CN118150741A

CN118150741A - Reference sample for Xiebaisan and preparation process thereof

Info

Publication number: CN118150741A
Application number: CN202311661079.8A
Authority: CN
Inventors: 黄武军; 杜剑松; 邹国辉; 涂海龙; 刘志华; 鄢章龙
Original assignee: Jiangxi Yaodu Zhangshu Pharmaceutical Co ltd
Current assignee: Jiangxi Yaodu Zhangshu Pharmaceutical Co ltd
Priority date: 2023-12-05
Filing date: 2023-12-05
Publication date: 2024-06-07

Abstract

The invention discloses a reference sample of Xiebaisan and a preparation process thereof. The invention carefully researches the preparation process of the reference sample of the Xiebai powder, researches the conditions of each link, and finally prepares the reference sample of the Xiebai powder with higher glycyrrhizin and glycyrrhizic acid content and moderate alcohol-soluble extract and moisture content, wherein; the content of the glycyrrhizin is 1.4-4.7 mg/g, the content of the glycyrrhizic acid is 1.5-4.9 mg/g, the proportion of the alcohol-soluble extract is 23.0-53.0%, the water content is not more than 9.0%, and the similarity between the fingerprint spectrum and the reference fingerprint spectrum is not less than 0.85. The preparation process provided by the invention is stable, quality control of each link is convenient, and mass production of stable quality of the reference sample of the Xiebaisan can be ensured, so that the production efficiency is improved, and various production losses and costs are reduced.

Description

Reference sample for Xiebaisan and preparation process thereof

Technical Field

The invention relates to the technical field of preparation of traditional Chinese medicine compositions, in particular to a reference sample of Xiebaisan and a preparation process thereof.

Background

The prescription of the Xiebai powder contains four medicines including cortex mori radicis, cortex lycii radicis, liquorice and polished round-grained rice. In the formula, the mulberry bark is sweet and cold in taste and enters the lung, clears the lung and purges heat, relieves asthma and relieves cough, and is a monarch drug while purges the lung without damaging the lung; cortex Lycii is sweet freshness cold, clear away the depressed fire in the lung, and has the function of nourishing yin, and is a ministerial drug; the licorice and the polished round-grained rice nourish the stomach and harmonize the middle, the earth is used for producing gold to strengthen the lung qi, and the stomach is not hurt by cold in the middle, and the medicines can be blended, are used as adjuvant medicines, and are used together to play roles of purging lung, clearing heat, relieving cough and asthma. It is used for treating cough caused by stagnated fire in lung and lung heat.

Specific decoction methods are explicitly described in the prescription, namely: cortex Lycii (washed off soil, baked), cortex Mori (fried yellow with thin file) and Glycyrrhrizae radix (baked) are respectively one or two. Taking water, mixing with semen oryzae Sativae, decocting with water for seven minutes, and taking before eating. However, the decoction process and the decoction form of the medicinal materials have great inconvenience, and the decoction process has great difference, so that the curative effect is easy to be inaccurate. Therefore, the traditional Chinese medicine preparation which is convenient to carry and take is developed by applying the modern science and technology, so that the traditional Chinese medicine preparation is not only the traditional Chinese medicine preparation, but also the clinical application of the classical prescription can be better promoted. Whether the extract is prepared into granules, pills, capsules and other dosage forms which are convenient to carry and take, the extract meeting the standard requirements is prepared first. However, at present, no clear research and standard are available for the preparation process and quality of the extract of Xiebai powder, so that the preparation process of the extract of Xiebai powder is necessary to be researched in order to provide the extract of Xiebai powder with stable quality and meeting the standard requirements.

Disclosure of Invention

The invention aims at providing a reference Xiebaisan sample aiming at the problem that no clear process research is carried out on the reference Xiebaisan sample in the prior art. The invention carefully researches the preparation process of the reference sample of the Xiebai powder, researches the conditions of each link, and finally prepares the reference sample of the Xiebai powder with higher glycyrrhizin and glycyrrhizic acid content and moderate alcohol-soluble extract and moisture content; and the preparation process is stable, the quality control of each link is convenient, and the mass production of stable quality of the reference sample of the Xiebaisan can be ensured, so that the production efficiency is improved, and various production losses and costs are reduced.

The invention further aims at providing a preparation method of a reference sample of Xiebaisan.

The prescription original text of the prescription of the children medicine syndrome straight recipe is: the infantile diarrhea Bai Sanzhi is excessive lung, and the cough and asthma is caused. Cortex Lycii (washed off soil, baked), cortex Mori (fried yellow with thin file) and Glycyrrhrizae radix (baked) are respectively one or two. Taking water, mixing with semen oryzae Sativae, decocting with water for seven minutes, and taking before eating. Through examination, the dosage of modern usage is determined as follows: cortex Lycii 41.30g, parched cortex Mori 41.30g, radix Glycyrrhizae Preparata 4.13g. The water is coarse powder, 4-8 g of the water is taken each time, one round of polished round-grained rice is added, 300mL of water is added, and the water is decocted to 180mL. The average weight of the polished round-grained rice is 2.0g after multiple times of weighing, so that the dosage of polished round-grained rice is 2.0g in terms of weight.

The above object of the present invention is achieved by the following means:

a reference sample for Xiebaisan contains glycyrrhizin (1.4-4.7 mg/g) and glycyrrhizic acid (1.5-4.9 mg/g).

Preferably, the content of the liquiritin is 1.4～4.5mg/g,1.4～4.0mg/g,1.5～3.5mg/g,1.5～3mg/g,1.5～2.7mg/g,1.7～4.5mg/g,1.8～4.5mg/g,1.9～4.5mg/g,1.9～4.3mg/g,1.9～4.0mg/g,1.9～3.8mg/g,2.0～3.8mg/g,2.0～3.6mg/g,2.0～3.4mg/g,2.1～3.3mg/g,2.2～3.3mg/g,2.4～3.3mg/g,2.4～3.2mg/g,2.4～3.1mg/g,2.4～3.0mg/g,2.5～3.0mg/g,2.6～3.0mg/g or 2.7-3.0 mg/g.

Preferably, the glycyrrhizic acid content is 1.5～4.5mg/g,1.5～4.0mg/g,1.5～3.5mg/g,1.5～3mg/g,1.6～2.8mg/g,1.7～4.5mg/g,1.8～4.5mg/g,1.9～4.7mg/g,1.9～4.5mg/g,1.9～4.3mg/g,1.9～4.0mg/g,2.0～4.0mg/g,2.1～3.9mg/g,2.3～3.9mg/g,2.4～3.9mg/g,2.4～3.9mg/g,2.5～3.9mg/g,2.5～3.8mg/g,2.5～3.7mg/g,2.6～3.7mg/g,2.6～3.6mg/g,2.6～3.5mg/g or 2.6-3.4 mg/g, 2.6-3.3 mg/g, 2.6-3.2 mg/g.

Preferably, the proportion of alcohol soluble extract in the reference sample of the xiebuia powder is 23.0% -53.0%; or, the proportion of the alcohol soluble extract is 23％～50％,23％～45％,23.2％～45％,23.5％～45％,25％～45％,27％～45％,29％～45％,29％～50％,31％～48％,33％～46％,33％～45％,34％～44％,35％～42％,36％～42％,37％～42％ or 38-42%.

Preferably, the moisture content of the reference xiebuia sample is no more than 9.0%, or the moisture content of the reference xiebuia sample is no more than 8.5%, no more than 8.0%, no more than 7.50%, no more than 7.0%, no more than 6.5%, no more than 6.0%.

More preferably, the moisture content of the reference Xiebaisan sample is 2.0% -9.0%; or the moisture content of the reference sample of the Xiebaisan is 2.0％～8.0％,2.0％～7.0％,2.0％～6.0％,3％～8％,3％～7.5％,4％～7.5％,4.5％～7.5％,5％～7.5％,5.2％～7.0％,5.2％～6.8％,5.2％～6.7％,5.2％～6.6％,5.2％～6.5％,5.2％～6.4％ or 5.2 to 6.3 percent.

Preferably, the fingerprint of the Xiebaisan reference sample is not less than 0.85 similar to the reference fingerprint (shown in fig. 6).

The mulberry bark is used as a monarch drug in the prescription of the Xiebai san. Cortex Lycii is used as ministerial drug. Glycyrrhrizae radix and semen oryzae Sativae are used as adjuvant drug. The monarch drug cortex mori has the effects of purging lung, relieving asthma and inducing diuresis to alleviate edema, wherein flavonoid components are the most main active components with highest content, and the morin is flavonoid component, has the pharmacological effects of anti-inflammatory and analgesic, has strong specificity and low content, and the content of the morin in 16 batches of cortex mori decoction pieces is measured, and the result is that the morin is detected in only 3 batches of cortex mori decoction pieces, so that the method does not serve as a content measurement index, and determines to carry out qualitative analysis on the cortex mori by adopting thin-layer identification; the ministerial drug cortex lycii radicis has the effects of cooling blood, removing steam, clearing lung and reducing fire, wherein alkaloid components are the most main active components, and cortex lycii radicis is one of representative alkaloid components, has higher content and strong specificity, but has poor stability, so the cortex lycii radicis is not included in content measurement indexes, and the qualitative analysis of cortex lycii radicis by thin-layer identification is determined; the licorice has the effects of invigorating spleen, regulating stomach, invigorating qi and restoring pulse. The glycyrrhizin belongs to flavonoid components and has various pharmacological effects of resisting inflammation, relieving cough, eliminating phlegm and the like; glycyrrhizic acid is triterpenoid saponin component, has various pharmacological effects such as anti-inflammatory, antiviral, antitussive and expectorant effects, is closely related to prescription drug effect, and glycyrrhizin and glycyrrhizic acid are quality control index of Glycyrrhrizae radix in Chinese pharmacopoeia of 2020 edition, so that glycyrrhizin and glycyrrhizic acid are used as quantitative research index component of Xiebai san. The auxiliary medicine polished round-grained rice has the efficacy of nourishing yin and relieving cough, and the main components of the auxiliary medicine polished round-grained rice are starch, lipid and protein, and are common food, and the related quality control components are not studied. On the basis, the quality of the Xiebaisan is comprehensively controlled by adding the fingerprint.

Preferably, the decoction pieces of the reference Xiebaisan sample are composed of: 41.3 parts of cortex lycii radicis (baked), 41.3 parts of fried cortex mori radicis, 4.13 parts of fried liquorice and 6-8 parts of polished round-grained rice.

Preferably, the liquid medicine is filtered and concentrated until the weight ratio of the liquid medicine to the feeding amount is 4-2:1, so as to obtain concentrated solution.

The invention also provides a preparation method of the reference sample of the Xiebaisan, which is characterized by comprising the following steps:

S1, the decoction pieces of a reference sample of Xiebaisan comprise the following components: 41.3 parts of cortex lycii radicis (baked), 41.3 parts of fried cortex mori radicis, 4.13 parts of fried liquorice and 6-8 parts of polished round-grained rice; wherein cortex Lycii, cortex Mori preparata and radix Glycyrrhizae Preparata are mixed, ground into coarse powder, 4-8 parts of coarse powder and 1.5-2.5 parts of semen oryzae Sativae are decocted with water, and filtered and centrifuged to obtain extractive solution;

s2, concentrating the obtained extracting solution until the weight ratio of the extracting solution to the feeding amount is 4-2:1, thereby obtaining concentrated solution;

S3, drying the obtained concentrated solution to obtain a reference sample of the Xiebaisan.

In mass production, the amounts of the components in step S1 may be scaled up in equal proportions.

Preferably, in the step S1, cortex lycii radicis, fried cortex mori radicis and roasted liquorice are mixed and ground into coarse powder, and the dosage of the coarse powder is 5.0-7.0 parts; more preferably, the coarse powder is used in an amount of 6.2.+ -. 0.5 parts; more preferably, the coarse powder is used in an amount of 6.2.+ -. 0.2 parts.

Preferably, in the step S1, the amount of the polished round-grained rice is 2+/-0.3 parts; more preferably, the amount of the polished round-grained rice is 2+/-0.2 parts; the dosage of the polished round-grained rice is 2+/-0.1 part.

Preferably, in the step S1, in the process of decocting in water, the water addition amount of the laxative powder is 200-300 parts; more preferably, the amount of water added is 300 parts.

In the present invention, the density of water was calculated according to 1g/cm ³. I.e. 100mL of water, is 100g.

Preferably, in step S1, the roasted white mulberry root-bark is cut into filaments; the fried licorice is cut into thick slices with the thickness of about 2-4 mm.

Preferably, in step S1, the coarse powder obtained by mixing cortex Lycii (baked), cortex Mori preparata and radix Glycyrrhizae preparata is powder which can pass through the first sieve (10 mesh) but can pass through the third sieve (50 mesh) for not more than 20%.

Preferably, the decocting process is that the boiling is carried out with strong fire firstly and then the decocting is carried out with slow fire for 15-30 min; more preferably, the slow fire decoction time is 20-30 min; more preferably, the slow fire decoction time is 20-25 min; more preferably, the slow fire decoction time is 20min.

Preferably, if the electric heating tool is used for decoction, the power of strong fire is 1600W, and the power of slow fire is 400W.

Preferably, in the step S1, the filter cloth used for filtering is 200 meshes; the rotational speed of centrifugation is 3000-5000 r/min, and the time is 5-15 min.

More preferably, in step S1, the rotational speed of the centrifugation is 4000 to 5000r/min for 5 to 10min.

More preferably, in step S1, the rotational speed of the centrifugation is 4000r/min for 10min.

Preferably, in step S2, the weight ratio of concentrate to feed is 4:1, 3:1 or 2:1.

Preferably, in the step S2, the concentration is reduced pressure concentration, the temperature is 50-70 ℃, and the vacuum degree is minus 0.08-minus 0.10Mpa. More preferably, the concentration is carried out under reduced pressure at a temperature of 60 to 70 ℃. More preferably, the concentration is carried out under reduced pressure at a temperature of 60 ℃.

Preferably, in step S3, drying is reduced pressure drying and freeze drying; wherein the pre-freezing temperature is-20 to-45 ℃ and the time is at least 40min; the cold trap temperature in the drying process is-50 to-70 ℃, the vacuum degree is less than 100Pa, and the freeze-drying time is at least 12 hours.

Preferably, in the prepared reference sample of the Xiebai powder, the content of the glycyrrhizin is 1.4-4.7 mg/g, the content of the glycyrrhizic acid is 1.5-4.9 mg/g, the proportion of the alcohol-soluble extract is 23.0-53.0%, and the moisture content is not more than 9.0%.

Preferably, the content of the glycyrrhizin in the prepared reference sample of the Xiebai powder is 1.4～4.5mg/g,1.4～4.0mg/g,1.5～3.5mg/g,1.5～3mg/g,1.7～4.5mg/g,1.8～4.5mg/g,1.9～4.5mg/g,1.9～4.3mg/g,1.9～4.0mg/g,1.9～3.8mg/g,2.0～3.8mg/g,2.0～3.6mg/g,2.0～3.4mg/g,2.1～3.3mg/g,2.2～3.3mg/g,2.4～3.3mg/g,2.4～3.2mg/g,2.4～3.1mg/g,2.4～3.0mg/g,2.5～3.0mg/g,2.6～3.0mg/g or 2.7-3.0 mg/g.

Preferably, the content of glycyrrhizic acid in the prepared reference sample of the Xiebai powder is 1.5～4.5mg/g,1.5～4.0mg/g,1.5～3.5mg/g,1.5～3mg/g,1.7～4.5mg/g,1.8～4.5mg/g,1.9～4.7mg/g,1.9～4.5mg/g,1.9～4.3mg/g,1.9～4.0mg/g,2.0～4.0mg/g,2.1～3.9mg/g,2.3～3.9mg/g,2.4～3.9mg/g,2.4～3.9mg/g,2.5～3.9mg/g,2.5～3.8mg/g,2.5～3.7mg/g,2.6～3.7mg/g,2.6～3.6mg/g,2.6～3.5mg/g or 2.6-3.4 mg/g, 2.6-3.3 mg/g and 2.6-3.2 mg/g.

Preferably, the proportion of the alcohol soluble extract in the prepared reference sample of the Xiebaisan is 23.0-53.0%; or, the proportion of the alcohol soluble extract is 23％～50％,23％～45％,23.2％～45％,23.5％～45％,25％～45％,27％～45％,29％～45％,29％～50％,31％～48％,33％～46％,33％～45％,34％～44％,35％～42％,36％～42％,37％～42％ or 38-42%.

Preferably, the reference sample of the laxative is prepared to have a moisture content of no more than 9.0%, or the reference sample of the laxative has a moisture content of no more than 8.5%, no more than 8.0%, no more than 7.50%, no more than 7.0%, no more than 6.5%, no more than 6.0%.

More preferably, the moisture content of the prepared reference sample of the Xiebai powder is 2.0-9.0%; or the moisture content of the reference sample of the Xiebaisan is 2.0％～8.0％,2.0％～7.0％,2.0％～6.0％,3％～8％,3％～7.5％,4％～7.5％,4.5％～7.5％,5％～7.5％,5.2％～7.0％,5.2％～6.8％,5.2％～6.7％,5.2％～6.6％,5.2％～6.5％,5.2％～6.4％ or 5.2 to 6.3 percent.

Compared with the prior art, the invention has the following beneficial effects:

The invention carefully researches the preparation process of the reference sample of the Xiebai powder, researches the conditions of each link, and finally prepares the reference sample of the Xiebai powder with higher glycyrrhizin and glycyrrhizic acid content and moderate alcohol-soluble extract and moisture content; the preparation process is stable, quality control of each link is convenient, the quality stability of the reference sample of the Xiebai powder can be ensured, the quality uniformity of the reference samples of multiple batches is ensured, controllable parameters are provided for the safety and the effectiveness of the bearing classical prescription, and meanwhile, a technical route and a quality control method are provided for mass production of Xiebai particles, so that the production efficiency is improved, various production losses and costs are reduced, and the product quality is effectively controlled.

Drawings

FIG. 1 shows the results of the detection of the total amount of glycyrrhizin in decoction pieces, extract solutions, concentrated solutions and reference samples in the above 3 batches according to the detection method of 5.1.

FIG. 2 shows the results of the detection of the total amount of glycyrrhizic acid in decoction pieces, extract solutions, concentrated solutions and reference samples of the above 3 batches according to the detection method of 5.1.

FIG. 3 shows the results of the detection of the total amounts of glycyrrhizin and glycyrrhizic acid in the decoction pieces, the extract, the concentrate and the reference sample of the above 1 lot according to the detection method of 5.2.

FIG. 4 is a fingerprint of the 1 lot extract, concentrate and reference sample measured according to the test method of 5.2. Wherein S1 is a reference sample, S2 is a concentrated solution, and S3 is an extracting solution.

Fig. 5 is a fingerprint of the 1 lot of herbal medicines, decoction pieces and reference samples measured according to the detection method of 5.2. Wherein 1 is a reference sample, 2 is a cortex mori radicis medicinal material, 3 is a cortex lycii radicis medicinal material, 4 is a liquorice medicinal material, 5 is a polished round-grained rice medicinal material (medicinal material and decoction pieces), 6 is a fried liquorice decoction piece, 7 is a fried cortex mori radicis decoction piece, and 8 is a cortex lycii radicis (baked) decoction piece.

Fig. 6 is a reference sample control fingerprint of xiebuia powder.

Detailed Description

The invention is further illustrated in detail below in connection with specific examples which are provided solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Quality control of medicinal materials

The stir-fried white mulberry root-bark is used as a monarch drug, cortex lycii radicis is used as a ministerial drug, liquorice and polished round-grained rice are used as an adjuvant drug, and the above drugs except liquorice are not found in the prior art, so that safety related chemical components are not limited. The content limit of exogenous components of Glycyrrhrizae radix is shown in Table A1.

TABLE A1 safety-related chemical Components and their limitations

The quality attributes of the prescription medicinal materials are summarized in a table A2.

Table A2 summary of quality attributes of medicinal materials

Pretreatment of decoction pieces

The prescription of the Xiebai powder contains four medicines, wherein cortex lycii radicis (baked), fried cortex mori radicis and fried liquorice are all required to be chipping into coarse powder, the coarse powder is the coarse powder (the coarse powder can pass through a first sieve (10 meshes) but is mixed with powder which can pass through a third sieve (50 meshes) and is not more than 20 percent), and the polished round-grained rice can be directly fed without pretreatment. Cortex Lycii is in the shape of cylinder or groove, the cortex Mori is in irregular wide thread shape, and radix Glycyrrhizae is in round or oval thick slice. Wherein cortex Lycii is fragile and can be directly smashed; parching Glycyrrhrizae radix to thick slice with thickness of about 2-4 mm, and directly mashing; pulverizing cortex Mori preparata, sampling by quartering method, pulverizing above medicinal materials into coarse powder, weighing according to prescription proportion, and mixing. The information of the decoction pieces for research is shown in Table A3.

Table A3 decoction piece information table for research

Based on the preliminary determined modern usage of examination paper, the method recorded in the classical name prescription directory is adopted for decoction, and specific process and condition parameters of the technology are researched. The extraction rate, the extraction total amount of glycyrrhizin, the extraction total amount of glycyrrhizic acid and the fingerprint are used as indexes to respectively examine the relevant parameters such as the water adding amount, the heating mode, the decoction time, the solid-liquid separation, the concentration ratio and the drying mode, so as to determine the reference sample process of the Xiebai powder. Specific studies and results are shown in the examples below.

EXAMPLE 1 investigation of the decoction Process

1. Inspection of the water addition

The water usage recorded in the classical name prescription catalogue is: the prescription water adding quantity is two small cups, and the decoction is divided into six minutes. According to examination, a small cup is 60-90 mL, so 120-180 mL of water is added, and the mixture is decocted to 84-126 mL.

Keeping the water adding quantity consistent with the records in the classical prescription catalogue, namely, the water adding quantity is 120mL, and decocting to 84mL, wherein the specific method is as follows:

The materials are added according to a single dosage, 6.2g of mixed coarse powder and 2.0g of polished round-grained rice are weighed and placed in a 300mL marmite, 120mL of drinking water is added, a cover is added, an electroceramic stove (with strong fire of 1600W and with slow fire of 400W) is adopted for heating, the mixture is firstly decocted with strong fire to be boiled, the mixture is decocted with slow fire to 84mL, and the extraction liquid volume and the decoction time are recorded, so that the results are shown in Table 1.1.

TABLE 1.1 decoction time results

From the above results, it is known that the Xiebai powder is decocted until the water adding amount is 120mL and six minutes, the time for decocting is 1 minute, and the polished round-grained rice is not thoroughly cooked, so that the decoction is carried out according to the water adding amount recorded in the original text, and the requirements of modern decoction conditions are not met, and the water adding amount is examined, and the specific method is as follows:

Weighing mixed coarse powder 6.2g according to single dose, placing 2.0g of polished round-grained rice in 500mL marmite (with the volume of 500mL, the bottom inner diameter of 11.4cm, the top inner diameter of 13.2cm, the height of 4.6cm, the wall thickness of the marmite of 0.7cm and the bottom thickness of the marmite of 0.8 cm), respectively adding 200mL, 300mL and 400mL of drinking water, capping, heating by a electroceramic furnace (with strong fire of 1600W and with slow fire of 200W), decocting with strong fire until boiling, respectively decocting with slow fire until the volume of 120mL, 180mL and 240mL are reached, preparing two parts in parallel with different water adding amounts, recording the volume of extracted liquid and the time of decoction, observing the decoction pieces, respectively measuring the content and the ointment yield of the extracted liquid glycyrrhizin and glycyrrhizin, and calculating the total amount of glycyrrhizin. The detection method adopts the method recorded in pharmacopoeia. The results are shown in Table 1.2.

The method for measuring the extract yield comprises the following steps: 25mL of the extract was precisely measured, placed in an evaporation dish W1 dried to a constant weight, evaporated to dryness in a water bath, dried at 105℃for 3 hours, cooled in a desiccator for 30 minutes, and rapidly and precisely weighed to weight W2.

TABLE 1.2 investigation of different Water addition amounts

As can be seen from the above table, the Xiebai powder is decocted to six minutes with water addition of 200mL, 300mL and 400mL, the time to reach the end point is 6 minutes, 20 minutes and 30 minutes respectively, the paste yield, the total extraction amount of glycyrrhizin and the total extraction amount of glycyrrhizic acid are obviously increased, but the prescription has the effects of clearing lung heat, relieving cough and asthma, belongs to the drugs for relieving exterior syndrome and clearing heat, and is specified in the medical institution traditional Chinese medicine decoction room management Specification: the exterior syndrome relieving and heat clearing herbs are not suitable for long-term decoction, and are usually decocted for 15-20 minutes after boiling, so 300mL of the decoction is selected as the water adding amount of the final Xiebai powder.

2. Investigation of heating modes

(1) Investigation of heating modes

The ancient decoction mode is open fire decoction, a gas cooker is used as an open fire heating mode, and the heating efficiency of an electroceramic furnace is controlled by power in consideration of the heating mode, so that the control fire effect is more stable, the paste yield is adopted, the total amount of glycyrrhizin and glycyrrhizic acid is used as an index, and the open fire (gas cooker) and electroceramic furnace heating modes are examined.

Weighing mixed coarse powder 6.2g according to single dose, placing 2.0g of polished round-grained rice in 500mL marmite (with the volume of about 500mL, the bottom inner diameter of 11.4cm, the top inner diameter of 13.2cm, the height of 4.6cm, the wall thickness of the marmite of 0.7cm and the bottom thickness of the marmite of 0.8 cm), adding 300mL of drinking water, covering, heating by open fire (gas stove) and electroceramic stove (with strong fire of 1600W and with slow fire of 200W) respectively, decocting with strong fire to boiling, decocting with slow fire to 180mL respectively, preparing two parts in parallel, recording the extraction liquid volume and the decoction time, observing decoction pieces, measuring the glycyrrhizin and glycyrrhizic acid content of the extract respectively, calculating the total glycyrrhizin and glycyrrhizic acid. The results are shown in Table 1.3.

TABLE 1.3 reference heating pattern comparison study results for Xiebai powder

The results show that the paste yield, the total glycyrrhizin extraction amount and the total glycyrrhizic acid extraction amount are all not obviously different, and the two heating modes are not obviously different by combining the above 3 investigation index analysis, so that the electric Tao Luhuo force is easier to control, and the electric ceramic furnace is selected as the final heating mode of the Xiebai powder.

(2) Determination of the time of decoction

The prescription is prepared by taking six minutes of decoction as the decoction end point of the Xiebai powder, but the real-time measurement of the volume of the extracted liquid in the test process needs a long time, needs to leave a fire source for operation, has complicated operation and can cause discontinuous decoction, so the time for the extracted liquid to be decocted to six minutes is measured, the control end point of the decoction time is simpler and is easy to operate, and the specific method is as follows:

Weighing mixed coarse powder 6.2g according to single dose, placing 2.0g of polished round-grained rice in a 500mL marmite (with the capacity of about 500mL, the bottom inner diameter of 11.4cm, the top inner diameter of 13.2cm, the height of 4.6cm, the wall thickness of the marmite is 0.7cm, the bottom thickness of the marmite is 0.8cm, the weight M1 of the marmite) into a 500mL marmite (with the capacity of about 500 mL), adding 300mL of drinking water, adding a cover, heating by an electroceramic furnace (with strong fire of 1600W and with slow fire of 200W), heating with strong fire until boiling, pouring out the liquid medicine when decocting with strong fire, weighing M2 (marmite+dregs) to obtain single dose dregs, and placing 180mL of the poured liquid medicine and dregs into a decoction container, wherein the weight is the theoretical decoction end point weight (M1+M3+180 mL of liquid medicine). Pouring the rest liquid medicine into a marmite, continuously decocting, monitoring the decoction process in real time, weighing until reaching the end of decoction, and recording the decoction time in detail. The result was found to be 1.4.

Table 1.4 study of the end-time of decoction

From the above results, the weight of the Xiebaisan in the water adding amount of 300mL is decocted to six minutes, and the end point of the decoction of 3 samples is respectively as follows: 940.30g, 890.89g, 898.89g; when the Chinese medicinal powder is decocted to the corresponding weight of 940.50g, 885.60g and 895.58g, the decoction time of 3 samples is 20 minutes, so the 20 minutes is determined as the final decoction time of the Xiebai powder.

Summary of the decoction Process

Through the above research, the reference decocting process of the temporary diarrhea white powder substance is as follows: pulverizing parched cortex Mori, cortex Lycii (baked) and parched Glycyrrhrizae radix into coarse powder respectively, weighing parched cortex Mori 41.30g, cortex Lycii (baked) 41.30g, and parched Glycyrrhrizae radix 4.13g, and mixing well for use; mixing above mixed powder 6.2g and semen oryzae Sativae 2.0g, placing in a casserole, adding 300mL of drinking water, heating with electroceramic stove (1600W with strong fire and 200W with slow fire), decocting with cover, heating with strong fire to boil, and decocting with slow fire for 20 min (about 180 mL).

EXAMPLE 2 study of solid-liquid separation Process

Adding 6.2g of mixed coarse powder and 2.0g of polished round-grained rice into 500mL marmite (with the capacity of about 500mL, the inner diameter of the bottom of the marmite is 11.4cm, the inner diameter of the top of the marmite is 13.2cm, the height of the marmite is 4.6cm, the wall thickness of the marmite is 0.7cm, the bottom of the marmite is 0.8 cm), adding 300mL of drinking water, adding a cover, heating by an electroceramic furnace (with the strong fire of 1600W and the slow fire of 200W), decocting with the strong fire for 20 minutes (with the strong fire of about 180 mL), filtering the decoction with 200-mesh filter cloth (material: nylon) while the decoction is hot, uniformly mixing the extracts, dividing the mixture into two parts, filtering one part of samples with 200 meshes, and centrifuging the other part (with the rotating speed of 4000 rpm) for 10 minutes to obtain a centrifugal sample. Recording the volume of the extracting solution, observing the clarity of the two samples, respectively measuring the content of glycyrrhizin and glycyrrhizic acid in the extracting solution and the extraction rate, and calculating the total amount of the glycyrrhizin and the glycyrrhizic acid. The result is shown in 2.

TABLE 2 results of different filtration modes

The results show that the total glycyrrhizin extraction amount and the total glycyrrhizic acid extraction amount have no obvious difference in solid-liquid separation process, the liquid medicine is clear after centrifugation, the ointment yield is slightly reduced, comprehensive analysis is carried out, 200-mesh filter cloth is selected, and the filter cloth is filtered while hot and centrifuged to be used as the final solid-liquid separation process of the Xiebai powder.

EXAMPLE 3 concentration Process Studies

Adding 6.2g of mixed coarse powder, 2.0g of polished round-grained rice, weighing 6 parts in parallel, respectively placing into 500mL of marmite, adding 300mL of drinking water, heating by adopting an electroceramic furnace (with strong fire of 1600W and slow fire of 200W), decocting with strong fire for 20 minutes (decocting to about 180 mL) after boiling, centrifuging 200-mesh filter cloth after hot filtration (with the rotating speed of 4000 rpm) for 10 minutes to obtain an extracting solution, merging 6 parts of extracting solution, equally dividing into 6 parts, concentrating to about 16.4g (with the feeding amount of 1:2 and the feeding amount of 24.6g (with the feeding amount of 1:3) and 32.8g (with the feeding amount of 1:4), concentrating under reduced pressure at 60 ℃ until the vacuum degree of-0.08 to-0.10 MPa), and recording the concentrating time. The pre-frozen material state and the drying condition are taken as investigation indexes, and the results are shown in Table 3.

The above-mentioned feeding amount is 6.2g of the total weight of coarse powder and 2.0g of polished round-grained rice.

Table 3 table of information for preparing reference sample data for concentration ratio study

From the above results, it is clear that 3 kinds of concentration ratios are good in prefreezing and drying effects, even in foaming, loose in dried matter and easy in paste production, so that 3 kinds of concentration ratios can be used as concentration ratios of the Xiebai powder.

EXAMPLE 4 study of drying Process

Adding 6.2g of mixed coarse powder according to single dose, weighing 2.0g of polished round-grained rice, parallel weighing 4 parts, respectively placing in 500mL marmite (with the capacity of about 500mL, the bottom inner diameter of 11.4cm, the top inner diameter of 13.2cm, the height of 4.6cm, the wall thickness of the marmite of 0.7cm, the bottom thickness of the marmite of 0.8 cm), adding 300mL of drinking water, heating by an electroceramic furnace (with the strong fire of 1600W and the slow fire of 200W), decocting with strong fire for 20 minutes (with the boiling temperature of about 180 mL), filtering with 200-mesh filter cloth while hot, merging the filtrates, centrifuging (with the rotating speed of 4000 rpm) for 10 minutes, equally dividing into 4 parts, concentrating under reduced pressure to 1:4 (g: g), merging the concentrated solution, evenly mixing into 2 parts, respectively drying under reduced pressure at 60 ℃ and freeze drying (with the pre-freeze temperature of-20 to-45 ℃ for at least 40 minutes, drying the freeze-drying the dry time of-50 to 70 ℃ and the vacuum of less than 100Pa, and at least 12 Pa, and recording the dry state, and weighing the obtained paste, and recording the dry state and drying time and the dry state; the glycyrrhizin and glycyrrhizic acid contents in the obtained reference samples were measured respectively, and the total amount of index components was calculated, and the results are shown in Table 4.

Table 4 drying mode study reference sample data preparation information table

From the above results, it was found that there was no significant difference between the 2 drying methods from the analysis of the extract rate, the total amount of glycyrrhizin extracted, and the total amount of glycyrrhizic acid extracted, and that the final drying method of Xiebai powder was determined by the vacuum freeze-drying method in view of the easiness of extraction of the extract by vacuum freeze-drying.

Through the research, the preparation process of the reference sample of the temporary diarrhea powder comprises the following steps: pulverizing parched cortex Mori, cortex Lycii (baked) and parched Glycyrrhrizae radix into coarse powder respectively, weighing parched cortex Mori 41.30g, cortex Lycii (baked) 41.30g, and parched Glycyrrhrizae radix 4.13g, and mixing well for use; taking 6.2g of mixed coarse powder and 2.0g of polished round-grained rice, placing the mixed coarse powder and the polished round-grained rice into a 500mL marmite, adding 300mL of drinking water, heating by adopting an electroceramic stove (with strong fire of 1600W and slow fire of 200W), covering and decocting, heating by strong fire until boiling, decocting by adopting slow fire for 20 minutes (decocting to about 180 mL), filtering by using 200-mesh filter cloth while the mixture is hot, centrifuging the filtrate (with the rotating speed of 4000 revolutions per minute) for 10 minutes, taking supernatant, concentrating under reduced pressure (with the temperature of 60 ℃ and the vacuum degree of-0.08 to-0.10 MPa) until the feeding amount is reached: the weight ratio of the concentrated solution is about 1:2-1:4 (g: g), and the standard sample is obtained by freeze drying (pre-freezing temperature is minus 20 to minus 45 ℃ for at least 40 minutes, drying is cold trap temperature is minus 50 to minus 70 ℃, vacuum degree is less than 100Pa, and freeze drying time is at least 12 hours).

Example 5 Process verification

1. And (5) three-batch verification. Process 3 runs were performed according to the procedure defined in examples 1-4 above, each run containing 25 individual doses.

Adding 6.2g of mixed coarse powder and 2.0g of polished round-grained rice into a 500mL marmite (with the capacity of about 500mL, the inner diameter of the bottom of the marmite is 11.4cm, the inner diameter of the top of the marmite is 13.2cm, the height of the marmite is 4.6cm, the wall thickness of the marmite is 0.7cm, the bottom of the marmite is 0.8 cm), adding 300mL of drinking water, heating by an electroceramic furnace (with the strong fire of 1600W and the slow fire of 200W), covering, decocting by strong fire until boiling, decocting by slow fire for 20 minutes (decocting to about 180 mL), preparing 25 parts of the mixture in parallel according to the process, filtering by 200-mesh filter cloth while hot, mixing the extract, centrifuging (with the rotating speed of 4000 turns per minute), uniformly mixing, reserving 50mL of liquid medicine (the extract in the detection method), measuring the glycyrrhizin, the glycyrrhizic acid content and the fingerprint of the liquid medicine, concentrating the rest liquid medicine under reduced pressure (with the temperature of 60 ℃ and the vacuum degree of-0.08 MPa to-0.10 MPa) until the feeding amount: the weight of the concentrated solution=1:2 (g: g), 20mL of the concentrated solution (the concentrated solution in the detection method) is reserved after shaking, the glycyrrhizin, the glycyrrhizic acid content and the fingerprint of the concentrated solution are measured, the rest concentrated solution is freeze-dried, and a reference sample (the reference sample in the detection method) is collected, so that 1 batch of reference samples of Xiebaisan are obtained.

According to the preparation process, 3 parts of the reference samples of 3 batches of Xiebaisan are prepared in parallel, the paste rate is calculated, the contents of moisture, extract, glycyrrhizin and glycyrrhizic acid in corresponding objects (reference samples) are measured, and the total amount of the extraction liquid, the concentrated liquid and the glycyrrhizin and the glycyrrhizic acid in the corresponding objects (reference samples) are calculated respectively.

The measuring method of the glycyrrhizin and glycyrrhizic acid content and the fingerprint in the extracting solution, the concentrated solution and the reference sample is as follows:

5.1 Process and three-batch Process verification content determination method

The measurement is carried out by high performance liquid chromatography (four general rules 0512 in 2020 edition of Chinese pharmacopoeia).

Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as filler (column length is 25cm, inner diameter is 4.6mm, and particle diameter is 5.0 μm); acetonitrile is taken as a mobile phase A, 0.05% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specifications in the following table; the detection wavelength was 237nm. The number of theoretical plates should be not less than 5000 calculated according to the glycyrrhizin peak.

Time (minutes)	Mobile phase a (%)	Mobile phase B (%)
			0～8	19	81
8～35	19→50	81→50
			35～36	50→100	50→0
36～40	100→19	0→81

Preparation of a control solution: taking appropriate amounts of glycyrrhizin reference substance and ammonium glycyrrhizinate reference substance, precisely weighing, and adding 70% ethanol to obtain mixed solution containing 50 μg of glycyrrhizin and 50 μg of ammonium glycyrrhizinate per 1mL, respectively (glycyrrhizic acid weight=ammonium glycyrrhizinate weight/1.0207).

Preparation of test solution:

Extracting solution: decocting, filtering, centrifuging, and filtering with 0.45 μm filter membrane to obtain filtrate.

And (3) concentrating: precisely measuring 1mL of the concentrated solution prepared by the method, placing in a 10mL measuring flask, adding water to dilute to a scale, shaking uniformly, filtering with a 0.45 μm filter membrane, and collecting the subsequent filtrate.

Reference sample: taking about 0.3g of the reference sample prepared by the method, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 70% ethanol, sealing, weighing, performing ultrasonic treatment (power 320W and frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% ethanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product.

Assay: respectively precisely sucking 10 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.

The measurement results are shown in Table 5.1, FIG. 1 and FIG. 2.

TABLE 5.1 reference Xiebaisan sample Process validation results-1

Note that: in Table 5.1, the values of each batch are the detection data obtained by mixing 25 single doses of the prepared extract.

Analysis of results: from the above results, according to the volume analysis of the extract, the volume average value of three batches of reference samples (each batch comprises 25 single doses) is 4387mL, and the volume average value is 175.5mL according to the single doses, and the volume average value is close to 180mL when 300mL of water is added, so that the determined decocting time of the process is reasonable.

According to the weight analysis of the concentrated solution, the average value of three batches of reference samples (each batch comprises 25 single dose) is 428.94g, and the average value of the three batches of reference samples is 17.16g according to the single dose, the single dose feeding amount is 8.2g, and the feeding amount is as follows: the weight of the concentrated solution is about 1:2 (g: g), the concentration of the concentrated solution is moderate, no obvious wall hanging phenomenon exists, and the quality control of the concentration process is feasible.

According to the analysis of the total amount of glycyrrhizin and the total amount of glycyrrhizic acid, three batches of verification show that the total amount of the glycyrrhizin is 108.30mg-95.89mg and the total amount of the glycyrrhizin is 228.00mg-133.79mg, so that the glycyrrhizin is easier to extract into the extracting solution than the glycyrrhizic acid; the total amount of glycyrrhizin and the total amount of glycyrrhizic acid in the extracting solution of each batch are basically parallel, which proves that the extraction process of the reference sample of the Xiebaisan is stable and feasible. The total amount of glycyrrhizin and glycyrrhizic acid in the extract-concentrate-reference sample is analyzed, the total amount of the glycyrrhizin is from 95.89mg to 91.18mg to 90.81mg, the total amount of the glycyrrhizic acid is from 133.79mg to 125.63mg to 124.36mg, and the total amount data of the glycyrrhizin and the glycyrrhizic acid in the extract-concentrate-reference sample are smaller in difference and basically parallel, so that the mass transfer loss of the glycyrrhizin and the glycyrrhizic acid in the process of the extract-concentrate-reference sample is smaller; the three batches of verification reference samples have no large difference in terms of paste yield, extract and moisture analysis.

In conclusion, the reference sample preparation process is reasonable, stable and controllable.

2. Single batch verification

The results are that the method under the item "5.1" is adopted for measurement, then the fingerprint spectrum and the content measurement method are combined into the same method through fumbling, and the UPLC is adopted for measurement, so that the analysis time is greatly shortened, a batch of samples are extracted by adopting the same preparation process, and the method under the items "5.2" and "5.3" are adopted for measurement, and the specific contents are as follows.

Adding 6.2g of mixed coarse powder and 2.0g of polished round-grained rice into a 500mL marmite (with the capacity of about 500mL, the inner diameter of the bottom of the marmite is 11.4cm, the inner diameter of the top of the marmite is 13.2cm, the height of the marmite is 4.6cm, the wall thickness of the marmite is 0.7cm, the bottom of the marmite is 0.8 cm), adding 300mL of drinking water, heating by an electroceramic furnace (with the strong fire of 1600W and the slow fire of 200W), covering, decocting with the strong fire until boiling, decocting with slow fire for 20 minutes (decocting to about 180 mL), preparing 3 parts in parallel, filtering with 200-mesh filter cloth while the mixture is hot, centrifuging (with the rotating speed of 4000 rpm) for 10 minutes after each 3 parts of decoction are combined, uniformly mixing, obtaining 50mL of extract (the extract in the detection method described below), measuring glycyrrhizin, glycyrrhizic acid content and fingerprint, and concentrating the rest extract under reduced pressure (with the temperature of 60 ℃ C., vacuum degree of-0.08-0.10 MPa) until the feeding amount: the weight of the concentrated solution=1:2 (g: g), 10mL of the concentrated solution (the concentrated solution in the detection method is shown below) is remained after shaking, the glycyrrhizin and glycyrrhizic acid content and the fingerprint of the concentrated solution are measured, the rest concentrated solution is freeze-dried (pre-freezing temperature-20 to-45 ℃ C. For at least 40 minutes, drying is carried out at a cold trap temperature of-50 to-70 ℃ C. And vacuum degree of less than 100Pa, and freeze-drying time is at least 12 hours), corresponding objects (reference samples) are collected, the ointment rate is calculated, the glycyrrhizin and glycyrrhizic acid content and the fingerprint of the corresponding objects (reference samples) are measured, and the total amount of the glycyrrhizin and the glycyrrhizic acid in the extracting solution, the concentrated solution and the reference samples are calculated respectively.

5.2 Process verification batch content determination method

Chromatographic conditions and System applicability test octadecylsilane chemically bonded silica was used as filler (column length 10cm, inner diameter 2.1mm, particle size 1.8 μm); acetonitrile is taken as a mobile phase A, 0.3% trifluoroacetic acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength was 277nm. The number of theoretical plates should be not less than 100000 calculated by glycyrrhizin peak.

Time (minutes)	Mobile phase a (%)	Mobile phase B (%)
			0～3	4→7	96→93
3～9	7	93
			9～11	7→12	93→88
11～18	12→21	88→79
			18～24	21→42	79→58
24～30	42→60	58→40

Preparation of a control solution: taking appropriate amounts of glycyrrhizin reference substance and ammonium glycyrrhizinate reference substance, precisely weighing, and adding 70% ethanol to obtain mixed solution containing 50 μg of glycyrrhizin and 75 μg of ammonium glycyrrhizinate per 1mL (glycyrrhizic acid weight=ammonium glycyrrhizinate weight/1.0207).

Preparation of test solution:

Extracting solution: precisely measuring 15mL of the extract, evaporating to dryness, dissolving the residue with 70% methanol, transferring to a 5mL measuring flask, diluting with 70% methanol to scale, shaking, centrifuging (at 13000 rpm) for 10min, and collecting supernatant.

And (3) concentrating: precisely measuring 3mL of concentrated solution, evaporating to dryness, dissolving the residue with 70% methanol, transferring to a 10mL measuring flask, diluting with 70% methanol to scale, shaking, centrifuging (at 13000 rpm) for 10min, and collecting supernatant.

Reference sample: about 0.5g of the product is precisely weighed, placed in a conical flask with a plug, precisely added with 25mL of 70% methanol, sealed, weighed, subjected to ultrasonic treatment (power 320W, frequency 40 kHz) for 30 minutes, cooled, weighed again, supplemented with 70% methanol for the weight loss, shaken uniformly, centrifuged (rotation speed 13000 revolutions per minute) for 10 minutes, and the supernatant is taken to obtain the product.

Assay: precisely sucking 2 μl of each of the reference solution and the sample solution, and measuring with ultra-high performance liquid chromatograph.

5.3 Fingerprint determination method

Chromatographic conditions and system suitability test: and 5.2.

Preparation of reference solution: taking appropriate amount of glycyrrhizin reference substance, precisely weighing, and adding 70% ethanol to obtain solution containing 50 μg of glycyrrhizin per 1 mL.

Preparation of test solution: the extract, concentrate and reference samples were prepared as 5.2.

Medicinal materials/decoction pieces: taking proper amount of each medicinal powder (the coarse powder) under the prescription (about 1.5g of white mulberry root-bark/stir-fried white mulberry root-bark, about 1.5g of cortex lycii radicis/cortex lycii radicis (baked), about 0.15g of liquorice/stir-fried liquorice and about 1g of polished round-grained rice), precisely weighing, placing the materials into a round-bottomed flask, adding 50mL of water, decocting for 30 minutes, filtering, evaporating filtrate, adding 25mL of 70% methanol into residues, carrying out ultrasonic treatment (the power is 320W and the frequency is 40 kHz) for 30 minutes, shaking uniformly, centrifuging (the rotating speed is 13000 rpm) for 10 minutes, and taking supernatant fluid to obtain the Chinese medicinal preparation.

Assay: respectively precisely sucking 2 μl of reference solution and 2 μl of sample solution, and measuring with ultra-high performance liquid chromatograph.

The results are shown in Table 5.2 and FIG. 3. And determining fingerprint of the same batch of medicinal materials and decoction pieces, and analyzing the relative retention time of the common peak, wherein the results are shown in Table 5.3, FIG. 4 and FIG. 5.

TABLE 5.2 reference sample Process verification results for Xiebaisan

According to total analysis of the extracting solutions, the average value of the total amount of the liquiritin in three extracting solutions (each batch comprises 25 single dose) is verified to be 95.89mg according to the method 5.1, and the average value of the total amount of the liquiritin in three extracting solutions is 3.84mg according to the single dose; the average value of the total amount of glycyrrhizic acid is 133.79mg, and the total amount of glycyrrhizic acid is 5.35mg according to the single dose; according to the method 5.2, the total amount of the glycyrrhizin in a batch of extracting solution (comprising 3 parts of single dose) is verified to be 10.25mg, and 3.42mg is verified to be 3.42mg according to the single dose; the average total glycyrrhizic acid amount is 14.10mg, and the total glycyrrhizic acid amount is 4.70mg according to the single dosage, and the total glycyrrhizin extraction amount and the total glycyrrhizic acid extraction amount are not greatly different in the single dosage. The difference between the two measurement methods is not great, and the analysis time can be shortened by adopting the UPLC method, so that the subsequent experimental study is carried out by selecting method 5.2.

TABLE 5.3 comparative retention time results of process validation of Xiebaisan reference samples

According to fingerprint analysis, peak 1 is new chlorogenic acid, and is common chromatographic peak of parched cortex Mori and cortex Lycii (baked), peaks 2, 3, and 4 are chromatographic peak of cortex Lycii, wherein peak 3 is cortex Lycii B, peaks 5, 6, and 7 are chromatographic peak of parched Glycyrrhrizae radix, wherein peak 5 is chromatographic peak of glycyrrhizin, and peak 7 is chromatographic peak of glycyrrhizic acid.

The chromatographic peak of the mark can be transmitted from decoction pieces, extracting solution and concentrated solution to a chromatogram of a reference sample, which shows that the decoction pieces, the extracting solution, the concentrated solution and the reference sample have better relativity, and 7 common peaks in the reference sample can find the attribution of the medicinal flavor, so the preparation process of the determined reference sample is reasonable, stable and feasible.

Proved by verification, the preparation process of the reference sample of the Xiebaisan comprises the following steps: pulverizing parched cortex Mori, cortex Lycii (baked), and parched Glycyrrhrizae radix into coarse powder respectively, weighing parched cortex Mori 41.30g, cortex Lycii 41.30g, and radix Glycyrrhizae Preparata 4.13g, and mixing well for use; mixing 6.2g of the mixed powder and 2.0g of polished round-grained rice, placing into a marmite (with the capacity of about 500ml, the inner diameter of the bottom of the marmite is 11.4cm, the inner diameter of the top of the marmite is 13.2cm, the height of the marmite is 4.6cm, the wall thickness of the marmite is 0.7cm, the thickness of the marmite bottom is 0.8 cm), adding 300ml of drinking water, heating by an electroceramic furnace (with strong fire of 1600W and with slow fire of 200W), covering, decocting by strong fire until boiling, decocting by slow fire for 20 minutes (decocting to about 180 ml), filtering by 200-mesh filter cloth while hot, centrifuging (rotating at 4000 rpm) for 10 minutes, concentrating under reduced pressure (the temperature of 60 ℃ and the vacuum degree of-0.08 to-0.10 MPa) until the feeding amount is reached: the weight ratio of the concentrated solution is 1:2-1:4 (g: g), and the standard sample is obtained after freeze drying (pre-freezing temperature is-20 to-45 ℃ for at least 40 minutes, drying is performed at a cold trap temperature of-50 to-70 ℃ and vacuum degree is less than 100Pa, and freeze drying time is at least 12 hours).

Example 6 verification of Process stability

The reference Xiebaisan sample was prepared according to the validated procedure of example 5, and 15 batches were repeated, 2 for each batch, and tested using the test method of example 5.2, with the results shown in 5.4.

TABLE 5.4 detection results for 15 batches of reference samples

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From the above data, in combination with various influencing factors existing in the actual production process, the extract, the glycyrrhizin content, the glycyrrhizic acid content, the moisture content and the fingerprint similarity of 15 batches (30 parts) of reference samples are concentrated, which indicates that the quality of the reference samples prepared by the preparation process is stable.

In order to control the quality of the reference sample, the quality standard of the reference sample is shown in Table 6 and FIG. 6 based on the above 15 batches of test data in combination with the actual conditions of the preparation process and factors of various influences existing in the actual production process

TABLE 6 quality attribute analysis Table

Fingerprint spectrum: a control fingerprint was generated using a chromatogram of 15 batches (30 parts) of reference samples, as shown in fig. 6. The sample fingerprint should show chromatographic peaks with the same retention time as that of the reference object, and the similarity is calculated according to the similarity evaluation system of the traditional Chinese medicine chromatographic fingerprint, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.85.

EXAMPLE 7 stability study of reference samples

The stability of a reference sample of Xiebaisan is researched by referring to the stability test guiding principle of four general rules 9001 of the edition of Chinese pharmacopoeia 2020, and the stability of different influencing factors, unsealing and reference samples are respectively inspected.

(1) Sample preparation

Following the procedure defined in example 5, 3 batches of reference samples were prepared. The feeding information of the 3 batches of reference samples is shown in Table 7.1.

TABLE 7.1 stability sample feeding information table

(2) Influence factor test

Taking 190114-01 batches of reference samples, examining the properties, the moisture (drying method), the glycyrrhizin and the glycyrrhizic acid content as data of 0 days, then respectively placing the reference samples under high temperature, high humidity and strong light conditions, respectively sampling on the 5 th day and the 10 th day, and examining the properties, the moisture, the glycyrrhizin and the glycyrrhizic acid content.

① High temperature test

Placing the above reference sample in a culture dish (without packaging, spreading into thin layer with thickness less than or equal to 5 mm), standing at 40deg.C for 10 days, sampling on 5 th and 10 th days, and examining properties, water content, glycyrrhizin and glycyrrhizic acid content.

② High humidity test

The above standard sample (with package) was left at 25℃for 10 days under a relative humidity of 75% + -5%, sampled on days 5 and 10, and examined for properties, moisture, glycyrrhizin and glycyrrhizic acid content.

③ Strong light irradiation test

Placing the reference sample in a culture dish (without packaging, spreading into a thin layer with the thickness less than or equal to 5 mm) in a strong light irradiation box with a fluorescent lamp under the condition that the temperature is 25 ℃ and the relative humidity is 20% +/-5%, placing for 10 days under the condition that the illuminance is 4500 lx+/-500 lx, sampling on the 5 th day and the 10 th day, and investigating the properties, the moisture, the glycyrrhizin and the glycyrrhizic acid content.

The results of the influence factor tests are shown in Table 7.2.

TABLE 7.2 influence factor test results

Conclusion: the standard Xiebaisan sample is placed for 10 days under the conditions of high temperature (40 ℃), high humidity (75% +/-5%) and strong light irradiation (4500 lx+/-500 lx) at the temperature of 25 ℃, and the properties and the content are not obviously changed compared with those of 0 days, so that the product has good stability under the conditions of high temperature, high humidity and strong light irradiation.

(3) Inspection of unsealing test

Taking 190114-01 batches of reference samples, simulating an actual weighing process, and respectively inspecting properties and hygroscopicity in different time periods within 1 day, wherein the specific operation is as follows: 1 bag of sample (about 10 g) was taken, unsealed, and weighed in a balance chamber having a relative humidity of not more than 45% at room temperature. About 1g of the sample is weighed and marked as M sample 1; after weighing, the packaging bag is added with a clip, so that air cannot be directly fed into the bag, the weight is weighed and recorded as M total 1, the packaging bag is placed in a dryer, and the properties of the sample 1 are recorded. After 1 hour, the sample was weighed again and packaged, designated M total 2, and about 1g of the sample was weighed, designated M sample 2, and the same procedure was followed.

And calculating the difference value of Mtotal 2-Mtotal 1, if the weight of Mtotal 2 is not increased or the difference value is within +/-5% of the weight of Mtotal 1, judging that the moisture is not absorbed, comparing the properties of the two samples, if the properties are consistent, continuing to stand for a period of time, and operating in the same way. The reference sample weights were weighed at 0h, 1h, 2h, 4h, 8h, 24h, respectively, and the results are shown in Table 7.3.

TABLE 7.3 results of unsealing test investigation

Time of placement (h)	Traits (3)	Hygroscopicity (DeltaM)
			0	The product is yellow powder; sweet and slightly bitter	0
1	The product is yellow powder; sweet and slightly bitter	0
			2	The product is yellow powder; sweet and slightly bitter	0.01％
4	The product is yellow powder; sweet and slightly bitter	0
			8	The product is yellow powder; sweet and slightly bitter	3.67％
24	The product is yellow powder; sweet and slightly bitter	0

Conclusion: the standard sample of Xiebaisan has stable sample property within 1 day and no moisture absorption phenomenon, which indicates that the product has good unsealing stability.

(4) Stability investigation

The stable samples are packaged and stored in a stable sample reserving chamber (temperature: 25 ℃ and humidity: 60%), and the samples are taken and inspected for properties, moisture, extract, thin layer, content measurement and fingerprint patterns respectively in 0 month, 1 month, 2 months, 3 months and 6 months. The results were compared to 0 months.

The long-term 6 monthly examination-observation results are shown in tables 7.4, 7.5 and 7.6.

TABLE 7.4 stability test examination results (lot number: 190114-01)

TABLE 7.5 stability test examination results (lot number: 190115-02)

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TABLE 7.6 stability test examination results (lot number: 190115-03)

Conclusion: the detection indexes of the 3 batches of reference samples of the Xiebaisan (reference samples) corresponding to the real objects meet the requirements of the quality standard text of the reference samples of the Xiebaisan in long-term test for 1 month, 2 months, 3 months and 6 months, and compared with 0 month, the reference samples of the Xiebaisan have no obvious difference, so that the stability samples of the reference samples of the Xiebaisan have stable quality in the examined time.

(5) Conclusion of stability investigation

The test results show that the standard Xiebaisan sample is placed for 10 days at high temperature (40 ℃), high humidity (75% +/-5%) and strong light irradiation (4500 lx+/-500 lx) at the temperature of 25 ℃, and the properties and the content are not obviously changed compared with those of the standard Xiebaisan sample in 0 days, so that the standard Xiebaisan sample has good stability under the conditions of high temperature, high humidity and strong light irradiation. Under the test conditions and operation of the unsealing test, the reference sample of the laxative powder has stable properties within 1 day and no moisture absorption phenomenon, and shows that the product has good unsealing stability. The detection indexes of 3 batches of reference Xiebaisan samples in long-term test for 1 month, 2 months, 3 months and 6 months all meet the requirements of the standard body of the quality standard of the reference Xiebaisan samples, and compared with 0 month, the standard sample has no obvious difference, so that the quasi-stable sample of the Xiebai reference sample has stable quality in the examined time.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims

1. A reference sample of Xiebaisan is characterized by comprising 1.4-4.7 mg/g of glycyrrhizin and 1.5-4.9 mg/g of glycyrrhizic acid.

2. The reference Xiebaisan sample according to claim 1, wherein the content of glycyrrhizin is 1.4～4.5mg/g,1.4～4.0mg/g,1.5～3.5mg/g,1.5～3.0mg/g,1.7～4.5mg/g,1.8～4.5mg/g,1.9～4.5mg/g,1.9～4.0mg/g,2.0～3.8mg/g,2.1～3.3mg/g,2.4～3.2mg/g,2.5～3.0mg/g,2.6～3.0mg/g or 2.7-3.0 mg/g;

The content of the glycyrrhizic acid is 1.5～4.5mg/g,1.5～4.0mg/g,1.5～3.5mg/g,1.5～3.0mg/g,1.7～4.5mg/g,1.8～4.5mg/g,1.9～4.7mg/g,2.0～4.2mg/g,2.3～3.9mg/g,2.5～3.7mg/g,2.6～3.5mg/g,2.6～3.3mg/g or 2.6-3.2 mg/g.

3. The reference xiebuia sample according to claim 1 or 2, wherein the proportion of alcohol-soluble extract in the reference xiebuia sample is 23.0% to 53.0%; or the proportion of the alcohol-soluble extract is 23% -50%, 23% -45%, 23.2% -45%, 23.5% -45%, 29% -50%, 33% -46%, 35% -42%, 37% -42% or 38% -42%.

4. The reference xiebuia sample of claim 1 or 2, wherein the reference xiebuia sample has a moisture content of no more than 9.0%, or wherein the reference xiebuia sample has a moisture content of no more than 8.5%, no more than 8.0%, no more than 7.50%, no more than 7.0%, no more than 6.5%, no more than 6.0%.

5. The reference xiebuia sample according to any one of claims 1 to 4, wherein the decoction pieces of the reference xiebuia sample have the following composition: 41.3 parts of cortex lycii radicis (baked), 41.3 parts of fried cortex mori radicis, 4.13 parts of fried liquorice and 1.5-2.5 parts of polished round-grained rice.

6. The reference sample of Xiebaisan according to claim 5, wherein the liquid medicine is filtered and concentrated to a weight ratio of 4-2:1 of the weight to the feeding amount to obtain a concentrated solution.

7. A method for preparing a reference sample of xiebuia powder according to any one of claims 1 to 6, comprising the steps of:

S1, medicinal material components of a reference Xiebaisan sample are as follows: 41.3 parts of cortex lycii radicis (baked), 41.3 parts of fried cortex mori radicis, 4.13 parts of fried liquorice and 1.5-2.5 parts of polished round-grained rice; wherein the cortex lycii radicis (baked), the fried cortex mori radicis and the fried liquorice are mixed and ground into coarse powder, 4.0 to 8.0 parts of coarse powder and 1.5 to 2.5 parts of polished round-grained rice are taken to be decocted with water, and the mixture is filtered and centrifuged to obtain an extracting solution;

8. The method for preparing a reference sample of Xiebaisan according to claim 7, wherein in the step S1, the water addition amount of the Xiebaisan is 200-300 parts in the process of decocting with water; more preferably, the amount of water added is 300 parts.

9. The method according to claim 7, wherein in the step S2, the weight ratio of the concentrated solution to the amount of the additive is 4:1, 3:1 or 2:1.

10. The method for preparing a reference sample of xie bai san according to any one of claims 7 to 9, wherein the prepared reference sample of xie bai san has a glycyrrhizin content of 1.4 to 4.7mg/g, a glycyrrhizic acid content of 1.5 to 4.9mg/g, an alcohol-soluble extract content of 23.0 to 53.0% and a moisture content of not more than 9.0%.