阅读说明：本技术 一种知母配方颗粒的质量标准检测方法 (Quality standard detection method of rhizoma anemarrhenae formula granules ) 是由 罗云 王朋辉 梁宇星 韩艳 赵峰 刘爱珍 于 2020-05-11 设计创作，主要内容包括：本发明公开了一种知母配方颗粒的质量标准检测方法,包括知母配方颗粒的鉴别、知母配方颗粒的检查、知母配方颗粒相关物质的含量测定。本发明针对知母配方颗粒提供了系统化,科学化的质量标准检测方法,包括其鉴别、各项理化指标的检查以及相关成分含量的测定,测定方法使用参数精准控制,能完全反应出知母配方颗粒的质量,更有效地控制产品质量,并可作为大规模生产的知母配方颗粒疗效一致性的评价依据,具有较高的科学指导和产业调控价值。(The invention discloses a quality standard detection method of rhizoma anemarrhenae formula granules, which comprises the steps of identifying the rhizoma anemarrhenae formula granules, checking the rhizoma anemarrhenae formula granules and measuring the content of related substances of the rhizoma anemarrhenae formula granules. The invention provides a systematic and scientific quality standard detection method aiming at rhizoma anemarrhenae formula granules, which comprises the steps of identification, check of various physicochemical indexes and determination of related component contents, and the determination method adopts accurate control of use parameters, can completely reflect the quality of the rhizoma anemarrhenae formula granules, more effectively control the product quality, can be used as an evaluation basis for the consistency of curative effects of the rhizoma anemarrhenae formula granules produced in a large scale, and has higher scientific guidance and industrial regulation and control values.)

1. A quality standard detection method of rhizoma anemarrhenae formula granules is characterized by comprising the following steps:

(1) identifying rhizoma anemarrhenae formula granules;

(2) checking rhizoma anemarrhenae formula granules;

(3) and (4) measuring the content of related substances of the rhizoma anemarrhenae formula granules.

2. The method for detecting the quality standard of the rhizoma anemarrhenae granules according to claim 1, wherein the identification method comprises the following steps:

(1) the characteristics are as follows: yellow-white to brown-yellow particles; light smell, slightly sweet and slightly bitter taste;

(2) taking 0.2g of rhizoma anemarrhenae formula particle powder, adding 10ml of 30% acetone, carrying out ultrasonic treatment for 20 minutes, and taking supernatant as a test solution;

(3) taking timosaponin BII reference substance, adding 30% acetone to obtain 1mg solution per 1ml as reference substance solution;

(4) and (3) according to thin-layer chromatography, sucking 4 μ l of each of the above solutions, respectively dropping on the same silica gel G thin-layer plate, developing with developing agent, taking out, air drying, spraying vanillin sulfuric acid test solution, heating at 105 deg.C until the spots are clearly developed, and displaying the spots with the same color in the sample chromatogram at the position corresponding to the control chromatogram.

3. The method for detecting the quality standard of the rhizoma anemarrhenae dispensing granule as claimed in claim 2, wherein the developing solvent in the step 4 is n-butanol, glacial acetic acid, and water in a volume ratio of 4: 1: 5, forming an upper layer solution of the mixed solution.

4. The method for detecting the quality standard of the rhizoma anemarrhenae granules according to claim 1, wherein the inspection method comprises the following steps:

(1) and (3) checking the granularity: according to the particle size and particle size distribution determination method, the sum of the first sieve and the fifth sieve which can not pass through the sieve can not exceed 15 percent;

(2) and (3) moisture inspection: the water content of the Chinese medicinal granule is not more than 8.0% by water content determination method;

(3) and (3) testing the dissolubility: taking 10g of a test sample, adding 200ml of hot water, stirring for 5 minutes, immediately observing, and completely dissolving or slightly turbidity without foreign matters such as coke breeze and the like;

(4) and (3) microbial limit inspection: according to the microbial limit test method, the number of aerobic bacteria is regulated to be less than 10³cfu/g, the number of mould and yeast is not more than 10²cfu/g, Escherichia coli could not be detected;

(5) heavy metal and harmful element inspection: according to the determination of the method for measuring lead, cadmium, arsenic, mercury and copper, lead can not exceed 5mg/kg, cadmium can not exceed 0.3mg/kg, arsenic can not exceed 2mg/kg, mercury can not exceed 0.2mg/kg and copper can not exceed 20 mg/kg;

(6) checking extract: it is measured by hot dipping method of alcohol-soluble extract measuring method, using ethanol as solvent, and should not be less than 19.0%.

5. The method for detecting the quality standard of the rhizoma anemarrhenae granules according to claim 1, wherein the content measurement comprises the content measurement of mangiferin and the content measurement of timosaponin BII.

6. The method for detecting the quality standard of the rhizoma anemarrhenae dispensing granule as claimed in claim 5, wherein the determination of the content of mangiferin comprises the following steps:

(1) chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as filler, acetonitrile-0.2% glacial acetic acid aqueous solution (15: 85) is used as mobile phase, the detection wavelength is 258nm, and the number of theoretical plates is not less than 6000 calculated according to mangiferin peak;

(2) preparation of control solutions: taking a proper amount of mangiferin reference substance, precisely weighing, and adding diluted ethanol to obtain a solution containing 50 μ g of mangiferin per 1 ml;

(3) preparation of a test solution: taking 0.1g of rhizoma anemarrhenae formula particle powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, weighing, performing ultrasonic treatment at the power of 400W and the frequency of 40kHz for 30 minutes, cooling, weighing again, supplementing the lost weight with the dilute ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the rhizoma anemarrhenae formula particle powder;

(4) the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring to obtain mangiferin content of not less than 2.3mg per 1g based on the dry product.

7. The method for detecting the quality standard of the rhizoma anemarrhenae granules according to claim 5, wherein the determination of the timosaponin BII content comprises the following steps:

(1) chromatographic conditions and system applicability test: using octyl silane bonded silica gel as filler, acetonitrile-water (25: 75) as mobile phase, detecting by an evaporative light scattering detector, wherein the number of theoretical plates is not less than 10000 calculated according to the peak of timosaponin BII;

(2) preparation of control solutions: accurately weighing appropriate amount of timosaponin BII reference substance, and adding 30% acetone to obtain solution containing 0.50mg per 1 ml;

(3) preparation of a test solution: taking 0.3g of rhizoma anemarrhenae formula particle powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 30% acetone, weighing, performing ultrasonic treatment at the power of 400W and the frequency of 40kHz for 30 minutes, taking out, cooling, weighing again, supplementing the weight loss by using 30% acetone, shaking up, filtering, and taking the subsequent filtrate to obtain the rhizoma anemarrhenae formula particle powder;

(4) the determination method comprises the following steps: precisely sucking 5 μ l and 10 μ l of reference solution and 5-10 μ l of test solution, respectively, injecting into liquid chromatograph, measuring, calculating with logarithm equation of external standard two-point method, and calculating according to dry product, wherein each 1g contains timosaponin BII not less than 10.0 mg.

Technical Field

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a quality standard detection method of rhizoma anemarrhenae formula granules.

Background

The rhizoma anemarrhenae granule is prepared from dried rhizome of Anemarrhena asphodeloides bge. It is bitter, sweet and cold in taste, enters lung, stomach and kidney channels, has functions of clearing heat and purging fire, nourishing yin and moistening dryness, and can be used for treating exogenous febrile disease, hyperpyrexia and polydipsia, lung heat dry cough, bone steaming tidal fever, internal heat diabetes, intestinal dryness and constipation.

In recent years, the development and research of the efficacy and the function of the rhizoma anemarrhenae, especially the processing of rhizoma anemarrhenae decoction pieces, are continuously carried out at home and abroad aiming at the efficacy and the function of the rhizoma anemarrhenae respectively, and a scheme of more matched treatment is hoped to be found. However, there is little research on the quality standard detection method of the rhizoma anemarrhenae formula granules, and the effective components of the rhizoma anemarrhenae formula granules cannot be correspondingly guaranteed, so that the medication safety and effectiveness of patients are difficult to guarantee.

Disclosure of Invention

The invention aims to make up for the defects of the prior art and provides a quality standard detection method of rhizoma anemarrhenae formula granules.

In order to achieve the above object, the present invention provides the following technical solutions:

a quality standard detection method of rhizoma anemarrhenae formula granules comprises the following steps:

(1) identifying rhizoma anemarrhenae formula granules;

(2) checking rhizoma anemarrhenae formula granules;

(3) and (4) measuring the content of related substances of the rhizoma anemarrhenae formula granules.

Further, the authentication method comprises the following steps:

(1) the characteristics are as follows: yellow-white to brown-yellow particles; light smell, slightly sweet and slightly bitter taste;

(2) taking 0.2g of rhizoma anemarrhenae formula particle powder, adding 10ml of 30% acetone, carrying out ultrasonic treatment for 20 minutes, and taking supernatant as a test solution;

(3) taking timosaponin BII reference substance, adding 30% acetone to obtain 1mg solution per 1ml as reference substance solution;

(4) and (3) according to thin-layer chromatography, sucking 4 μ l of each of the above solutions, respectively dropping on the same silica gel G thin-layer plate, developing with developing agent, taking out, air drying, spraying vanillin sulfuric acid test solution, heating at 105 deg.C until the spots are clearly developed, and displaying the spots with the same color in the sample chromatogram at the position corresponding to the control chromatogram.

Preferably, the developing solvent in the step 4 is n-butanol, glacial acetic acid and water according to a volume ratio of 4: 1: 5, forming an upper layer solution of the mixed solution.

Further, the inspection method includes the steps of:

(1) and (3) checking the granularity: according to the particle size and particle size distribution determination method, the sum of the first sieve and the fifth sieve which can not pass through the sieve can not exceed 15 percent;

(2) and (3) moisture inspection: the water content of the Chinese medicinal granule is not more than 8.0% by water content determination method;

(3) and (3) testing the dissolubility: taking 10g of a test sample, adding 200ml of hot water, stirring for 5 minutes, immediately observing, and completely dissolving or slightly turbidity without foreign matters such as coke breeze and the like;

(4) and (3) microbial limit inspection: according to the microbial limit test method, the number of aerobic bacteria is regulated to be less than 10³cfu/g, the number of mould and yeast is not more than 10²cfu/g, Escherichia coli could not be detected;

(5) heavy metal and harmful element inspection: according to the determination of the method for measuring lead, cadmium, arsenic, mercury and copper, lead can not exceed 5mg/kg, cadmium can not exceed 0.3mg/kg, arsenic can not exceed 2mg/kg, mercury can not exceed 0.2mg/kg and copper can not exceed 20 mg/kg;

(6) checking extract: it is measured by hot dipping method of alcohol-soluble extract measuring method, using ethanol as solvent, and should not be less than 19.0%.

Further, the content determination comprises the content determination of mangiferin and the content determination of timosaponin BII.

Further, the content determination of mangiferin comprises the following steps:

(1) chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as filler, acetonitrile-0.2% glacial acetic acid aqueous solution (15: 85) is used as mobile phase, the detection wavelength is 258nm, and the number of theoretical plates is not less than 6000 calculated according to mangiferin peak;

(2) preparation of control solutions: taking a proper amount of mangiferin reference substance, precisely weighing, and adding diluted ethanol to obtain a solution containing 50 μ g of mangiferin per 1 ml;

(3) preparation of a test solution: taking 0.1g of rhizoma anemarrhenae formula particle powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, weighing, performing ultrasonic treatment at the power of 400W and the frequency of 40kHz for 30 minutes, cooling, weighing again, supplementing the lost weight with the dilute ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the rhizoma anemarrhenae formula particle powder;

(4) the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, measuring, and measuring by dry product, wherein each 1g contains mangiferin (C)₁₉H₁₈O₁₁) Not less than 2.3 mg.

Furthermore, the content determination of timosaponin BII comprises the following steps:

(1) chromatographic conditions and system applicability test: using octyl silane bonded silica gel as filler, acetonitrile-water (25: 75) as mobile phase, detecting by an evaporative light scattering detector, wherein the number of theoretical plates is not less than 10000 calculated according to the peak of timosaponin BII;

(2) preparation of control solutions: accurately weighing appropriate amount of timosaponin BII reference substance, and adding 30% acetone to obtain solution containing 0.50mg per 1 ml;

(3) preparation of a test solution: taking 0.3g of rhizoma anemarrhenae formula particle powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 30% acetone, weighing, performing ultrasonic treatment at the power of 400W and the frequency of 40kHz for 30 minutes, taking out, cooling, weighing again, supplementing the weight loss by using 30% acetone, shaking up, filtering, and taking the subsequent filtrate to obtain the rhizoma anemarrhenae formula particle powder;

(4) the determination method comprises the following steps: precisely sucking 5 μ l and 10 μ l of reference solution and 5-10 μ l of test solution, respectively, injecting into liquid chromatograph, measuring, calculating with external standard two-point method logarithmic equation, and calculating according to dry product, each 1g contains timosaponin BII (C)₄₅H₇₆O₁₉) Not less than 10.0 mg.

The invention has the advantages that:

the invention provides a systematic and scientific quality standard detection method aiming at rhizoma anemarrhenae formula granules, which comprises the steps of identification, check of various physicochemical indexes and determination of related component contents, and the determination method adopts accurate control of use parameters, can completely reflect the quality of the rhizoma anemarrhenae formula granules, more effectively control the product quality, can be used as an evaluation basis for the consistency of curative effects of the rhizoma anemarrhenae formula granules produced in a large scale, and has higher scientific guidance and industrial regulation and control values.

Drawings

Fig. 1 shows a thin-layer chromatogram for identifying rhizoma anemarrhenae granule, wherein: 1. rhizoma anemarrhenae granulate 201805012; 2. rhizoma anemarrhenae granulate 201805022; 3. rhizoma anemarrhenae granulate 201805032; 4. a negative sample; 5. timosaponin BII control.

FIG. 2 is a graph showing the absorbance standard curve of lead standards at different concentrations.

FIG. 3 is a graph showing the absorbance standard curve of cadmium standard at different concentrations.

FIG. 4 is a graph showing the absorbance standard of arsenic standards at different concentrations.

FIG. 5 is a graph showing absorbance standards for various concentrations of mercury standards.

FIG. 6 is a graph showing absorbance standards for various concentrations of copper standards.

FIG. 7 shows a comparison spectrum of a reference substance, a sample, an auxiliary material and a blank sample HP L C in mangiferin content measurement.

Fig. 8 shows a mangiferin standard curve.

FIG. 9 shows L N B17064 column durability test.

FIG. 10 shows L N B18123 column durability test.

FIG. 11 shows L N B18102 column durability test.

FIG. 12 shows the comparative spectrum of control, sample, adjuvant, and blank HP L C in determination of timosaponin BII content.

FIG. 13 is a BII standard curve diagram of timosaponin.

FIG. 14 shows L OT: 15224 column durability test.

FIG. 15 shows L OT 15308 column durability test.

FIG. 16 shows L OT 14106 column durability test.

Detailed Description

The technical scheme of the invention is further explained by combining the specific examples as follows: