in the original liquid preparation identification experiment

[0203]

[0204]

[0205] Table 5 Materials and equipment for the original liquid preparation identification experiment

[0206]

[0207] 1.1 Investigation of identification ingredients

[0208] (1) Take 5 mL of this product and scutellaria baicalensis negative reagent, respectively, and dilute them with 5 mL of distilled water. Extract the diluted solution twice with 20 mL of ethyl acetate, collect the ethyl acetate layer solution, evaporate to dryness, and dissolve the residue in 5 mL of ethyl acetate, which are used as the test solution and negative control solution, respectively. Separately take baicalin, baicalein, and wogonin reference standards, add methanol to make solutions containing 1 mg baicalin, 0.5 mg baicalein, and 0.5 mg wogonin per 1 mL of solution, respectively, as reference solution. Take 2 μL of each of the above solutions, spot them on the same silica gel GF254, use toluene-ethyl acetate-methanol-formic acid (10:3:1:2) as the developing agent, develop, take out, dry, and examine under ultraviolet light (254 nm). See the results. Figure 1 In the chromatogram of the test sample, there are corresponding color spots with baicalin and baicalein, but both have negative interference. The same color spot appears at the corresponding position of the chromatogram of wogonin.

[0209] (2) Take 5 mL of this product and 5 mL of betel nut negative reagent, respectively, and dilute them with 5 mL of distilled water. Extract the dilution twice with 20 mL of ethyl acetate, collect the ethyl acetate layer solution, evaporate to dryness, and dissolve the residue in 5 mL of ethyl acetate, as the test solution and negative control solution, respectively. Take 1 g of betel nut control medicinal material coarse powder, add 25 mL of distilled water, reflux extract for 1 hour, cool, filter, and take the filtrate. Extract the filtrate twice with 25 mL of ethyl acetate, collect the ethyl acetate layer solution, evaporate to dryness, and dissolve the residue in 5 mL of ethyl acetate, as the control medicinal material solution. Take 2 μL of each of the above two solutions, spot them on the same silica gel G thin layer plate, use toluene-ethyl acetate-formic acid (4:1:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C to develop color until the spots are clear, and place under ultraviolet light (365nm) for inspection. Results are shown in Figure 2 In the test sample chromatogram, fluorescent spots of the same color appeared at the corresponding positions in the control medicinal material chromatogram, but the thin layer chromatography spots were not clear and the separation was poor.

[0210] (3) Take 5 mL of this product and 5 mL of patchouli negative reagent, respectively, and pass them through ODS-C18 extraction columns. Rinse the columns with 5 mL of distilled water, 20% ethanol, 60% ethanol and 80% ethanol, respectively. Collect the 60% ethanol layer eluate, evaporate to dryness in a water bath, and dissolve the residue in 2 mL of methanol as the test solution and negative control solution. Take another 2.0 g of patchouli control medicinal material powder, add 25 mL of distilled water and reflux for extraction for 30 minutes, cool and filter, concentrate the filtrate to 5 mL, and then treat it according to the above method to prepare the control medicinal material solution. Take 3 μL of each of the above solutions and spot them on the same silica gel plate, use toluene-ethyl acetate-n-hexane-formic acid (2:4:8:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat until the spots are clear, and place under ultraviolet light (365 nm) for inspection. Results are shown in Figure 3In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position in the chromatogram of the control medicinal material, and there is no interference from the negative sample.

[0211] (4) Take a macroporous adsorption resin column (wet column loading), add 5 mL of the test sample and the northern bupleurum negative reagent respectively, rinse the column with 30 mL of distilled water, 20% ethanol, 60% ethanol and 80% ethanol respectively, collect the 80% ethanol layer eluate, evaporate it in a water bath, and dissolve the residue in 2 mL of methanol as the test solution and negative control solution. Take another 2.0 g of the northern bupleurum control medicinal material crude powder, add 25 mL of distilled water and reflux extract for 30 minutes, cool and filter, concentrate the filtrate to 5 mL, and then treat it according to the above method to obtain the control medicinal material solution. Take 2 μL of each of the above solutions and spot them on the same silica gel plate, use ethyl acetate-ethanol-water-formic acid (10:2:1:0.1) as the developing solvent, develop, take out, dry, spray with 2% p-dimethylaminobenzaldehyde in 10% sulfuric acid solution, heat until the spots are clear, and place under ultraviolet light (365nm) for inspection. The results are as follows Figure 4 In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position in the chromatogram of the control medicinal material, and there is no interference from the negative sample.

[0212] (5) Take 5 mL of this product and 5 mL of negative reagent of Sarcandra syringae, dilute them with 5 mL of distilled water respectively, extract the dilution with ethyl acetate by shaking twice, 20 mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue with 5 mL of ethyl acetate, and use them as the test solution and negative control solution respectively. Take another 2.0 g Sarcandra syringae control medicinal material, add 50 mL of distilled water and ultrasonicate for 30 minutes, filter, extract the filtrate with ethyl acetate by shaking twice, 20 mL each time, combine the ethyl acetate layer solution, evaporate to dryness in a water bath, dissolve the residue with 2 mL of methanol, and use it as the control medicinal material solution. Take another isoflurane reference substance, add methanol to make a solution containing 0.5 mg of isoflurane per 1 mL, and use it as the reference substance solution. Take 2 μL of each of the above solutions, spot them on the same silica gel plate, use the upper layer solution of toluene-ethyl acetate-methanol-water (10:4:2:0.5) as the developing agent, develop, take out, dry, and place under ultraviolet light (365 nm) for inspection. Figure 5 In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatograms of the reference medicinal material and the reference sample.

[0213] (6) Take 5 mL of this product and red peony root negative reagent, dilute with 5 mL of distilled water respectively, add saturated n-butanol to shake and extract twice, 20 mL each time, combine the n-butanol extracts, evaporate to dryness, add 5 mL of methanol to dissolve the residue, and use them as the test solution and negative control solution respectively. Take another paeoniflorin reference substance, add ethanol to make a solution containing 2 mg per 1 mL, as the reference substance solution. Take 2 μL of each of the above solutions, spot them on the same silica gel plate, use chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2) as the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, heat until the spots are clear, and place under sunlight for inspection. The results are as follows Figure 6 In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material. The chromatographic spots have no negative interference, but the separation is poor.

[0214] (7) Take 5 mL of this product and 5 mL of the negative reagent of bran-deficient fried green peel, add them to the treated polyamide column (4 g), elute with 40 mL of water, 20% ethanol, and 80% ethanol, collect the 80% ethanol eluate, evaporate to dryness, add 5 mL of methanol to the residue to dissolve it, and use it as the test solution and negative control solution. Take the hesperidin reference substance, add methanol to make a saturated solution, and use it as the reference substance solution. Take 2 μL of each of the above solutions, and spot them on the same polyamide film, use ethyl acetate-methanol-water (100:17:13) as the developing agent, develop to 3 cm, take out, dry, and then use toluene-ethyl acetate-formic acid-water (20:10:1:1) as the developing agent, develop 8 cm, take out, dry, spray with aluminum chloride test solution, dry, and examine under ultraviolet light (365 nm). The results are as follows Figure 7 In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material, and the separation degree of the chromatographic spots is poor.

[0215] From the above results, it can be seen that when using thin layer chromatography to identify the Dayuan liquid preparation, Pogostemon cablin, Bupleurum chinense and Sarcandra chinensis should be used as identification ingredients.

[0216] 1.2 Methodological validation of identification methods

[0217] (1) Methodological verification of sample size

[0218] a. Verification of the method of spotting the amount of Sargassum syringae: According to the sample processing and development method in the above Sargassum syringae identification method, 1μL, 2μL, 4μL and 6μL of the test sample were spotted on the same silica gel G plate, and the results were examined by the above development method. Figure 8 When the sample volume is 2μL or above, the spots at the corresponding positions of the test sample and the control medicinal material are clear and there is no other interference. Therefore, the sample volume for identification of Sarcandra glabra is 2μL.

[0219] b. Verification of the method of spotting amount of patchouli: According to the sample processing and development method in the above patchouli identification method, 2μL, 3μL, 4μL and 5μL of the test sample were spotted on the same silica gel G plate, and the results were examined by the above development method. Fig. 9 When the spotting volume is 3 μL or more, the spots at the corresponding positions of the test sample and the control medicinal material are clear and there is no other interference. After comprehensive consideration, the spotting volume is selected as 3 μL.

[0220] c. Verification of the method of spotting amount of Bupleurum chinense: According to the sample processing and development method in the above identification method of Bupleurum chinense, 1μL, 2μL, 4μL and 6μL of the test sample were spotted on the same silica gel G plate, and the results were examined by the above development method. Fig.10 When the spotting volume is 3 μL or more, the spots at the corresponding positions of the test sample and the control medicinal material are clear and there is no other interference. After comprehensive consideration, the spotting volume is selected as 3 μL.

[0221] (2) Investigation of different thin layer plates

[0222] a. Qingdao Ocean Silica G Plate, Qingdao Ocean High-efficiency G Plate, Sinopharm Group Silica G Plate and German Merck Plate were used as carriers, and the above-mentioned method of swollen knotweed was used to examine the durability of different thin layer plates. The results are shown in Fig.11 , using different thin layer plates (Qingdao Ocean Silica G plate, Qingdao Ocean High-efficiency G plate, Sinopharm Group Silica G plate, German Merck plate), the chromatograms of the test samples of Sarcandra syringae showed spots of the same color at the corresponding positions of the chromatograms of the Sarcandra syringae reference medicinal materials and the chromatograms of the isoflurane reference substance, with clear color development, good separation, no tailing phenomenon, and no background interference.

[0223] b. Qingdao Ocean Silica G Plate, Qingdao Ocean High-efficiency G Plate, Sinopharm Group Silica G Plate and German Merck Plate were used as carriers, and the above-mentioned patchouli development method was used to examine the durability of different thin layer plates. The results are shown in Fig.12 Different thin layer plates (Qingdao Ocean Silica G plate, Qingdao Ocean High-efficiency G plate, Sinopharm Group Silica G plate, German Merck plate) were used for development. The chromatograms of the patchouli test products showed spots of the same color as the corresponding positions of the chromatograms of the patchouli control medicinal materials. The color was clear, the separation was good, there was no tailing phenomenon, and there was no background interference.

[0224] c. Qingdao Ocean Silica G Plate, Qingdao Ocean High-efficiency G Plate, Sinopharm Silica G Plate and German Merck Plate were used as carriers, respectively, and the above-mentioned Bupleurum chinense development method was used to examine the durability of different thin layer plates. The results are shown in Fig.13, the spots were not clear and the separation was poor when developed using the Sinopharm Silica G plate. When developed using other different thin layer plates (Qingdao Ocean Silica G plate, Qingdao Ocean High-Efficiency G plate, German Merck plate), the chromatograms of the test samples of Bupleurum chinense showed spots of the same color as the chromatograms of the reference medicinal materials of Bupleurum chinense at the corresponding positions. The color was clear, the separation was good, there was no tailing phenomenon, and there was no background interference.

[0225] Therefore, when identifying the Chinese medicinal material ingredients of the Dayuan liquid preparation, Qingdao Ocean Silica G Plate, Qingdao Ocean High-Efficiency G Plate and German Merck Plate can be selected as carriers.

[0226] (3) Temperature investigation: Investigate the applicability of the identification method at 5°C, 25°C, and 35°C.

[0227] a. Use the above identification method to carry out the inspection, the results are shown in Fig.14 Under different temperature conditions, although the chromatogram of the test sample of Sarcandra sylvestris showed spots of the same color at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the isoflurane reference substance, the Rf values ​​at 5℃ and 35℃ were low, and the Rf value at 25℃ was appropriate, so the temperature needs to be controlled at 25℃ when this method is carried out.

[0228] b. The above patchouli identification method was used to carry out the inspection, and the results are shown in Fig.15 Under different temperature conditions, spots of the same color appeared at the corresponding positions of the test sample chromatogram and the control medicinal material chromatogram, and the main spots were clearly colored, with good separation, no tailing phenomenon, and no background interference, indicating that temperature had no obvious effect on the thin layer identification of patchouli in Dayuan Disinfection Mixture.

[0229] c. The results are shown in the following table using the above identification method for Bupleurum chinense. Fig.16 Under different temperature conditions, spots of the same color appeared at the corresponding positions of the test sample chromatogram and the control medicinal material chromatogram, and the main spots were clearly colored, with good separation, no tailing phenomenon, and no background interference, indicating that temperature had no obvious effect on the thin layer identification of Bupleurum chinense in Dayuan Disinfection Mixture.

[0230] Therefore, when identifying the medicinal ingredients of the Dayuan liquid preparation, the identification temperature is selected to be 25°C.

[0231] (4) Different humidity investigation: The applicability of the method was investigated at 33%, 66% and 88% humidity.

[0232] a. Carry out inspection according to the above-mentioned method for identification of Sarcandra serrata, and the results are as follows Fig.17Under different humidity conditions, although the chromatograms of the test sample of Sarcandra scabra, the chromatograms of the reference medicinal materials and the chromatograms of the isoflurane reference substance showed spots of the same color at the corresponding positions, the Rf value under 33% humidity conditions was low and the separation was poor, while the Rf values ​​under 66% and 88% humidity were appropriate and the separation was good, so the humidity needed to be controlled at 66% or above when this method was implemented.

[0233] b. Use the above-mentioned patchouli identification method to carry out the inspection, the results are as follows Fig.18 Under different humidity conditions, spots of the same color appeared at the corresponding positions of the test sample chromatogram and the control medicinal material chromatogram, and the main spots were clearly colored, with good separation, no tailing phenomenon, and no background interference. Humidity had no obvious effect on the thin layer identification of patchouli in the Dayuan liquid preparation.

[0234] c. Use the above identification method to check the results of Bupleurum chinense. Fig.19 The test sample chromatogram and the control medicinal material chromatogram showed spots of the same color at the corresponding positions, and the main spots were clearly colored, with good separation, no tailing phenomenon, and no background interference, indicating that humidity had no obvious effect on the thin layer identification of Bupleurum chinense in the Dayuan liquid preparation.

[0235] Therefore, when identifying the medicinal material ingredients in the Dayuan liquid preparation, the humidity should be controlled above 66%.

[0236] 2. Detection method of main component content

[0237] In the study of the main component content detection method, the drugs used are shown in Table 6 and the equipment used are shown in Table 7.

[0238] Table 6 List of drugs used in the detection method research

[0239]

[0240]

[0241] Table 7 List of equipment used for detection method research

[0242]

[0243] 2.1 Investigation of testing conditions

[0244] (1) Investigation of absorption wavelength

[0245] The UV absorption spectra of the reference solution of paeoniflorin, hesperidin, baicalin and the sample solution at 190nm to 400nm were recorded respectively. Figure 20-1 to Figure 20-6The chromatographic peak of the paeoniflorin reference solution has the maximum ultraviolet absorption at about 230nm, and the chromatographic peaks of the hesperidin and baicalin reference solutions have the maximum ultraviolet absorption at about 280nm; the corresponding chromatographic peaks of the test sample paeoniflorin have the maximum ultraviolet absorption at about 230nm, and hesperidin and baicalin have the maximum absorption at about 280nm, so 230nm and 280nm are confirmed to be the optimal absorption wavelengths for the determination of paeoniflorin, hesperidin and baicalin in the Dayuan liquid preparation, respectively.

[0246] (2) Investigation of chromatographic conditions

[0247] Octadecylsilane bonded silica gel was used as filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid water was used as mobile phase B, and elution was performed according to Table 8 below; temperature was 30°C, flow rate was 0.3 mL / min; the detection wavelength for paeoniflorin was 230 nm, and the detection wavelength for hesperidin and baicalin was 280 nm; injection volume was 1 μL. The theoretical plate number calculated based on the paeoniflorin peak should be no less than 13,000.

[0248] Table 8 Elution gradient table

[0249]

[0250]

[0251] (3) Investigation of extraction solvent

[0252] Accurately measure 3 mL of the test sample and place it in a 25 mL volumetric flask. Add appropriate amounts of extraction solvents such as methanol, 70% methanol, 50% methanol, 30% methanol, ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and water in sequence, shake well, add appropriate amounts of solvents to 25 mL in sequence, shake well, and filter. Accurately pipette 1 μL of each sample solution and inject it into the liquid chromatograph to calculate the content of baicalin, hesperidin, and paeoniflorin. The experimental results are shown in Table 9.

[0253] Table 9 Results of investigation of extraction solvents

[0254]

[0255] In the experiment, the test solution extracted with water as the solvent is difficult to filter and the test solution will produce precipitation after filtration. Considering the impact on the chromatographic column, water is not considered in the comparison range. From the above results, it can be seen that when methanol and ethanol are used as extraction solvents, the content of each component gradually decreases with the increase of water content; among them, when methanol is used as the extraction solvent, the content of each measured component is the highest, so methanol is selected as the best extraction solvent.

[0256] (4) Investigation of extraction methods

[0257] Take 3 mL of the test sample, accurately measure two groups, 2 portions each, and place them in a 25 mL volumetric flask. Add an appropriate amount of methanol to one group, shake well, ultrasonicate for 30 min, add methanol to 25 mL, and shake well; add an appropriate amount of methanol to the other group, shake well, make up to 25 mL, shake well, filter, and get it. Accurately draw 1 μL, inject into the liquid chromatograph, measure, and calculate the content of baicalin, hesperidin, and paeoniflorin. The results are shown in Table 10.

[0258] Table 10 Results of the investigation of extraction methods

[0259]

[0260] It can be seen from the table that ultrasound or shaking has no significant effect on the extraction content. Considering that the direct shaking extraction method is simpler, shaking is chosen.

[0261] 2.2 Methodological investigation of detection methods

[0262] (1) Specificity inspection

[0263] Take 1 μL of the test solution, the reference solution and the methanol blank solvent respectively, and inject them into the liquid chromatograph for detection. The results are as follows: Figure 21-1 to Figure 21-6 In the blank solvent chromatogram, there were no chromatographic peaks at the corresponding retention times of paeoniflorin, hesperidin and baicalin; the negative sample was prepared according to the same process, and there was no interference in the determination of its corresponding chromatographic peaks, indicating that the solvent had no interference in the determination of the sample content. This method is specific for determining the contents of paeoniflorin, hesperidin and baicalin in Dayuan Disinfection Mixture.

[0264] (2) Linear investigation

[0265] Take appropriate amount of paeoniflorin, hesperidin and baicalin reference substances, dissolve in methanol to make up to volume, and prepare paeoniflorin reference substance solutions with concentrations of 0.12106 mg, 0.10592 mg, 0.09079 mg, 0.07566 mg, 0.06053 mg and 0.03026 mg per 1 mL of solution; and paeoniflorin reference substance solutions with concentrations of 0.12200 mg, 0.10675 mg, 0.09150 mg, 0.07625 mg and 0.06 100mg, 0.03050mg hesperidin reference solution; concentration of baicalin reference solution containing 0.39855mg, 0.34873mg, 0.29891mg, 0.24909mg, 0.19927mg, 0.09964mg baicalin per 1mL solution, accurately pipette 1μL of each into the liquid chromatograph, and draw a standard curve with the peak area as the ordinate and the concentration (mg / mL) of paeoniflorin, hesperidin, and baicalin as the abscissa. The results are as follows: Figure 22-1 to Figure 22-3, the linear regression equation of paeoniflorin is y=4140.7x+0.9533, and the correlation coefficient R 2 =0.9992; the linear regression equation of hesperidin is y=5903.8x+8.1371, and the correlation coefficient R 2 =0.9994; the linear regression equation of baicalin is y=10841x+21.095, and the correlation coefficient R 2 =0.9991. The concentrations of the above three components have a good linear relationship with the peak area within the concentration range.

[0266] (3) Precision inspection

[0267] The test solution was taken and the sample was injected 6 times repeatedly to determine the contents of paeoniflorin, hesperidin and baicalin in the test solution, and the RSD% values ​​were calculated. The results are shown in Table 11 below.

[0268] Table 11 Investigation of the precision of the detection method

[0269]

[0270] It can be seen that the RSD% of paeoniflorin and hesperidin content is less than 3%, and the RSD% of baicalin content is less than 2%. According to the "Guidelines for Validation of Analytical Methods" of the 2020 edition of the "Chinese Pharmacopoeia", when the content of the tested component in the sample is greater than 0.1 mg / g, the precision RSD% limit is less than 3%, indicating that the instrument has good precision.

[0271] (4) Stability test

[0272] Six test solutions were prepared and injected at 0 h, 2 h, 4 h, 6 h, 8 h, 16 h and 24 h, respectively. The contents and RSD% values ​​of paeoniflorin, hesperidin and baicalin in the test solutions were determined. The results are shown in Table 12 below.

[0273] Table 12 Stability study of detection method

[0274]

[0275]

[0276] As shown in Table 12, the RSD% of the contents of paeoniflorin and hesperidin were less than 3%, and the RSD% of the contents of baicalin were all less than 2%. The test solution of the Dayuan liquid preparation was relatively stable within 24 hours.

[0277] (5) Repeatability study

[0278] The Dayuan liquid preparation was used to prepare 6 test solutions in parallel, with an injection volume of 1 μL. The contents and RSD% values ​​of paeoniflorin, hesperidin and baicalin in the test solutions were determined, and the results are shown in Table 13 below.

[0279] Table 13 Repeatability of detection method

[0280]

[0281]

[0282] As shown in Table 13, the RSD% of the contents of paeoniflorin and hesperidin were less than 3%, and the RSD% of the content of baicalin was less than 2%, indicating that the analytical method had good repeatability.

[0283] 2.3 Durability test

[0284] (1) Investigation of different chromatographic columns

[0285] Prepare the test solution of Dayuan liquid preparation, accurately measure 1 μL and inject it into the high performance liquid chromatograph to investigate the effect of different chromatographic columns on the determination of paeoniflorin, hesperidin and baicalin in Dayuan liquid preparation. The information of the chromatographic columns used is shown in Table 14.

[0286] Table 14 Chromatographic column information

[0287]

[0288] The test results of different chromatographic columns are shown in Table 15 below, and the chromatograms are shown in Figure 23-1 to Figure 23-4 .

[0289] Table 15 Effect of different chromatographic columns on the determination of index components in preparations

[0290]

[0291] According to Table 15 and Figure 23-1 to Figure 23-4 It can be seen that the durability of different chromatographic columns varies. The separation and symmetry of the chromatographic column 4-Agilent ZORBAX SB C18 (2.1mm×150mm, 1.8μm) are better, so this chromatographic column was used for detection.

[0292] (2) Investigation of different column temperatures

[0293] Accurately take 1 μL of the test sample solution and put it into the high performance liquid chromatograph. The test sample solution was detected at column temperatures of 25°C, 30°C and 35°C respectively to investigate the effects of different column temperatures on the detection contents of paeoniflorin, baicalin and hesperidin in the samples. The measurement results are shown in Table 16 below.

[0294] Table 16 Column temperature investigation

[0295]

[0296] The results showed that the analysis method had poor durability for the content of paeoniflorin at different column temperatures. The separation and symmetry were better when the column temperature was 30℃, so the optimal column temperature of 30℃ was selected.

[0297] (3) Investigation of different flow rates

[0298] Accurately take 1 μL of the test sample solution into the high performance liquid chromatograph, and change the flow rate to 0.28 mL / min, 0.30 mL / min, and 0.32 mL / min respectively. Compare the effects of different flow rates on the detection content of paeoniflorin, hesperidin, and baicalin in the test samples. The test results are shown in Table 17.

[0299] Table 17 Flow rate investigation

[0300]

[0301] The experimental results show that the analytical method has poor durability at different flow rates, and the flow rates of 0.28mL / min and 0.32mL / min have poor separation of paeoniflorin, so the optimal flow rate is 0.30mL / min.

[0302] The content of the active ingredient in the prepared product detected by the detection method can reach an average of 0.421 mg / mL for paeoniflorin, and a range of ±30% of the average of 0.30 to 0.55 mg / mL; the average of baicalin is 1.98 mg / mL, and the range of ±30% of the average is 1.39 to 2.58 mg / mL; the average of hesperidin is 0.415 mg / mL, and the range of ±30% of the average is 0.29 to 0.54 mg / mL.

[0303] 3. Product stability test

[0304] 3.1 Experiment on influencing factors

[0305] (1) High temperature stability test

[0306] The pilot sample opening was placed in a constant temperature device at 60℃±2℃. Sampling was taken on the 0th day, 5th day, 10th day and 30th day respectively to examine the sample density, pH value and active ingredient content. The results are shown in Table 18.

[0307] Table 18 High temperature stability test results

[0308]

[0309] Under high temperature conditions, the density, pH value and active ingredient content of the samples were stable within 30 days, and the clarity and microbial limit were in compliance with the provisions of the General Rules for Preparations of the Chinese Pharmacopoeia.

[0310] (2) Strong light irradiation experiment

[0311] Take the pilot sample and place the opening in a container with an illumination of 4500±500lx, and the total illumination of the light source is not less than 1.2×10 6 lux·hr, the energy of near-ultraviolet lamp is not less than 200W·hr / m2. Samples were taken on the 0th, 5th, 10th and 30th days to investigate the sample density, pH value and active ingredient content. The results are shown in Table 19.

[0312] Table 19 Strong light irradiation stability test results

[0313]

[0314] Under strong light irradiation, the density, pH value and active ingredient content of the samples were stable within 30 days, and the clarity and microbial limit were in compliance with the provisions of the General Rules for Preparations of the Chinese Pharmacopoeia.

[0315] 3.2 Accelerated stability test

[0316] The pilot samples were placed at a temperature of 40°C ± 2°C and a relative humidity of 75% ± 5% for 6 months. Samples were taken at the end of the first, second, third and sixth months of the test to examine the sample density, pH value and active ingredient content. The results are shown in Table 20.

[0317] Table 20 Accelerated stability test results

[0318]

[0319] When stored under high temperature and humidity conditions, the density, pH value and active ingredient content of the samples were stable within 6 months, and the clarity and microbial limits were in compliance with the requirements of the General Rules for Preparations of the Chinese Pharmacopoeia.

[0320] The Dayuan liquid preparation prepared by the method of the present invention is convenient for carrying and transportation as a mixture. After stability experiments, it is proved that the product has stable properties, and the content of the effective ingredients paeoniflorin, baicalin and hesperidin is maintained at a high level. It is not easy to deteriorate and is easy to take. It is suitable for patients with mild new coronavirus infection, people in epidemic areas and people in self-isolation at home for epidemic prevention.

Claims

1. A method for identifying a Dayuan liquid preparation, characterized in that: Specifically, the Bupleurum chinense, Pogostemon cablin and Sarcandra chinensis in the preparation are identified by thin layer chromatography; The identification comprises the following steps: (1) preparing a test sample solution; (2) preparing a control medicinal material solution; and (3) a control chromatogram.

2. The identification method of the Dayuan liquid preparation according to claim 1, wherein: The preparation methods of the test sample solution are different according to the components to be identified: when identifying Pogostemon cablin and Bupleurum chinense, the test sample solution is prepared by column chromatography, and when identifying Sarcandra glabra, the test sample solution is prepared by dissolution method; Preferably, in the identification method, when preparing the patchouli test sample solution, the chromatographic column used is an ODS-C18 extraction column, and the column is washed with distilled water, 20% ethanol, 60% ethanol and 80% ethanol in sequence, and the 60% ethanol layer eluate is collected and evaporated to dryness, and the residue is dissolved with methanol; When preparing the sample solution for identification of Radix Bupleuri, a macroporous adsorption resin is used to load the column (wet column loading), and the test sample and Radix Bupleuri negative reagent are added respectively, and the column is washed with distilled water, 20% ethanol, 60% ethanol and 80% ethanol in sequence, and the 80% ethanol layer eluate is collected and evaporated to dryness, and the residue is dissolved with methanol; Preferably, in the identification method, when preparing the test sample solution of Sarcandra salsa, the solvent used in the dissolution method is ethyl acetate.

3. The identification method of the Dayuan liquid preparation according to claim 1 or 2, wherein: The method for preparing the control drug solution comprises the following steps: (1) preparing a control drug extract solution: taking coarse powders of patchouli and bupleurum chinense respectively and extracting them by reflux with distilled water, and taking herb of sarmentosa lappa by ultrasonic extraction with water; (2) preparing a control drug solution: taking the control drug obtained in the previous step and The extracts were used to prepare control drug solutions according to the preparation method of the test sample solutions.

4. The identification method of the Dayuan liquid preparation according to any one of claims 1 to 3, wherein: In the chromatography experiment, the developing solvent for identifying patchouli is toluene-ethyl acetate-n-hexane-formic acid (2:4:8:0.1), the developing solvent for identifying Bupleurum chinense is ethyl acetate-ethanol-water-formic acid (10:2:1:0.1), and the developing solvent for identifying Sarcandra scabra is the upper layer solution of toluene-ethyl acetate-methanol-water (10:4:2:0.5); the color developer for identifying patchouli is 10% sulfuric acid ethanol solution, and the color developer for identifying Bupleurum chinense is 2% p-dimethylaminobenzaldehyde in 10% sulfuric acid solution.

5. The identification method of the Dayuan liquid preparation according to any one of claims 1 to 4, wherein: The unfolding humidity is controlled above 66%, preferably 66%-88%; And / or when identifying Sarcandra glabra, the temperature is controlled between 5-35°C, preferably at 25°C.

6. The method for identifying the Dayuan liquid preparation according to any one of claims 1 to 5, wherein: The liquid preparation is prepared by concentrating, preparing, filtering and finally filling the Dayuan decoction slice extract, and the Dayuan decoction slice extract is prepared by decocting the raw medicinal material decoction slices.

7. The identification method of the Dayuan liquid preparation according to claim 6, wherein: The raw medicinal material slices include the following components by weight: 26.75-27.25 parts of Bupleurum chinense, 26.75-27.25 parts of Patchouli, 17.67-18.5 parts of Perilla frutescens, 19.75-20.50 parts of Platycodon grandiflorum, 12.50-14.00 parts of Honey Ephedra, 26.75-27.25 parts of Polygonatum odoratum, 12.50-14.00 parts of White Wild Marsh, 12.50-14.00 parts of Fried Atractylodes lancea, 26.75-27.25 parts of Magnolia officinalis, 19.75-20.50 parts of Areca nut, 26.75-27.25 parts of Salt Anemarrhena. 25 parts, 32.50-34.00 parts of red peony root, 32.50-34.00 parts of scutellaria, 32.50-34.00 parts of thunbergii, 12.50-14.00 parts of cirrhosa, 8.5-9.5 parts of tsaoko, 32.50-34.00 parts of lotus leaf, 19.75-20.50 parts of stir-fried green peel with bran, 32.50-34.00 parts of stir-fried malt, 26.75-27.25 parts of stewed bitter almond, 19.75-20.50 parts of jianqu, 32.50-34.00 parts of swollen joint herb and 21.50-23.00 parts of notopterygium.

8. The method for identifying the Dayuan liquid preparation according to claim 6, wherein: By weight, the Dayuan liquid preparation comprises 27.00-27.25 parts of northern bupleurum, 27.00-27.25 parts of patchouli, 18.00-18.50 parts of perilla leaves, 20.00-20.25 parts of platycodon, 13.00-13.50 parts of honey ephedra, 27.00-27.25 parts of polygonatum, 13.00-13.50 parts of white mulberry, 13.00-13.50 parts of bran-fried atractylodes, 27.00-27.25 parts of ginger magnolia bark, 20.00-20.25 parts of betel nut, 27.00-27.25 parts of salt anemarrhena, and 27.00-27.25 parts of salt anemarrhena. The invention discloses a novel herbal medicine comprising the following: 33.00-33.75 parts of red peony root, 33.00-33.75 parts of scutellaria baicalensis, 33.00-33.75 parts of thunbergii bulbs, 13.00-13.50 parts of cirrhosa bulbs, 9-9.5 parts of tsaoko, 33.00-33.75 parts of lotus leaves, 20.00-20.25 parts of stir-fried green peels with bran, 33.00-33.75 parts of stir-fried malt, 27.00-27.25 parts of stewed bitter almonds, 20.00-20.25 parts of jianqu, 33.00-33.75 parts of scutellaria baicalensis, 33.00-33.75 parts of thunbergii bulbs, 13.00-13.50 parts of tsaoko, 9.5 parts of 9. The method for identifying the Dayuan liquid preparation according to any one of claims 1 to 8, wherein: After the Dayuan decoction piece extract is concentrated, a flavoring agent and a preservative are added.

10. The identification method of Dayuan liquid preparation according to claim 9, wherein: The flavoring agent is xylitol, and the added amount is 6-10% by weight of the concentrated Dayuan decoction piece extract, preferably 8-10%.

11. The identification method of the Dayuan liquid preparation according to claim 9 or 10, wherein: The preservative is potassium sorbate or ethylparaben; Preferably, the added amount is 0.04-0.05% by weight of the original decoction piece extract concentrate.

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