CN110618231A

CN110618231A - Preparation process and quality standard research of Baihu decoction granules

Info

Publication number: CN110618231A
Application number: CN201911012778.3A
Authority: CN
Inventors: 闫雪生; 于蓓蓓; 王平; 刘瑾; 孙丹丹; 生立嵩; 李富欣; 王文静; 杜立遥
Original assignee: Shandong Academy of Chinese Medicine
Current assignee: Shandong Academy of Chinese Medicine
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2019-12-27

Abstract

The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a preparation method of a Baihu decoction granule and a quality standard detection method. A method for preparing BAIHUTANG granule with CaSO is provided₄·2H₂0 retention rate, new mangiferin retention rate, mangiferin retention rate and impurity removal rate are considered indexes, the preparation method is determined, the quality of the prepared white tiger soup particles is stable, and the utilization rate of active ingredients is high; the invention establishes a quality control method of the Baihu decoction granules, carries out thin-layer chromatography identification on rhizoma anemarrhenae and radix glycyrrhizae preparata in the preparation, carries out content measurement on the rhizoma anemarrhenae and the radix glycyrrhizae preparata in the preparation, has simple and convenient operation, strong specificity and accurate result, and can be used for the quality control of the Baihu decoction granules.

Description

Preparation process and quality standard research of Baihu decoction granules

Technical Field

The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a preparation method of a Baihu decoction granule and a quality standard detection method.

Background

The Jing Fang is the ancestor of the medical prescriptions, which refers to a prescription recorded in the treatise on the exogenous febrile diseases (classified as the treatise on exogenous febrile diseases and the general lack of supply) by Zhang Zhongjing in the Han Dynasty, and has the characteristics and advantages of "general, simple, cheap and effective". Wherein the Baihu decoction is issued from Shanghai treatise on Shanghai, is a typical prescription for treating yang-Ming-Qi-branch heat excess syndrome, is composed of 4 medicinal materials including gypsum, rhizoma anemarrhenae, radix Glycyrrhizae Preparata and semen oryzae Sativae, and has the effects of clearing heat and promoting fluid production.

As a classic famous prescription in Shanghai treatise on Cold-induced diseases, the white tiger decoction mainly focuses on the influence of different compatibility combinations on the dissolution of chemical components and the clinical application of the white tiger decoction, and the white tiger decoction is prepared into a proper dosage form, so that the aim of new use of ancient prescriptions is achieved.

Currently, there is little doctor in applying the menstruation prescription in modern clinical treatment, which causes the current situation: firstly, the medicine is not used, because the user needs to be familiar with and master the mutual compatibility and dosage of the medicines on the basis of the basic theory of the traditional Chinese medicine, and the experience of clinical practice is realized, so that the medicine has stronger practical and empirical properties; the menstruation is dare not to use, the menstruation side has good effect but is not a universal side, the correct application has good effect on treating diseases, if the menstruation side is misused, the menstruation side has no effect, and the menstruation side can also hurt patients, so the menstruation side is not used as the menstruation side is responsible for the disease; most prescriptions are less in flavor and have economic benefits than traditional prescriptions, especially for the first reason. Due to the rapid progress and development of modern scientific technology and modern medicine, the clinical symptoms of the current are greatly different from the ancient times, and the exertion of the clinical effects of the menstruation recipe is greatly impacted and challenged, so that the basic material research and the clinical practice research of the menstruation recipe are carried out by combining the gist of the original Zhang Zhongjing theory and the current science and technology, and more reference values can be laid for the further development and improvement of the menstruation recipe.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a preparation method of the white tiger decoction particles and a detection method of quality standards, and the quality of the white tiger decoction particles is ensured by using the detection method.

In order to realize the purpose, the invention adopts the following technical scheme:

the preparation method of the white tiger decoction particles comprises the following steps:

(1) weighing gypsum, rhizoma anemarrhenae, radix Glycyrrhizae Preparata and semen oryzae Sativae according to the formula ratio, adding 8 times of water in the 1 st time, soaking for 0.5h, decocting, adding 6 times of water in the 2 nd and 3 rd times of decoction respectively, decocting for 0.5h in the three times, filtering with gauze, mixing the filtrates, and concentrating until the relative density is 1.02;

(2) adding a carboxymethyl chitosan solution into the concentrated filtrate obtained in the step (1) at 60 ℃, stirring for 20 min, standing for 12 h, centrifuging, filtering, and concentrating the filtrate to obtain a thick paste with the relative density of 1.10-1.12;

(3) adding adjuvant A, drying under reduced pressure, pulverizing into fine powder, adding adjuvant B, adding 88% ethanol to make soft mass, granulating, drying, grading, and packaging to obtain BAIHUTONG granule.

Preferably, the mass ratio of the gypsum, the rhizoma anemarrhenae, the radix glycyrrhizae preparata and the polished round-grained rice in the step (1) is 50: 18: 6: 9.

preferably, the mass concentration of the carboxymethyl chitosan solution in the step (2) is 0.5%; the volume ratio of the concentrated filtrate to the carboxymethyl chitosan solution is 1: 5.

Preferably, the auxiliary materials A and B in the step (3) form a total auxiliary material; the mass ratio of the thick paste in the step (2) to the total auxiliary materials is 1: 0.3; wherein the mass ratio of the auxiliary material A to the auxiliary material B in the step (3) is 2: 1; the ratio of dextrin to soluble starch in the auxiliary materials is 3: 2.

The detection method for the quality standard of the Baihu decoction granules comprises the following steps: qualitatively identifying rhizoma anemarrhenae and radix Glycyrrhizae Preparata by TLC, and determining the content of rhizoma anemarrhenae and radix Glycyrrhizae by HPLC.

Further, the qualitative identification of rhizoma anemarrhenae and radix glycyrrhizae preparata is carried out by the following method:

A. weighing 2 g of BAIHU decoction, grinding, adding 10 mL of methanol, respectively, and performing ultrasonic treatment for 30 min to obtain supernatant as sample solution; precisely weighing mangiferin and new mangiferin reference substances respectively, adding methanol to obtain mangiferin with concentration of 0.25 mg/mL^-1The concentration of the new mangiferin is 0.23 mg/mL^-1The solution of (4) as a control solution; weighing 0.5 g of rhizoma anemarrhenae powder, and preparing into rhizoma anemarrhenae reference solution by the same method as the test solution; weighing other prescription medicinal materials except rhizoma anemarrhenae according to the proportion of the white tiger decoction particles, preparing rhizoma anemarrhenae deficiency negative particles according to the preparation process of the particles, and preparing rhizoma anemarrhenae deficiency negative control solution according to the preparation method of the test solution; performing thin layer chromatography (2015 version of the fourth general rule 0502 of Chinese pharmacopoeia)Respectively dropping 5 μ L of mangiferin reference solution, neomangiferin reference solution, rhizoma anemarrhenae deficiency negative reference solution, and test solution on the same polyamide film, developing with ethanol-water as developing agent at volume ratio of 1: 1, taking out, air drying, and inspecting under ultraviolet lamp 365 nm to obtain test sample, rhizoma anemarrhenae reference solution, and reference solution chromatogram with the same color fluorescent spots, and no rhizoma anemarrhenae deficiency negative particles;

B. weighing 15 g of BAIHU decoction granule, adding diethyl ether 40 mL, heating and refluxing for 1 h, filtering, discarding ether solution, adding methanol 30 mL into the residue, heating and refluxing for 1 h, filtering, evaporating the filtrate to dryness, adding water 40 mL into the residue to dissolve, extracting with n-butanol for 3 times, 20 mL each time, mixing n-butanol solutions, washing with water for 3 times, discarding water solution, evaporating n-butanol solution to dryness, adding methanol 5 mL into the residue to dissolve, and using as sample solution; taking liquiritin reference substance, adding methanol to prepare solution containing 2 mg per 1mL as reference substance solution; weighing other prescription medicinal materials except Glycyrrhrizae radix according to the proportion of BAIHUTANG granule, preparing into negative granule without Glycyrrhrizae radix according to the preparation process of the granule, and preparing into negative control solution without Glycyrrhrizae radix according to the preparation method of the sample solution; performing thin-layer chromatography (2015 version of Chinese pharmacopoeia, general 0502 of the fourth ministry of pharmacopoeia of China), sucking 5 μ L of a licorice-lacking negative control solution, a liquiritin control solution and three batches of test solution, respectively dropping the solution on a same silica gel G thin-layer plate made of 1% sodium hydroxide solution, developing by taking ethyl acetate-formic acid-glacial acetic acid-water as a developing agent with the volume ratio of 15: 1: 2, taking out, drying in the air, uniformly spraying 10% sulfuric acid ethanol solution for color development, heating at 105 ℃ for 5-10 min, and inspecting under an ultraviolet lamp (365 nm), wherein fluorescent spots with the same color are formed on the positions corresponding to the color spectrums of the three batches of test solution and the control solution, and spots do not exist at the positions where the licorice-lacking negative particles.

Further, the method A can also adopt the following method:

weighing 1 g of BAIHU decoction, grinding, adding 30% acetone 10 mL, ultrasonic treating for 30 min, and collecting supernatant as sample solution; accurately weighing timosaponin BII reference substance, adding 30% acetone to obtain a solution with a concentration of 0.56 mg/mL^-1The solution of (4) as a control solution; according to the positionWeighing other prescription medicinal materials except rhizoma anemarrhenae according to the proportion, preparing rhizoma anemarrhenae deficiency negative granules according to the preparation process of the granules, and preparing rhizoma anemarrhenae deficiency negative control solution according to the preparation method of the test solution; performing thin layer chromatography (2015 edition of Chinese pharmacopoeia, general rule of the fourth part 0502), respectively sucking 5 μ L of a control solution, a rhizoma anemarrhenae deficiency negative control solution and a test solution, respectively dropping on the same silica gel G thin layer plate, developing with n-butanol-glacial acetic acid-water as a developing agent at a volume ratio of 4: 1:5, taking out, air drying, spraying a 5% vanillin sulfuric acid ethanol test solution, heating at 105 ℃ until the spots are clearly developed, and as a result, the spots with the same color are developed on the positions corresponding to the chromatogram of the test solution and the control solution, and no spots are formed in the positions of rhizoma anemarrhenae deficiency negative particles.

Further, the method for determining the content of rhizoma anemarrhenae and radix glycyrrhizae by using the HPLC method comprises the following steps:

(1) chromatographic conditions are as follows: lichrospher C18 (5 μm, 4.6 mm. times.250 mm) high performance liquid chromatography column; mobile phase: acetonitrile-B25 mmol. L^-1Potassium dihydrogen phosphate, gradient eluting (0-2 min, 98% B; 2-8 min, 98% -85% B; 8-15min, 85% -78% B; 15-20 min, 78% -72% B; 20-30 min, 72% -50% B), column temperature 30 deg.C, flow rate 1 mL/min^-1The detection wavelength is 257 nm, and the sample injection amount is 10 mu L;

(2) preparation of mixed control solution: weighing 4 kinds of reference substances including neomangiferin, mangiferin, liquiritin and monoammonium glycyrrhizinate, dissolving with methanol, and diluting to desired volume to obtain mixed reference solution, wherein the concentration of neomangiferin is 26.22 μ g/mL^-1The mangiferin concentration is 20.38 mug.mL^-1The concentration of liquiritin is 26.46 mug. multidot.mL^-1The concentration of the monoammonium glycyrrhizinate is 30.67 mu g/mL^-1Filtering with 0.45 μm microporous membrane, and collecting the filtrate.

(3) Preparation of a test solution: grinding BAIHU decoction granule, collecting 1 g, adding 25 mL of methanol, weighing, performing ultrasonic treatment at 250W and 40KHz frequency for 30 min, cooling, weighing, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate;

(4) preparation of negative control solution

Weighing other medicinal materials except rhizoma anemarrhenae and radix Glycyrrhizae Preparata, and preparing into negative control solution without rhizoma anemarrhenae and radix Glycyrrhizae Preparata according to the preparation method of BAIHU decoction granule and the preparation method of test solution, wherein the negative control solution has no color spectrum peak at the position corresponding to the color spectrum peak of the control solution, which indicates that Gypsum Fibrosum and semen oryzae Sativae in the prescription has no interference to measurement;

(5) respectively sucking 10 μ L of the mixed reference solution, the test solution and the negative reference solution, injecting into a high performance liquid chromatograph, and detecting at 257 nm according to the above chromatographic conditions, wherein the mixed reference solution and the test solution show absorption peaks in the same retention time, and no chromatographic peak appears in the negative reference of rhizoma anemarrhenae and radix Glycyrrhizae;

(6) the determination method comprises the following steps: respectively sucking 10 μ L of the mixed reference solution, and injecting into high performance liquid chromatograph, wherein each 1 g of the product contains rhizoma anemarrhenae based on the content of neomangiferin and mangiferin, the content of neomangiferin should not be less than 0.4 mg, and the content of mangiferin should not be less than 0.5 mg; the product contains Glycyrrhrizae radix in each 1 g, and calculated by content of glycyrrhizin and monoammonium glycyrrhizinate, the content of glycyrrhizin should not be less than 0.3 mg, and the content of monoammonium glycyrrhizinate should be 0.3 mg.

Advantageous effects

(1) The invention provides a preparation method of white tiger soup particles, which is determined by taking CaSO 4.2H 20 retention rate, neomangiferin retention rate, mangiferin retention rate and impurity removal rate as examination indexes.

(2) The invention establishes a quality control method of the Baihu decoction granules, carries out thin-layer chromatography identification on rhizoma anemarrhenae and radix glycyrrhizae preparata in the preparation, carries out content measurement on the rhizoma anemarrhenae and the radix glycyrrhizae preparata in the preparation, has simple and convenient operation, strong specificity and accurate result, and can be used for the quality control of the Baihu decoction granules.

Drawings

FIG. 1 is a process for preparing BAIHUTANG granule;

FIG. 2 is a TLC chart of Zhimu and Zhigan Cao; wherein, FIG. 2A is a TLC diagram of mangiferin and new mangiferin, FIG. 2A-1 is a TLC diagram of mangiferin reference, FIG. 2A-2 is a TLC diagram of new mangiferin reference, FIG. 2A-3 is a TLC diagram of mangiferin and new mangiferin as reference medicinal material of rhizoma anemarrhenae, and FIG. 2A-4 is a TLC diagram of mangiferin and new mangiferin as negative granules lacking rhizoma anemarrhenae; FIG. 2A-5, 2A-6, 2A-7 are TLC images of three batches of Baihu decoction granules to be tested for mangiferin and neomangiferin; wherein FIG. 2B is a TLC chart of timosaponin BII, FIG. 2B-1 is a reference sample of timosaponin BII, FIG. 2B-2 is a TLC chart of timosaponin BII lacking rhizoma anemarrhenae negative granules, and FIGS. 2B-3, 2B-4, and 2B 5 are TLC charts of timosaponin BII to be tested from three batches of BAIHUTANG granules; FIG. 2C is a TLC image of Glycyrrhiza glabra, FIG. 2C-1 lacks Glycyrrhiza glabra negative particles; FIG. 2C-2 glycyrrhizin control; fig. 2C-3, fig. 2C-4, and fig. 2C-5 show three batches of Baihu decoction granules to be tested.

FIG. 3 is an HPLC chart of 4 mixed control (A), anemarrhena negative control (B), and test sample (C);

FIG. 4 is a HPLC chart of a control, wherein 1 is neomangiferin, 2 is mangiferin, 3 is liquiritin, and 4 is monoammonium glycyrrhizinate;

FIG. 5 is finger print of BAIHUGUO granule, wherein 6 is neomangiferin, 8 is mangiferin, 12 is liquiritin, and 18 is monoammonium glycyrrhizinate;

FIG. 6 shows the finger prints of 10 batches of BAIHUTANG granule samples, wherein S1-S10 is 10 batches of BAIHUTANG granule samples, and R is the control.

Detailed Description

The present invention is further illustrated by the following examples, which should be construed as merely illustrative and not limitative.

The following examples used starting materials and reagents, respectively: gypsum Fibrosum (Shanxi, batch No. 170502), rhizoma anemarrhenae (Hebei, batch No. 161101), radix Glycyrrhizae Preparata (Gansu, batch No. 170201), all of which are purchased from Shandong Baiweitang Chinese medicinal decoction pieces Limited, and semen oryzae Sativae is commercially available from Ningpo rice of Jinlongyu. Rhizoma anemarrhenae as reference material (batch No. 121070-. Mangiferin reference substance (batch number: B20837-20 mg, not less than 98%), neomangiferin reference substance (batch number: B21397-20 mg, not less than 98%), liquiritin reference substance (batch number: B20414-20 mg, not less than 98%), monoammonium glycyrrhizinate reference substance (batch number: B20418-20 mg, not less than 98%), and all the reference substances are purchased from Shanghai-sourced leaf Biotechnology Co., Ltd. Monopotassium phosphate (liquid phase, 100 g of chemical reagent of Kemiou, Tianjin, Inc.), acetonitrile and methanol for the liquid phase, Wahaha purified water for the liquid phase, and analytically pure other reagents.

Example 1

Accurately weighing 500 g of gypsum (coarse powder), 180 g of rhizoma anemarrhenae, 90 g of japonica rice and 60 g of radix glycyrrhizae preparata according to the proportion of a prescription, totaling 830 g, adding 8 times of water for 1 time, soaking for 0.5h, respectively adding 6 times of water for 2 and 3 times, decocting for 0.5h each time, filtering with gauze, combining the three filtrates, concentrating the liquid medicine to the relative density of 1.02, and concentrating at 60 ℃ according to the ratio of 20 mL to 100mL^-1Adding 0.5% carboxymethyl chitosan solution into the liquid medicine, stirring for 20 min, standing for 12 h, centrifuging, filtering, concentrating the filtrate to obtain thick paste with the relative density of 1.10-1.12 (60 ℃), and mixing the thick paste with dextrin: adding an appropriate amount of mixed auxiliary materials into the soluble starch in a ratio of 3:2, drying under reduced pressure at 60 ℃, crushing into fine powder, adding the rest auxiliary materials according to the amount which is 0.3 time of that of the thick paste, adding 88% ethanol to prepare a soft material, granulating by using a 12-mesh sieve, drying at 60 ℃, granulating, packaging, and obtaining 12 g/bag.

Example 2

TLC (thin layer chromatography) identification of mangiferin and neomangiferin in rhizoma anemarrhenae

Preparation of a test solution: weighing 2 g of each of the three batches of Baihu decoction granules, grinding, adding 10 mL of methanol respectively, performing ultrasonic treatment for 30 min, and taking supernatant as a test solution.

Preparing a mangiferin and new mangiferin reference solution: precisely weighing mangiferin and new mangiferin reference substances, respectively, adding methanol to obtain solutions with mangiferin concentration of 0.25 mg/mL-1 and new mangiferin concentration of 0.23 mg/mL-1, respectively, as reference substance solutions.

Preparing a rhizoma anemarrhenae reference medicinal material solution: weighing rhizoma anemarrhenae 0.5 g, and making into rhizoma anemarrhenae reference solution by the same method as the test solution.

Preparation of anemarrhena negative control solution: weighing other prescription medicinal materials except rhizoma anemarrhenae according to the prescription proportion, preparing rhizoma anemarrhenae deficiency negative granules according to the preparation process of the granules, and preparing rhizoma anemarrhenae deficiency negative control solution according to the preparation method of the test solution.

The thin layer chromatography method comprises the following steps: performing thin layer chromatography (2015 edition of Chinese pharmacopoeia, Tong rule 0502, fourth part of pharmacopoeia of China), respectively sucking 5 μ L of mangiferin control solution, neomangiferin control solution, rhizoma anemarrhenae deficiency negative control solution, and test solution, respectively dropping on the same polyamide film, developing with ethanol-water (1: 1) as developing agent, taking out, air drying, and inspecting under 365 nm ultraviolet lamp. As a result, the three batches of samples and the rhizoma anemarrhenae contrast medicinal material have fluorescent spots with the same pigment color at the corresponding positions of the chromatogram and the reference substance chromatogram, and no spots are generated at the positions lacking the rhizoma anemarrhenae negative particles. The results are shown in FIG. 2A-1.

TLC identification of timosaponin BII in rhizoma anemarrhenae

Preparation of a test solution: weighing 1 g of each of the three batches of Baihu decoction particles, grinding, adding 10 mL of 30% acetone for 30 min, and taking supernatant as a test solution.

Preparing a timosaponin BII reference substance solution: precisely weighing timosaponin BII reference substance, adding 30% acetone to obtain solution with concentration of 0.56 mg/mL-1 as reference solution.

The thin layer chromatography method comprises the following steps: performing thin layer chromatography (2015 edition of Chinese pharmacopoeia, fourth general rule 0502), respectively sucking 5 μ L of control solution, rhizoma anemarrhenae deficiency negative control solution, and test solution, respectively dropping on the same silica gel G thin layer plate, developing with n-butanol-glacial acetic acid-water (4: 1: 5) as developing agent, taking out, air drying, spraying 5% vanillin sulfuric acid ethanol test solution, and heating at 105 deg.C until the spots are clearly developed. As a result, the three batches of the test samples and the reference sample show spots with the same pigment color at the corresponding positions of the chromatograms, and no spots exist at the positions lacking the rhizoma anemarrhenae negative particles. The results are shown in FIG. 2B.

TLC identification of liquorice (roasted)

The sample solution is prepared by weighing 15 g of each of the three batches of BAIHUTANG granule, adding 40 mL of diethyl ether, refluxing under heating for 1 h, filtering, discarding ether solution, adding 30 mL of methanol into the residue, refluxing under heating for 1 h, filtering, evaporating the filtrate, dissolving the residue with 40 mL of water, extracting with n-butanol for 3 times (20 mL each time), mixing n-butanol solutions, washing with water for 3 times, discarding water solution, evaporating n-butanol solution, dissolving the residue with 5 mL of methanol, and making into sample solution.

The liquiritin reference substance solution is prepared by adding methanol into liquiritin reference substance to obtain solution containing 2 mg per 1mL as reference substance solution.

The Glycyrrhrizae radix deficiency negative control solution is prepared by weighing other medicinal materials except Glycyrrhrizae radix according to the prescription proportion, preparing into Glycyrrhrizae radix deficiency negative granule according to the granule preparation process, and preparing into Glycyrrhrizae radix deficiency negative control solution according to the test solution preparation method.

The thin layer chromatography method comprises performing thin layer chromatography (2015 version of the fourth general rule of Chinese pharmacopoeia 0502), collecting Glycyrrhrizae radix-deficient negative control solution, glycyrrhizin control solution, and three test solutions 5 μ L, respectively dropping on the same silica gel G thin layer plate prepared from 1% sodium hydroxide solution, developing with ethyl acetate-formic acid-glacial acetic acid-water (15: 1: 2) as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution for color development, heating at 105 deg.C for 5-10 min, and inspecting under ultraviolet lamp (365 nm). As a result, the three test samples showed fluorescent spots of the same pigment color at the positions corresponding to the chromatograms of the control sample and the licorice negative particles, and no spots were observed at the positions lacking the licorice negative particles. The results are shown in FIG. 2C.

Example 3

The three batches of white tiger soup granules prepared in example 1 were examined for differences in particle size, moisture, solubility, and loading according to the requirements of 0104 granules in the general rules of the four divisions of the chinese pharmacopoeia 2015 edition, and the results are shown in table 1.

TABLE 1 examination of three batches of Baihu decoction granules

Example 4

1. HPLC chromatographic content determination of rhizoma anemarrhenae and radix Glycyrrhizae

1.1 chromatographic conditions

A chromatographic column: lichrospher C18 (5 μm, 4.6 mm. times.250 mm) high performance liquid chromatography column; mobile phase: acetonitrile-B25 mmol. L^-1Potassium dihydrogen phosphate, gradient eluting (0-2 min, 98% B; 2-8 min, 98% -85% B; 8-15min, 85% -78% B; 15-20 min, 78% -72% B; 20-30 min, 72% -50% B), column temperature 30 deg.C, flow rate 1 mL/min^-1The detection wavelength is 257 nm, and the sample injection amount is 10 mu L.

1.2 preparation of Mixed control solutions

Precisely weighing 4 reference substances including neomangiferin, mangiferin, liquiritin and monoammonium glycyrrhizinate, dissolving with methanol, diluting to desired volume, and making into mixed reference solution with neomangiferin concentration of 26.22 μ g/mL^-1The mangiferin concentration is 20.38 mug.mL^-1The concentration of liquiritin is 26.46 mug. multidot.mL^-1The concentration of the monoammonium glycyrrhizinate is 30.67 mu g/mL^-1Filtering with 0.45 μm microporous membrane, and collecting the filtrate.

1.3 preparation of test solutions

Grinding BAIHU decoction granule, collecting 1 g, accurately weighing, accurately adding 25 mL methanol, weighing, ultrasonic treating (power 250W, frequency 40 KHz) for 30 min, cooling, weighing again, supplementing lost weight with methanol, shaking, filtering, and collecting filtrate.

1.4 preparation of negative control solution

Weighing other medicinal materials except rhizoma anemarrhenae and radix Glycyrrhizae Preparata, and preparing into negative control solution without rhizoma anemarrhenae and radix Glycyrrhizae Preparata according to the preparation method of the product and the preparation method of the test solution. The negative control solution has no chromatographic peak at the position corresponding to the chromatographic peak of the control solution, which indicates that gypsum and polished round-grained rice in the prescription have no interference to the measurement.

1.5 systematic compliance test

Precisely sucking 10 μ L of each of the mixed control solution, the sample solution and the negative control solution, injecting into a high performance liquid chromatograph, and detecting at 257 nm according to the above chromatographic conditions. The result shows that the mixed reference solution and the test solution show absorption peaks in the same retention time, and the negative control without rhizoma anemarrhenae and radix Glycyrrhizae has no chromatographic peak. The HPLC chromatogram is shown in FIG. 3. The method has no interference and good specificity.

1.6 examination of Linear relationship

Taking the above mixed reference solution, injecting 2, 4, 6, 8, 10, 12 μ L respectively, performing HPLC analysis according to chromatographic condition under item "1", and analyzing peak area of each referenceY(A) As ordinate, by sample sizeX(μ g) is the abscissa, and linear regression analysis is performed to obtain a regression equation and correlation coefficients as shown in Table 2. The result shows that the sampling amounts of the new mangiferin, the liquiritin and the monoammonium glycyrrhizinate are respectively 0.05244-0.3146 mu g, 0.04076-0.2446 mu g, 0.05292-0.3175 mu g and 0.06134-0.3680 mu g, and the linear relation is good. The results are shown in tables 3, 4, 5 and 6, respectively.

TABLE 2 examination of the Linear relationship

TABLE 3 Linear examination of New mangiferin results

TABLE 4 Linear examination of mangiferin results

TABLE 5 Glycyrrhiza glycoside Linearity examination results

TABLE 6 Linear examination of monoammonium glycyrrhizinate

Peak area (A)

0.755

1.439

2.119

2.682

3.443

4.089

1.7 precision test

Respectively and precisely sucking 10 μ L of mixed reference solution, injecting into high performance liquid chromatograph, continuously measuring for 6 times, measuring peak area integral value, and obtaining peak areas of new mangiferin, liquiritin, and monoammonium glycyrrhizinateRSD0.063%, 0.172%, 0.271%, 0.293%, respectively (n = 6). The results are shown in Table 7.

TABLE 7 examination of the results of the precision

1.8 stability test

Precisely sucking 10 μ L of sample solution under item "3", introducing sample for 0, 2, 4, 8, 12, and 24 hr, respectively, measuring for 6 times, and measuring peak area integral value to obtain peak areas of new mangiferin, liquiritin, and monoammonium glycyrrhizinateRSD0.434%, 0.645%, 0.895%, 0.599%, respectively (n = 6). Can know the supply ofThe stability of the test solution is good within 24 h. The results are shown in Table 8.

TABLE 8 stability test results

1.9 repeatability test

Taking the same batch of BAIHUTANG granule samples, preparing 6 parts of test solution in parallel according to the method under item 1.3, measuring peak area integral value, and calculating its content to obtain new mangiferin, liquiritin, and monoammonium glycyrrhizinateRSD1.147%, 1.734%, 2.752%, 2.123% (n = 6), respectively, the results indicate that the method is well reproducible. The results are shown in Table 9.

TABLE 9 results of the repeatability tests

1.10 sample recovery test

Taking 6 portions of the same batch of white tiger soup particle samples, each portion of which is 0.5 g, precisely weighing, precisely adding the new mangiferin, liquiritin and monoammonium glycyrrhizinate reference substance solution, placing the solution into a 25 mL volumetric flask with methanol, analyzing according to the chromatographic condition under the item of 1.1, respectively measuring the contents of the new mangiferin, liquiritin and monoammonium glycyrrhizinate, and calculating the respective recovery rates. The result shows that the average recovery rate of the new mangiferin is 100.31 percent,RSD= 1.731%; the average recovery rate of mangiferin is 101.84%,RSD= 1.567%; the average recovery rate of the liquiritin is 99.16 percent,RSD= 2.093%; the average recovery rate of the mono-ammonium glycyrrhizinate is 99.41 percent,RSD=2.204%, indicating good recovery for this assay. The results are shown in Table 10.

TABLE 10 sample recovery test results (n = 6)

1.11 assay of three samples

Taking 3 batches of samples, preparing a test solution according to the method under the item 1.3, measuring for 2 times in each batch, and determining the average content of each component shown in the table 11.

TABLE 4-113 measurement results of the contents of Baihu decoction granules

The Baihu decoction particles are prepared from 4 medicinal materials including gypsum, rhizoma anemarrhenae, radix Glycyrrhizae Preparata and polished round-grained rice, and in the research on the quality standard of the Baihu decoction particles, CaSO in the gypsum is also added₄·2H₂0 content was measured, and the results showed that the average content of the three batches of Baihu decoction particles was 3.9%, while the content of the gypsum-deficient negative particles was 1.8%, which accounted for about 46% of the sample content, and thus CaSO was not added₄·2H₂The content of 0 is taken as the quality standard of the granule.

When HPLC method is adopted for content determination and mobile phase selection, acetonitrile-25 mmol.L is selected for component determination in rhizoma anemarrhenae^-1Potassium dihydrogen phosphate and acetonitrile-0.1% formic acid water are used as mobile phase, new mangiferin and mangiferin can be detected, and acetonitrile-25 mmol.L is selected for determining components in Glycyrrhrizae radix^-1Potassium dihydrogen phosphate and acetonitrile-0.05% phosphoric acid as mobile phase were found to be able to detect glycyrrhizin and monoammonium glycyrrhizinate, therefore, acetonitrile-25 mmol.L was selected for cost saving^-1Four components were measured with potassium dihydrogen phosphate as the mobile phase.

In conclusion, a TLC method is used for establishing an identification method of rhizoma anemarrhenae and liquorice, and an HPLC method is used for determining the content determination method of neomangiferin, mangiferin, liquiritin and mono-ammonium glycyrrhizinate, and the method is used as a quality standard of the particles, is suitable and can better control the quality of the Baihu soup particles.

2. Determination of finger print

2.1 establishment of assay methods

2.1.1 chromatographic conditions

A chromatographic column: lichrospher C18 column (4.6 mm. times.250 mm, 5 μm). Mobile phase: 0.1% aqueous formic acid (A) -acetonitrile (C), gradient elution: 0-4 min, 98% A; 4-8 min, 98% -85% A; 8-15min, 85% -78% A; 15-20 min, 78% -72% A; 20-30 min, 72% -50% A; 30-50 min, 50% -35% A; 50-60 min, 35% -15% A. Flow rate: 1.0 mL/min^-1(ii) a Detection wavelength: 258 nm; column temperature: 30 ℃; sample introduction amount: 10 μ L.

2.1.2 preparation of test solutions

Grinding BAIHU decoction, collecting 5 g, precisely weighing, precisely adding 25 mL of methanol, weighing, ultrasonic treating (power 250W, frequency 40 kHz) for 30 min, cooling, weighing, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate.

2.1.3 preparation of control solutions

The mixed control solution under the item "2.1.2" was taken as the control solution.

2.1.4 precision test

Precisely absorbing the same batch of BAIHUTANG granule sample solution, injecting into high performance liquid chromatograph, continuously injecting sample for 6 times (10 μ L each time), recording chromatogram, determining relative retention time and relative peak area of each common peak with peak No. 6 as reference peak, and determining the result for 6 timesRSDThe values are all less than 3 percent, which shows that the precision of the instrument is good and meets the technical requirements of the fingerprint spectrumRSD< 5.0%), the results are shown in tables 12 and 13.

TABLE 12 results of precision examination (relative retention time)

TABLE 13 results of precision examination (relative peak area)

2.1.5 stability test

Precisely sucking the same batch of BAIHUTANG granule sample solution, injecting sample once each for 0, 3, 6, 9, 12, and 24 hr, 10 μ L each time, taking peak No. 6 as reference peak, and determining relative retention time of each common peakAnd relative peak area, results of 6 determinationsRSDThe values are all less than 3 percent, which shows that the stability of the white tiger decoction granule sample is good in 24 hours and meets the technical requirements of fingerprint spectrums, and the results are shown in tables 14 and 15.

TABLE 14 stability test results (relative Retention time)

TABLE 15 stability test results (relative peak area)

2.1.6 repeatability test

Taking the same lot of BAIHU decoction granule samples, preparing 6 test solutions according to the method under item "2.1.2", taking No. 6 peak as reference peak, respectively determining relative retention time and relative peak area of each common peak, and determining 6 times as a resultRSDThe values are all less than 3 percent, which shows that the method has good repeatability and meets the technical requirements of the fingerprint, and the results are shown in tables 16 and 17.

TABLE 16 results of the reproducibility tests (relative retention time)

TABLE 17 repeatability test results (relative peak area)

2.2 creation of fingerprint

10 batches of white tiger decoction particles are taken and ground into fine powder, 5 g of white tiger decoction particles are taken from each batch, the fine powder is precisely weighed, 10 batches of white tiger decoction particle sample solutions are respectively prepared according to the method under the item of 2.1.2, the chromatographic conditions under the item of 2.1.1 are measured to obtain 10 batches of white tiger decoction particle HPLC fingerprints, a Chinese pharmacopoeia committee traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2004A) is adopted, the 10 batches of samples are subjected to multipoint correction by a median method, the time window is 0.10, automatic matching is carried out to form a white tiger decoction particle HPLC fingerprint common mode, the similarity of each spectrum is more than 96%, and the results are shown in a table 18.

TABLE 1810 sample similarity results

20 common peaks are determined by analysis, the No. 6 peak is taken as a reference peak, the relative retention time and the relative peak area of the common peaks are calculated, and the results are shown in tables 19 and 20. By comparing with HPLC chromatogram of control, peak 6 is identified as neomangiferin, peak 8 is identified as mangiferin, peak 12 is identified as liquiritin, and peak 18 is identified as monoammonium glycyrrhizinate, as shown in FIG. 4. The mobile phase is examined by acetonitrile-25 mmol. L^-1Potassium dihydrogen phosphate and acetonitrile-0.1% formic acid water, and the peak number is more and the peak shape is better under the condition of acetonitrile-0.1% formic acid water, so the acetonitrile-0.1% formic acid water solution is selected as the mobile phase. The extraction solvent was selected from methanol, dilute ethanol, 70% ethanol, and 70% methanol, because methanol was used as the extraction solvent because the number of peaks was larger when methanol was used as the extraction solvent. The detection wavelength is examined, the wavelengths of 220 nm, 230 nm, 240 nm, 250 nm, 258 nm, 268nm, 280 nm and 300 nm are respectively selected for detection, and the number of peaks at the wavelength of 258 nm is large and the peak shape is good, so 258 nm is taken as the detection wavelength.

According to the research of the preparation quality standard, the thin-layer chromatography is adopted to respectively identify the neomangiferin, the mangiferin and the timosaponin BII in the rhizoma anemarrhenae and the liquiritin in the honey-fried licorice root, and the result shows that the TLC identification negativity of the rhizoma anemarrhenae and the honey-fried licorice root is not interfered, the spot color development is clear, and the repeatability is good. The content of the neomangiferin, the mangiferin, the liquiritin and the monoammonium glycyrrhizinate in the Baihu decoction particles are simultaneously measured by an HPLC method, and the method established by the result is simple, convenient, sensitive, accurate and good in repeatability, and can be used as the content measuring method of the preparation. The fingerprint of the white tiger decoction particles is established, and 20 common peaks are determined, wherein the peak 6 is new mangiferin, the peak 8 is mangiferin, the peak 12 is liquiritin, the peak 18 is monoammonium glycyrrhizinate, and the similarity of 10 batches of white tiger decoction particles is more than 96%. The initial stability of the preparation is studied, and a sample observation method is adopted to carry out a 6-month initial stability test, so that three batches of particles have no obvious change and have stable quality.

TABLE 1910 batch Baihu decoction granule fingerprint relative retention time

Relative peak area of fingerprint of 2010 batch of Baihu decoction granules

Claims

1. The preparation method of the white tiger soup particles is characterized by comprising the following steps of:

2. The preparation method according to claim 1, wherein the mass ratio of the gypsum, the rhizoma anemarrhenae, the radix glycyrrhizae preparata and the polished round-grained rice in the step (1) is 50: 18: 6: 9.

3. the preparation method according to claim 1, wherein the carboxymethyl chitosan solution of step (2) has a mass concentration of 0.5%; the volume ratio of the concentrated filtrate to the carboxymethyl chitosan solution is 1: 5.

4. The preparation method according to claim 1, wherein the auxiliary materials A and B in the step (3) constitute a total auxiliary material; the mass ratio of the thick paste in the step (2) to the total auxiliary materials is 1: 0.3; wherein the mass ratio of the auxiliary material A to the auxiliary material B in the step (3) is 2: 1; the ratio of dextrin to soluble starch in the auxiliary materials is 3: 2.

5. The method for detecting quality standards of BAIHUTANG granule as claimed in any one of claims 1-4, comprising: qualitatively identifying rhizoma anemarrhenae and radix Glycyrrhizae Preparata by TLC, and determining the content of rhizoma anemarrhenae and radix Glycyrrhizae by HPLC.

6. The detection method according to claim 5, wherein the qualitative identification of Anemarrhena asphodeloides and prepared licorice root is carried out by the following method:

A. weighing 2 g of BAIHU decoction, grinding, adding 10 mL of methanol, respectively, and performing ultrasonic treatment for 30 min to obtain supernatant as sample solution; precisely weighing mangiferin and new mangiferin reference substances respectively, and adding methanol to obtain solutions with mangiferin concentration of 0.25 mg/mL-1 and new mangiferin concentration of 0.23 mg/mL-1 as reference substance solutions; weighing 0.5 g of rhizoma anemarrhenae powder, and preparing into rhizoma anemarrhenae reference solution by the same method as the test solution; weighing other prescription medicinal materials except rhizoma anemarrhenae according to the proportion of the white tiger decoction particles, preparing rhizoma anemarrhenae deficiency negative particles according to the preparation process of the particles, and preparing rhizoma anemarrhenae deficiency negative control solution according to the preparation method of the test solution; performing test according to thin-layer chromatography (2015 edition of Chinese pharmacopoeia, Tonghe 0502), respectively sucking 5 μ L of mangiferin control solution, neomangiferin control solution, rhizoma anemarrhenae lacking negative control solution, and test solution, respectively dropping on the same polyamide film, developing with ethanol-water as developing agent at volume ratio of 1: 1, taking out, air drying, and inspecting under ultraviolet lamp 365 nm to obtain test sample, rhizoma anemarrhenae control solution chromatogram and control chromatogram at corresponding positions, wherein the fluorescent spots of the same color are displayed, and the rhizoma anemarrhenae lacking negative particles have no spots;

7. The detection method according to claim 6, wherein the method A further comprises the following steps:

weighing 1 g of BAIHU decoction, grinding, adding 30% acetone 10 mL, ultrasonic treating for 30 min, and collecting supernatant as sample solution; accurately weighing timosaponin BII reference substance, adding 30% acetone to obtain solution with concentration of 0.56 mg/mL-1 as reference solution; weighing other prescription medicinal materials except rhizoma anemarrhenae according to the prescription proportion, preparing rhizoma anemarrhenae deficiency negative granules according to the preparation process of the granules, and preparing rhizoma anemarrhenae deficiency negative control solution according to the preparation method of the test solution; performing thin layer chromatography (2015 edition of Chinese pharmacopoeia, general rule of the fourth part 0502), respectively sucking 5 μ L of a control solution, a rhizoma anemarrhenae deficiency negative control solution and a test solution, respectively dropping on the same silica gel G thin layer plate, developing with n-butanol-glacial acetic acid-water as a developing agent at a volume ratio of 4: 1:5, taking out, air drying, spraying a 5% vanillin sulfuric acid ethanol test solution, heating at 105 ℃ until the color development of the spots is clear, and as a result, the spots with the same color appear on the corresponding positions of the chromatogram of the test solution and the control solution, and no spots exist in the positions of rhizoma anemarrhenae deficiency negative particles.

8. The detection method according to claim 5, wherein the HPLC method for detecting the content of rhizoma anemarrhenae and radix Glycyrrhizae comprises:

(1) chromatographic conditions are as follows: lichrospher C18 (5 μm, 4.6 mm. times.250 mm) high performance liquid chromatography column; mobile phase: acetonitrile A, 25 mmol.L-1 potassium dihydrogen phosphate B, gradient elution (0-2 min, 98 percent B, 2-8 min, 98-85 percent B, 8-15min, 85-78 percent B, 15-20 min, 78-72 percent B, 20-30 min, 72-50 percent B), column temperature of 30 ℃, flow rate of 1 mL.min-1, detection wavelength of 257 nm and sample injection amount of 10 mu L;

(2) preparation of mixed control solution: weighing 4 reference substances of neomangiferin, mangiferin, liquiritin and monoammonium glycyrrhizinate, dissolving with methanol, fixing the volume, and preparing into a mixed reference substance solution, wherein the concentration of neomangiferin is 26.22 mug.mL < -1 >, the concentration of mangiferin is 20.38 mug.mL < -1 >, the concentration of liquiritin is 26.46 mug.mL < -1 >, the concentration of monoammonium glycyrrhizinate is 30.67 mug.mL < -1 >, and filtering with a 0.45 mu m microporous membrane to obtain a subsequent filtrate;

(4) preparation of negative control solution