阅读说明：本技术 一种烫狗脊标准汤剂质量检测方法 (Method for detecting quality of standard decoction of rhizoma cibotii ) 是由 何述金 周乐学 黄黎明 周代俊 朱美成 喻艳 何承东 于 2021-09-13 设计创作，主要内容包括：本发明提供一种烫狗脊标准汤剂质量检测方法,包括通过对烫狗脊标准汤剂的性状、干浸膏出膏率、薄层鉴别、浸出物、特征图谱及原儿茶酸含量测定,将标准汤剂含量标准限定为每1g含原儿茶酸为0.80-5.20mg,其中,干浸膏出膏率测定采用煎煮法进行测定；薄层鉴别采用照薄层色谱法进行鉴别；浸出物采用热浸法测定；特征图谱及原儿茶酸含量测定均采用液相色谱法测定。本发明的烫狗脊标准汤剂质量检测方法,通过多方面测量,来评定烫狗脊标准汤剂的质量,为产品的质量稳定奠定坚实的基础,能够建立烫狗脊汤剂可行的质量标准,实现烫狗脊标准汤剂质量的有效控制。(The invention provides a method for detecting the quality of a standard decoction of scalded rhizoma cibotii, which comprises the steps of limiting the standard decoction content to 0.80-5.20mg of protocatechuic acid in each 1g of the standard decoction of the scalded rhizoma cibotii by the properties of the standard decoction of the scalded rhizoma cibotii, the paste yield of a dry extract, thin-layer identification, a leachate, a characteristic spectrum and the determination of the protocatechuic acid content, wherein the determination of the paste yield of the dry extract is determined by adopting a decocting method; thin-layer identification is carried out by adopting thin-layer chromatography; measuring the extract by adopting a hot dipping method; the characteristic spectrum and the protocatechuic acid content are measured by liquid chromatography. According to the method for detecting the quality of the standard decoction of the scalded rhizoma cibotii, the quality of the standard decoction of the scalded rhizoma cibotii is evaluated through multi-aspect measurement, a solid foundation is laid for the stable quality of products, a feasible quality standard of the decoction of the scalded rhizoma cibotii can be established, and the effective control of the quality of the standard decoction of the scalded rhizoma cibotii is realized.)

1. A method for detecting the quality of standard decoction of rhizoma Cibotii comprises the following steps,

the standard decoction content is limited to 0.80-5.20mg of protocatechuic acid per 1g by properties of standard decoction of the scalded rhizoma cibotii, dry extract yield, thin-layer identification, extract, characteristic spectrum and protocatechuic acid content measurement, wherein the dry extract yield measurement adopts a decoction method for measurement; thin-layer identification is carried out by adopting thin-layer chromatography; measuring the extract by adopting a hot dipping method; measuring the characteristic spectrum and the protocatechuic acid content by adopting a liquid chromatography;

the characteristic spectrum determination by liquid chromatography comprises the following steps: performing liquid chromatograph analysis, taking solution prepared from rhizoma Cibotii reference medicinal material as reference solution, protocatechuic acid reference solution as reference solution, taking solution prepared from rhizoma Cibotii standard decoction sample as test solution, respectively precisely sucking reference solution, reference solution and test solution, respectively injecting into liquid chromatograph, and measuring; wherein, the adopted chromatographic conditions are C18(250mmx4.6mm, 5um) (Waters XSelec HSS T3); mobile phase: gradient elution was performed as specified in table a using methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B;

TABLE a gradient elution procedure

Flow rate: 1.0 mL/min; column temperature: 35 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: 254 nm.

2. The method for detecting the quality of the standard rhizoma cibotii decoction of claim 1, wherein the decocting method comprises the following steps: soaking rhizoma Cibotii decoction pieces in water for 30-40min, decocting twice, the first time for 30-40min and the second time for 25-30min, performing solid-liquid separation while hot, mixing filtrates, concentrating, and drying to obtain standard rhizoma Cibotii decoction dry extract powder.

3. The method for detecting the quality of the standard decoction of the hot dog bone as claimed in claim 1, wherein the thin layer chromatography comprises the following steps:

(1) preparing a test solution a: taking a sample of 2g of the standard decoction of the scalded rhizoma cibotii, adding 50mL of methanol, carrying out ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, and dissolving residues in 1mL of methanol to prepare a test sample solution a;

(2) preparation of control solution a: dissolving protocatechuic acid reference substance in methanol to obtain reference substance solution a with concentration of 1 mg/mL;

(3) performing thin layer chromatography analysis: the thin layer chromatography conditions were thin layer plates: silica gel G thin layer plate; sample amount of spotting: 1uL of each of the test solution a and the reference solution a; developing agent: chloroform-ethyl acetate-methanol-formic acid in a volume ratio of 12:2:1: 0.8; color developing agent: temporarily preparing a 2% ferric trichloride solution-1% potassium ferricyanide solution, wherein the volume ratio of the 2% ferric trichloride solution to the 1% potassium ferricyanide solution is 1:1, and inspecting under sunlight.

4. The method for detecting quality of rhizoma Cibotii standard decoction of claim 1, wherein the hot dipping method uses ethanol as solvent, and adopts the hot dipping method under the alcohol-soluble extract measuring method to measure extract range.

5. The method for detecting the quality of the standard decoction of the hot dog bone as claimed in claim 1, wherein the step of determining the characteristic map by adopting the liquid chromatography further comprises the following steps:

(1) preparation of reference solution b: taking 1.0g of rhizoma Cibotii reference material, adding 25mL of water, decocting for 1 hour, filtering, evaporating filtrate to dryness, adding 5mL of methanol into residue, filtering, and taking filtrate as reference material solution b;

(2) preparation of control solution b: taking a proper amount of protocatechuic acid reference substance, precisely weighing, and adding methanol for dissolving to obtain a reference substance solution b with the concentration of 0.1 mg/mL;

(3) preparing a test solution b: taking 1.0g of a scalded rhizoma cibotii standard decoction sample, precisely weighing, placing the sample in a conical flask with a plug, adding 25mL of precisely weighed methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, and taking the filtrate as a test sample solution b.

6. The method for detecting the quality of the standard decoction of rhizoma cibotii as claimed in claim 1, wherein the determination of the protocatechuic acid content by liquid chromatography comprises: performing liquid chromatograph analysis, taking solution prepared from protocatechuic acid reference substance as reference substance solution c, taking solution prepared from scalded rhizoma Cibotii standard decoction sample as test substance solution c, precisely absorbing reference substance solution c and test substance solution c respectively, injecting into liquid chromatograph, and measuring; wherein, the adopted chromatographic conditions are C18(250mmx4.6mm, 5um) (Waters XSelec HSS T3); mobile phase: taking methanol as a mobile phase A and 0.1% phosphoric acid solution as a mobile phase B, and performing gradient elution according to the specification; flow rate: 1.0 mL/min; column temperature: 35 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: 254 nm.

7. The method for detecting the quality of the standard decoction of rhizoma cibotii as claimed in claim 6, wherein the step of determining the content of protocatechuic acid by liquid chromatography further comprises the following steps:

(1) preparation of control solutions: taking a proper amount of protocatechuic acid reference substance, precisely weighing, and adding methanol to prepare a solution containing protocatechuic acid with the concentration of 0.1mg/ml as a reference substance solution c;

(2) preparing a test solution: taking about 1.0g of a scalded rhizoma cibotii standard decoction sample, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, complementing the weight loss reduction amount with methanol, shaking uniformly, and filtering to obtain a test solution c.

Technical Field

The invention relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a method for detecting the quality of a standard decoction of rhizoma cibotii.

Background

Modern medicines need to have three characteristics of stability, uniformity, safety and effectiveness, and Chinese patent medicines are difficult to be compared with western medicines in the aspects, so that various means are needed for detection, and the reliability and stability of detection results are ensured. The scalded rhizoma Cibotii is prepared from dried rhizome of Cibotium baromettz (L.) J.Sm of Unionidae, and has effects of nourishing liver and kidney, removing rheumatism, strengthening waist and foot, benefiting articulation, and treating soreness of waist and back, gonalgia, weakness of foot, cold dampness, periarthris, urination frequency, nocturnal emission, and leucorrhea. At present, the detection method of the scalded rhizoma cibotii medicinal material is mainly liquid chromatography, but the existing liquid chromatography is only adopted to detect the scalded rhizoma cibotii decoction, which is defective and can not meet the quality control requirement of the traditional Chinese medicine formula granule. Therefore, it is necessary to establish a standard decoction quality detection method for rhizoma Cibotii.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provide a method for detecting the quality of the standard decoction of the rhizoma cibotii, so as to better control the quality of the rhizoma cibotii decoction, characterize the quality of the medicine and improve the stability of the medicine.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

the invention provides a method for detecting the quality of standard decoction of rhizoma cibotii, which comprises the following steps,

the standard decoction content is limited to 0.80-5.20mg of protocatechuic acid per 1g by properties of standard decoction of the scalded rhizoma cibotii, dry extract yield, thin-layer identification, extract, characteristic spectrum and protocatechuic acid content measurement, wherein the dry extract yield measurement adopts a decoction method for measurement; thin-layer identification is carried out by adopting thin-layer chromatography; measuring the extract by adopting a hot dipping method; measuring the characteristic spectrum and the protocatechuic acid content by adopting a liquid chromatography;

the characteristic spectrum determination by liquid chromatography comprises the following steps: performing liquid chromatograph analysis, taking solution prepared from rhizoma Cibotii reference medicinal material as reference solution, protocatechuic acid reference solution as reference solution, taking solution prepared from rhizoma Cibotii standard decoction sample as test solution, respectively precisely sucking reference solution, reference solution and test solution, respectively injecting into liquid chromatograph, and measuring; wherein, the adopted chromatographic conditions are C18(250mmx4.6mm, 5um) (Waters XSelec HSS T3); mobile phase: gradient elution was performed as specified in table a using methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B;

TABLE a gradient elution procedure

Flow rate: 1.0 mL/min; column temperature: 35 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: 254 nm.

In one embodiment, the cooking method comprises: the decocting method comprises the following steps: soaking rhizoma Cibotii decoction pieces in water for 30-40min, decocting twice, the first time for 30-40min and the second time for 25-30min, performing solid-liquid separation while hot, mixing filtrates, concentrating, and drying to obtain standard rhizoma Cibotii decoction dry extract powder.

In one embodiment, the thin layer chromatography comprises the following steps:

(1) preparing a test solution a: taking a sample of 2g of the standard decoction of the scalded rhizoma cibotii, adding 50mL of methanol, carrying out ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, and dissolving residues in 1mL of methanol to prepare a test sample solution a;

(2) preparation of control solution a: dissolving protocatechuic acid reference substance in methanol to obtain reference substance solution a with concentration of 1 mg/mL;

(3) performing thin layer chromatography analysis: the thin layer chromatography conditions were thin layer plates: silica gel G thin layer plate; sample amount of spotting: 1uL of each of the test solution a and the reference solution a; developing agent: chloroform-ethyl acetate-methanol-formic acid in a volume ratio of 12:2:1: 0.8; color developing agent: temporarily preparing a 2% ferric trichloride solution-1% potassium ferricyanide solution, wherein the volume ratio of the 2% ferric trichloride solution to the 1% potassium ferricyanide solution is 1:1, and inspecting under sunlight.

In one embodiment, the hot dipping method uses ethanol as a solvent, and adopts the hot dipping method under the item of alcohol-soluble extract measuring method to measure the extract range.

In one embodiment, the step of determining the characteristic profile by liquid chromatography further comprises the following steps:

(1) preparation of reference solution b: taking 1.0g of rhizoma Cibotii reference material, adding 25mL of water, decocting for 1 hour, filtering, evaporating filtrate to dryness, adding 5mL of methanol into residue, filtering, and taking filtrate as reference material solution b;

(2) preparation of control solution b: taking a proper amount of protocatechuic acid reference substance, precisely weighing, and adding methanol for dissolving to obtain a reference substance solution b with the concentration of 0.1 mg/mL;

(3) preparing a test solution b: taking 1.0g of a scalded rhizoma cibotii standard decoction sample, precisely weighing, placing the sample in a conical flask with a plug, adding 25mL of precisely weighed methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, and taking the filtrate as a test sample solution b.

In one embodiment, the determination of the protocatechuic acid content by liquid chromatography comprises: performing liquid chromatograph analysis, taking solution prepared from protocatechuic acid reference substance as reference substance solution c, taking solution prepared from scalded rhizoma Cibotii standard decoction sample as test substance solution c, precisely absorbing reference substance solution c and test substance solution c respectively, injecting into liquid chromatograph, and measuring; wherein, the adopted chromatographic conditions are C18(250mmx4.6mm, 5um) (Waters XSelec HSS T3); mobile phase: taking methanol as a mobile phase A and 0.1% phosphoric acid solution as a mobile phase B, and performing gradient elution according to the specification; flow rate: 1.0 mL/min; column temperature: 35 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: 254 nm.

In one embodiment, the step of determining the content of protocatechuic acid by liquid chromatography further comprises the following steps:

(1) preparation of control solutions: taking a proper amount of protocatechuic acid reference substance, precisely weighing, and adding methanol to prepare a solution containing protocatechuic acid with the concentration of 0.1mg/ml as a reference substance solution c;

(2) preparing a test solution: taking about 1.0g of a scalded rhizoma cibotii standard decoction sample, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, complementing the weight loss reduction amount with methanol, shaking uniformly, and filtering to obtain a test solution c.

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

(1) the quality of the standard decoction of the scalded rhizoma cibotii is evaluated by researching the properties of the standard decoction of the scalded rhizoma cibotii, the dry extract yield, the thin-layer identification, the extract, the characteristic map and the protocatechuic acid content measurement, a solid foundation is laid for the quality stability of products, a feasible quality standard of the standard decoction of the scalded rhizoma cibotii can be established, the effective control of the quality of the standard decoction of the scalded rhizoma cibotii is realized, and the chromatographic condition of the application is adopted for liquid phase analysis, so that a chromatogram with better and clearer resolution can be obtained.

(2) The rhizoma cibotii decoction pieces are prepared into the rhizoma cibotii decoction piece standard decoction by a decoction method, the average protocatechuic acid content is 1.92mg/g, the measured content range is 1.10-4.00mg/g, the SD (standard deviation) is 1.12, the allowable range of the protocatechuic acid content is 1.35-2.50mg/g according to the average value of 70-130%, the allowable range of the protocatechuic acid content of the standard decoction is 0.80-5.28 mg/g according to the low limit of minus SD and the high limit of plus 3 SD. The average protocatechuic acid transfer rate is 64.02%, the transfer rate range is 37.8-87.8%, the SD is 19.43, according to technical requirements for quality control and standard formulation of Chinese medicinal granules, the allowable range of the protocatechuic acid content transfer rate is calculated according to 70-130% of the transfer rate average value, and the allowable range of the protocatechuic acid content transfer rate is 44.81-83.23%. The allowable range of the protocatechuic acid content transfer rate is 25.16-102.88% calculated according to +/-2 SD. According to the data, the determination limit of the protocatechuic acid content of the standard decoction is drawn as follows: 0.80 mg/g-5.20 mg/g, and the allowable range of the content transfer rate is 25.0-100.0%. The results show that the protocatechuic acid content and the transfer rate thereof in the standard decoction of a plurality of batches are in an allowable range, so the invention can provide reference basis for the quality standard research of the rhizoma cibotii formula granules.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a thin-layer diagram of a standard decoction of 15 batches of rhizoma Cibotii decoction pieces according to an embodiment of the present invention; wherein, 1 group of maps are negative control sample thin-layer maps, 2 groups of maps are protocatechuic acid control solution thin-layer maps, and 3-17 groups of maps are 15 batches of standard decoction thin-layer maps of rhizoma cibotii decoction pieces.

FIG. 2 is a comparison of different extraction methods in the investigation of the extraction method of the present invention; wherein S1 is a characteristic spectrum of the sample solution extracted by ultrasound; s2 is the characteristic spectrum of the test solution extracted by reflux.

FIG. 3 is a comparison of different extraction solvents in the present invention; wherein S1 is a characteristic map of a test solution prepared by 20% methanol extraction; s2 is a characteristic spectrum of a test solution prepared by 50% ethanol extraction; s3 is a sample solution feature map prepared by methanol extraction.

FIG. 4 is a graph comparing different sample amounts in the sample amount taking examination according to the present invention; wherein S1 is a characteristic diagram of the sample solution with the sample taking amount of 0.5 g; s2 is a characteristic diagram of the sample solution with the sample dosage of 1.0 g; s3 is the sample solution characteristic map with the sample taking amount of 1.5 g.

FIG. 5 is a comparison of different extraction times in the present invention; wherein S1 is a characteristic spectrum of the sample solution extracted by ultrasound for 20 min; s2 is a characteristic spectrum of the sample solution extracted by ultrasonic for 30 min; s3 is a sample solution characteristic spectrum extracted by ultrasonic for 40 min.

FIG. 6 is a comparison of blank solvents for the specificity test of the present invention; wherein S1 is a blank solvent (methanol) characteristic map; s2 is a characteristic map of the reference solution; s3 is the characteristic map of the test solution.

FIG. 7 is a common peak superposition signature for the repeatability tests of the present invention; wherein S1 is a common peak superposition characteristic spectrum of the test solution under the repeatability 1; s2 is a common peak superposition characteristic spectrum of the test solution under the repeatability 2; s3 is a common peak superposition characteristic spectrum of the test solution under repeatability 3; s4 is a common peak superposition characteristic spectrum of the test solution under repeatability 4; s5 is a common peak superposition characteristic spectrum of the test solution under the repeatability 5; s6 is a common peak superposition characteristic spectrum of the test solution under the repeatability 6; s7 is a control map.

FIG. 8 is a precision test common peak overlap profile of the present invention; wherein, S1 is a common peak superposition characteristic spectrum of the test solution under the precision 1; s2 is a common peak superposition characteristic spectrum of the test solution under precision 2; s3 is a common peak superposition characteristic spectrum of the test solution under precision 3; s4 is a common peak superposition characteristic spectrum of the test solution under the precision of 4; s5 is a common peak superposition characteristic spectrum of the test solution under the precision of 5; s6 is a common peak superposition characteristic spectrum of the test solution under the precision of 6; s7 is a control map.

FIG. 9 is a common peak superposition signature for the stability test of the present invention; wherein S1 is a common peak superposition characteristic map of the test sample solution measured in 0 h; s2 is a common peak superposition characteristic map of the test sample solution measured in 2 h; s3 is a common peak superposition characteristic map of the test sample solution measured in 4 h; s4 is a common peak superposition characteristic map of the test sample solution measured in 8 h; s5 is a common peak superposition characteristic map of the test sample solution measured in 12 h; s6 is a common peak superposition characteristic map of the test sample solution measured in 24 h; s7 is a control map.

FIG. 10 is the protocatechuic acid reference substance map in the standard decoction feature map determination of the invention.

FIG. 11 is a standard decoction feature map of the present invention for determination of the control drug map of rhizoma Cibotii.

FIG. 12 is a superimposed graph of 15 batches of rhizoma Cibotii Chinese medicinal materials in standard decoction feature map determination of the invention; wherein S1-S15 represent the superposition spectrum of 1-15 batches of the Chinese medicinal herb of the scalded rhizoma cibotii.

FIG. 13 is a graph showing the overlay of the characteristic spectra of 15 batches of standard decoction, a reference substance and a reference medicinal material; wherein S1 represents the characteristic map of the reference substance, S2 represents the characteristic map of the reference medicinal material, and S3-S17 represent the superimposed maps of 1-15 batches of standard decoction of rhizoma Cibotii.

FIG. 14 is a graph of a standard decoction fit of 15 batches of scalded rhizoma Cibotii in the standard decoction profile determination of the present invention.

FIG. 15 is a linear plot of the different concentrations of protocatechuic acid control in the linear range test of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.

The invention provides a method for detecting the quality of a standard decoction of scalded rhizoma cibotii, which comprises the following detection method, wherein the standard content of the standard decoction is limited to 0.80-5.20mg of protocatechuic acid in each 1g of the standard decoction of the scalded rhizoma cibotii by the characteristics of the standard decoction, the paste yield of a dry extract, the identification of a thin layer, the determination of a extract, a characteristic spectrum and the determination of the protocatechuic acid content, wherein the determination of the paste yield of the dry extract is determined by adopting a decocting method; thin-layer identification is carried out by adopting thin-layer chromatography; measuring the extract by adopting a hot dipping method; the characteristic spectrum and the protocatechuic acid content are measured by liquid chromatography.

In this embodiment:

preparing a standard decoction of the scalded rhizoma cibotii: referring to a decocting method in the administration Standard of Chinese medicine decoction Chamber of medical institution (No. 2009) 3 of the State administration of traditional Chinese medicine), 15 batches of rhizoma Cibotii decoction pieces are taken, added with water until the rhizoma Cibotii decoction pieces submerge for about 4-5cm, soaked for 30-40min, decocted for two times, the first decocting time is 30-40min, the second decocting time is 25-30min, and subjected to solid-liquid separation while hot, the filtrates are combined, concentrated and dried to prepare 15 batches of rhizoma Cibotii decoction standard dry extract powder.

1. Trait survey

According to the physical characteristics of the 15 batches of the scalded rhizoma cibotii standard decoction, the granules are described as yellow brown to tan granules, and the granules have light smell, light taste and slight astringent.

2. Dry extract yield test

Taking the 15 batches of scalded rhizoma cibotii decoction pieces, preparing 15 batches of standard decoction dry extract powder according to the preparation method, calculating the dry extract yield (see table 1) by using the dry extract powder, calculating the average yield to be 17.813%, calculating the allowable range of the paste yield (the average value is 70-130%) according to the standard decoction paste yield, and setting the paste yield of the product to be 12.5-23.0%.

Table 1: rate of paste discharge

The results show that the yield of 15 batches of standard decoction dry extract is 15.8-20.2%, and the ranges of the standard decoction dry extract and the standard decoction dry extract meet the set limit range of 12.5-23.0%.

3. Thin layer authentication

The product is a dry extract of single-component decoction piece scalded rhizoma cibotii, a protocatechuic acid reference substance is used as a reference by referring to a method under the item of 'thin-layer identification' of scalded rhizoma cibotii in Chinese pharmacopoeia, a thin-layer identification method of the product is established, spots of a test sample are clear after 15 batches of sample tests, so the product is drawn as the thin-layer identification method of the product, the spots of the test sample are clear after 15 batches of sample tests, and a negative reference sample is free of interference, so the product is drawn as the identification item. The test methods and results are as follows:

the test method comprises the following steps: performing thin layer chromatography (China pharmacopoeia 2020 edition four-part general rule 0502)

Preparing a test solution: taking 2g of the product, adding 50ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1ml of methanol to dissolve residues to obtain a test solution.

Preparation of a reference solution: taking a proper amount of protocatechuic acid reference substance, adding methanol for dissolving, and preparing a reference substance solution with the concentration of 1 mg/mL.

Thin-layer chromatography conditions: thin-layer plate: silica gel G thin layer plate; sample amount of spotting: the sample solution and the reference solution are both 1 uL; developing agent: chloroform-ethyl acetate-methanol-formic acid (12: 2:1: 0.8); color developing agent: sprayed with 2% ferric trichloride solution-1% potassium ferricyanide solution (1: 1) (prepared in the clinical practice), and examined in the daylight.

As a result: spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution. In detail, see FIG. 1 for a TLC pattern of a standard decoction of 15 batches.

4. Measurement of extract

Taking 15 batches of standard decoction, taking ethanol as solvent, and performing hot-dipping assay under alcohol-soluble extract assay (2201 in 2020 th edition of Chinese pharmacopoeia), and the results are shown in Table 2.

Table 2: measurement results of extract

The results show that the mean value of 15 batches of standard decoction extract is 52.46%, the alcohol-soluble extract of the product is determined to be not less than 37.0% by referring to the lower limit of the allowable range of the standard limit (mean value is 70-130%), and the measurement results of 15 batches of standard decoction all meet the requirement of the determined limit.

5. Feature map testing

5.1 liquid chromatography

Chromatographic conditions are as follows: a chromatographic column: c18(250mmx4.6mm, 5um) (Waters XSelec HSS T3); mobile phase: gradient elution was performed as specified in table 3 using methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; flow rate: 1.0 mL/min; column temperature: 35 ℃; detection wavelength: 254 nm.

Table 3:

(1) preparation of reference solutions: taking 1.0g of rhizoma Cibotii reference material, adding 25mL of water, decocting for 1 hour, filtering, evaporating filtrate to dryness, adding 5mL of methanol into residue, filtering, and taking filtrate as reference solution;

(2) preparation of control solutions: taking a proper amount of protocatechuic acid reference substance, precisely weighing, and adding methanol for dissolving to obtain a reference substance solution with the concentration of 0.1 mg/mL;

(3) preparing a test solution: taking 1.0g of a scalded rhizoma cibotii standard decoction sample, precisely weighing, placing in a conical flask with a plug, adding 25mL of precisely weighed methanol, sealing the plug, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, and taking the filtrate as a test sample solution.

The determination method comprises the following steps: precisely sucking 10 μ L of reference solution, reference solution and sample solution respectively, injecting into liquid chromatograph, and measuring.

5.2 methodological considerations

Investigation of extraction method: the test solutions were prepared by different extraction methods, including ultrasonic 30min extraction, reflux 30min extraction, and measured according to the 5.1 test method described above. The results show that as shown in fig. 2 and table 4, the number of main peaks of 30min of ultrasonic treatment and 30min of reflux treatment is consistent, the number of main peaks is consistent, and the total peak area RSD of the main peaks is 0.29%, so that the sample extraction mode is selected to be safer and more convenient ultrasonic treatment for 30 min.

Table 4:

investigation of extraction solvent: the test solutions were prepared with different extraction solvents and tested according to the 5.1 test method. The details are shown in table 5, and the results show that, as shown in fig. 3, the number of main peaks is consistent, and when the extraction solvent is methanol, the total peak area of the main peaks is the largest, so that methanol is determined to be used as the extraction solvent.

Table 5:

sample taking amount investigation: taking different sample quantities respectively to prepare test solution, and measuring according to a 5.1 test method. As shown in FIG. 4, the sample amount is preferably 1.0g, so that the sample amount is determined to be 1.0g (see Table 6).

Table 6: investigation results of different sample taking quantities

Examination of extraction time: the test solutions were prepared at different ultrasonic extraction times and tested as described above for test 5.1. The results show that, as shown in fig. 5, the difference of different extraction times is not large, the RSD is 1.42%, and the total area of the main peak is slightly larger in 30 minutes of ultrasound (see table 7 for details), so that the extraction time of the test sample is determined to be 30 minutes of ultrasound.

Table 7: different extraction time contrast investigation results

In summary, the main parameters for determining the preparation method of the test solution are as follows: taking about 1.0g of scalded rhizoma Cibotii standard decoction sample, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of methanol, sealing the plug, weighing, performing ultrasonic treatment for 20min, cooling, weighing again, supplementing weight loss with methanol, shaking, and filtering.

5.3 feature Pattern analysis method verification

And (3) special investigation: the sample is taken and 10 mu L of methanol is used as a solvent, and the measurement is carried out according to 5.1 chromatographic conditions, and the test shows that the blank solvent has no interference as shown in figure 6.

And (3) repeatability test: about 1.0g of the same sample (batch number T210701) is taken, 6 parts are taken, and the measurement is carried out according to 5.1 chromatographic conditions, and the results show that 6 common peaks exist in the characteristic spectrum of 6 test samples, and the relative retention time RSD is less than 3%, which indicates that the method has good reproducibility (detailed pictures in figures 7, 8 and 9).

Table 8: relative peak area of characteristic spectrum of repeatability test

Peak number	S1	S2	S3	S4	S5	S6	RSD％
								S1	0.205	0.209	0.208	0.206	0.205	0.205	0.849
S2	0.125	0.126	0.127	0.126	0.125	0.125	0.650
								S3	3.447	3.470	3.491	3.414	3.427	3.412	0.928
S4(S)	1.000	1.000	1.000	1.000	1.000	1.000	0.000
								S5	0.928	0.928	0.928	0.928	0.928	0.928	0.000
S6	0.083	0.084	0.083	0.083	0.082	0.082	0.909

Table 9: relative retention time of characteristic spectrum of repeatability test

Peak number	S1	S2	S3	S4	S5	S6	RSD％
								S1	0.531	0.531	0.531	0.532	0.532	0.532	0.103
S2	0.708	0.708	0.708	0.710	0.710	0.710	0.155
								S3	0.777	0.777	0.778	0.778	0.778	0.778	0.066
S4(S)	1.000	1.000	1.000	1.000	1.000	1.000	0.000
								S5	1.106	1.106	1.107	1.107	1.107	1.107	0.047
S6	1.465	1.465	1.464	1.467	1.467	1.467	0.091

And (3) precision test: about 1.0g of the same batch of samples are taken, the samples are measured according to the 5.1 chromatographic condition, 6 needles are continuously injected for measurement, the peak shape and the peak number are basically consistent, a similarity evaluation system (2012 version) of a traditional Chinese medicine chromatographic fingerprint image is adopted to evaluate the similarity of the specified 6 common characteristic peaks, and the relative retention time RSD is less than 3 percent (shown in the detailed chart 8, the table 10 and the table 11), which indicates that the precision of the instrument is good.

Table 10: relative peak area of characteristic spectrum of precision test

Peak number	S1	S2	S3	S4	S5	S6	RSD％
								S1	0.186	0.186	0.186	0.187	0.186	0.186	0.219
S2	0.113	0.113	0.113	0.112	0.112	0.113	0.458
								S3	3.102	3.101	3.100	3.102	3.096	3.094	0.109
S4(S)	1.000	1.000	1.000	1.000	1.000	1.000	0.000
								S5	0.117	0.117	0.117	0.119	0.117	0.118	0.712
S6	0.077	0.077	0.077	0.077	0.077	0.077	0.000

Table 11: relative retention time of characteristic spectrum of precision test

Peak number	S1	S2	S3	S4	S5	S6	RSD％
								S1	0.532	0.532	0.532	0.532	0.532	0.531	0.077
S2	0.710	0.710	0.710	0.709	0.709	0.709	0.077
								S3	0.778	0.779	0.778	0.778	0.778	0.778	0.052
S4(S)	1.000	1.000	1.000	1.000	1.000	1.000	0.000
								S5	1.107	1.107	1.107	1.107	1.107	1.107	0.000
S6	1.467	1.467	1.467	1.467	1.467	1.467	0.000

And (3) stability test: about 1.0g of the same sample is taken, the sample is measured according to the 5.1 chromatographic condition, the sample injection measurement is carried out respectively at 0h, 2h, 4h, 8h, 12h and 24h, the peak shape and the peak number of the characteristic spectrum are basically stable, and the relative retention time RSD is less than 3 percent, which indicates that the sample solution is stable within 24 hours. (see Table 12, Table 13 and FIG. 9 for details), it is shown that the test solutions are stable over 24 hours.

Table 12: relative peak area of characteristic spectrum of stability test

Peak number	0	2h	4h	8h	12h	24h	RSD％
								S1	0.187	0.187	0.186	0.187	0.185	0.186	0.438
S2	0.112	0.113	0.114	0.113	0.113	0.112	0.667
								S3	3.091	3.105	3.130	3.122	3.093	3.098	0.517
S4(S)	1.000	1.000	1.000	1.000	1.000	1.000	0.000
								S5	0.103	0.103	0.104	0.102	0.103	0.102	0.732
S6	0.077	0.077	0.077	0.077	0.077	0.077	0.000

Table 13: stability test feature profile relative retention time

Peak number	0	2h	4h	8h	12h	24h	RSD％
								S1	0.532	0.531	0.531	0.531	0.532	0.531	0.097
S2	0.709	0.709	0.708	0.708	0.709	0.709	0.073
								S3	0.778	0.777	0.777	0.777	0.777	0.778	0.066
S4	1.000	1.000	1.000	1.000	1.000	1.000	0.000
								S5	1.107	1.106	1.106	1.107	1.106	1.107	0.050
S6	1.467	1.466	1.465	1.465	1.466	1.467	0.061

5.4 Standard decoction characteristic atlas characterization analysis

Determination of standard decoction characteristic map

According to a characteristic spectrum analysis method drawn up by 5.3, the characteristic spectrums of 15 batches of scalded rhizoma cibotii decoction pieces and 15 batches of medicinal materials used for preparing the standard decoction pieces are measured, and the result shows that 6 common peaks exist in the characteristic chromatograms of the standard decoction pieces and the traditional Chinese medicine decoction pieces used for preparing the standard decoction pieces and correspond to the retention time of 6 characteristic peaks in the reference chromatogram of the reference medicinal material, wherein the peak corresponding to the protocatechuic acid reference substance solution is peak 6, and the common peak characteristic spectrums are shown in detail in figures 10 to 14.

Evaluation of similarity of characteristic chromatogram

The similarity evaluation system (2012 edition) is adopted to evaluate the similarity of the 6 selected common characteristic peaks, and the result shows that the similarity of the characteristic chromatograms of the standard decoction of 15 batches of the scalded rhizoma cibotii decoction pieces is more than 0.9, which indicates that the quality of the standard decoction is relatively stable. The peak (4) corresponding to the protocatechuic acid reference peak was used as the S peak, and the relative retention times of the common peak and the S peak were calculated, and the relative retention times and ranges thereof are shown in table 14.

Table 14: 15 batches of standard decoction shared peak relative retention time

In conclusion, the method for determining the standard decoction characteristic spectrum established by the high performance liquid chromatography is adopted, and the established method is verified in precision, repeatability and stability according to the analysis method verification guiding principle (general rule 9101) of the four parts of the Chinese pharmacopoeia 2020 edition, and meets the requirements. 6 common characteristic peaks were labeled, with peak 6 being protocatechuic acid. Taking the peak corresponding to the protocatechuic acid reference substance as an S peak, calculating the relative retention time of other 5 characteristic peaks, and drawing up the average value of the relative retention time of the peaks of 15 batches of samples as specified values: 0.53 (peak 1), 0.71 (peak 2), 0.78 (peak 3), 1.11 (peak 5), 1.47 (peak 6), considering the error of test operation, instrument, reagent and other multifactorial factors, the relative retention time allowed range is defined as +/-10%.

6. Determination of content

6.1 test methods

According to the content detection item of the scalded cibotium barometz, protocatechuic acid is used as a content index component.

Test methods liquid chromatography as in the above-described characteristic spectrum test.

The chromatographic conditions are as follows: c18(250mmx4.6mm, 5um) (Waters XSelec HSS T3); mobile phase: gradient elution was performed as specified in table 15 using methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; flow rate: 1.0ml per minute; column temperature: 35 ℃; detection wavelength: 254 nm.

Table 15:

preparation of control solutions: taking appropriate amount of protocatechuic acid reference substance, precisely weighing, and adding methanol to obtain solution containing protocatechuic acid with concentration of 0.1mg/ml as reference substance solution.

Preparing a test solution: taking about 1.0g of a scalded rhizoma cibotii standard decoction sample, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, complementing the weight loss reduction amount with methanol, shaking uniformly, and filtering to obtain a test solution.

6.2 methodological investigation

Investigation of extraction method: the sample solution was prepared by reflux and ultrasonic extraction, and the assay was performed as described above for test 6.1. The results show that the difference between the reflux of the sample for 30min and the ultrasound for 30min is not great (power 250W, frequency 25KHZ), and the RSD is 0.80%. (see table 16 for details), the more convenient and safe sample extraction method is ultrasonic treatment for 30 minutes.

Table 16: comparison of protocatechuic acid content in different extraction methods

Examination of extraction time: the test solutions were prepared at different extraction times and tested as described above for test 6.1. The results show that the difference between the ultrasonic treatment of the sample for 20min and the ultrasonic treatment for 30min and 40min is small (power 250W, frequency 25KHZ), the ultrasonic treatment for 30min is slightly higher, and the RSD is 0.76% (see table 17 for details), so that the ultrasonic treatment for 30min is adopted as a more convenient and safer sample extraction method.

Table 17: comparison of protocatechuic acid content at different extraction times

Investigation of extraction solvent: the test solutions were prepared with different extraction solvents and tested according to the test method 6.1 above. The results showed that the highest protocatechuic acid content and RSD of 0.76% were obtained when the extraction solvent was methanol (see table 18 for details), so methanol was determined to be the extraction solvent.

Table 18: comparison of protocatechuic acid content in different extraction solvents

Sample amount investigation: the test solutions were prepared by taking different sample amounts, and the assay was performed according to the test method 6.1 described above. The results show that the sample size of the sample is 1.0g, which is the best, and the protocatechuic acid content is relatively high (see table 19 for details), so that the sample size of the sample is determined to be 1.0 g.

Table 19: comparison of protocatechuic acid content at different sample quantities

6.2 assay methodology validation

And (3) repeatability test: about 1.0g of the same batch of standard decoction samples is taken, 6 parts in total are measured according to the 6.1 test method, the average value of the protocatechuic acid content in the samples is calculated to be 3.72mg/g, the RSD value is 054 percent, and the test shows that the method has good reproducibility (see Table 20 for details).

Table 20:

and (3) precision test: the protocatechuic acid reference substance solution shown in 6.1 is continuously injected into a sample 6 needles, the peak area is measured according to the test method of 9.1, and the RSD value of the calculated protocatechuic acid peak area is 0.89%, which indicates that the precision of the instrument is good (see Table 21 for details).

Table 21:

and (3) stability test: about 1.0g of a batch of standard decoction samples are taken, sample injection is carried out for 0h, 3h, 6h, 9h, 12h and 24h respectively according to the test method of the 9.1, the peak areas are measured, the RSD value of the peak areas is calculated to be 0.33%, and tests show that the test solution is stable within 24 hours (see table 22 for details).

Table 22:

linear range test: taking protocatechuic acid reference substance solution (concentration is 0.105mg/ml), respectively adding 1ul, 5ul, 10ul, 15ul, 20ul and 25 ul. The measurement was carried out under the chromatographic conditions under item 6.1. Taking the area of the protocatechuic acid peak as a vertical coordinate and the sample amount as a horizontal coordinate, drawing a standard curve, and performing linear regression, wherein the regression equation is as follows: y 3E +06x +34842, R²When the amount of protocatechuic acid was 0.9998, it was found that protocatechuic acid had a good linear relationship with its peak area in the range of 0.2625mg to 6.5625mg (see table 23 and fig. 15).

Table 23: protocatechuic acid linear relation investigation result

Sample recovery rate test: precisely weighing about 0.5g of sample (protocatechuic acid content is 3.72mg/g), and 6 parts in total, adding 10ml of protocatechuic acid reference substance solution (0.105mg/ml) with known concentration into each 6 parts of sample, preparing test sample solution according to the method under item 6.1, and measuring according to chromatographic conditions under item 6.1, and calculating the average sample recovery rate of the protocatechuic acid is 94.08% and the RSD is 0.73% (see table 24).

Table 24: protocatechuic acid sample adding recovery rate test result

6.3 Standard decoction and Chinese medicinal material content determination

The rhizoma cibotii decoction pieces are processed into the rhizoma cibotii decoction pieces after being cleaned, the protocatechuic acid content of the rhizoma cibotii decoction pieces is not changed by washing and baking in the cleaning process and only by screening, so the characteristic chromatogram and the protocatechuic acid content of the rhizoma cibotii decoction pieces refer to the medicinal material data.

The contents of 15 batches of rhizoma Cibotii standard decoction and 15 batches of decoction pieces of Chinese medicinal materials used for preparation thereof, protocatechuic acid, were determined according to the proposed content analysis method, and the results are detailed in tables 25 and 26.

Table 25: measurement result of protocatechuic acid of 15 batches of rhizoma cibotii decoction pieces

Table 26: measurement result of protocatechuic acid in 15 batches of standard decoction of rhizoma Cibotii

Protocatechuic acid content transfer rate: according to the detection method determined by standard decoction methodology research, the protocatechuic acid content transfer rate is calculated for 15 batches of standard decoction and the measurement results of the traditional Chinese medicine decoction pieces used for preparation, the mass transfer condition is mastered, and a basis is provided for formulating the internal control standard of the materials and the allowable range of the characterization parameters. The rhizoma Cibotii decoction piece standard decoction is prepared by decocting rhizoma Cibotii decoction piece with water for 2 times, concentrating the filtrate, and freeze drying. The protocatechuic acid content transfer rate is detailed in Table 27.

Table 27: protocatechuic acid content transfer rate of standard decoction of 15 batches of scalded rhizoma Cibotii

According to the data, the rhizoma cibotii decoction pieces are decocted according to the scheme to prepare the rhizoma cibotii standard decoction, the protocatechuic acid average content of the rhizoma cibotii standard decoction is 1.92mg/g, the content value range is determined to be 1.10-4.00mg/g, SD: 1.12. calculated according to the average value of 70-130%, the allowable range of the protocatechuic acid content is 1.35-2.50mg/g, calculated according to the low limit of minus SD and the high limit of plus 3SD, and the allowable range of the protocatechuic acid content of the standard decoction is 0.80-5.28 mg/g.

According to the data, the rhizoma cibotii decoction pieces are decocted according to the scheme to prepare the rhizoma cibotii decoction piece standard decoction, the average protocatechuic acid transfer rate is 64.02%, the transfer rate measurement value range is 37.8-87.8%, SD: 19.43 according to the technical requirements for quality control and standard formulation of Chinese medicinal granules, the allowable range of the protocatechuic acid content transfer rate is 44.81-83.23 percent according to the average value of 70-130 percent. The allowable range of the protocatechuic acid content transfer rate is 25.16-102.88% calculated according to +/-2 SD. The results show that the protocatechuic acid content and the transfer rate thereof in the 15 batches of standard decoction are both in the allowable range, and can provide reference basis for the quality research of the rhizoma cibotii formula granules.

According to the method for detecting the quality of the standard decoction of the scalded rhizoma cibotii, the properties of the standard decoction of the scalded rhizoma cibotii, the dry extract yield, the thin layer identification, the extract, the characteristic spectrum and the protocatechuic acid content are researched, the quality of the standard decoction of the scalded rhizoma cibotii is evaluated through multi-aspect measurement, a solid foundation is laid for the quality stability of products, the feasible quality standard of the decoction of the scalded rhizoma cibotii can be established, the effective control of the quality of the standard decoction of the scalded rhizoma cibotii is realized, and in addition, the chromatographic conditions are adopted for liquid phase analysis, so that a chromatogram with better and clearer separation degree can be obtained. The rhizoma cibotii decoction pieces are prepared into the rhizoma cibotii decoction piece standard decoction by a decoction method, the average protocatechuic acid content is 1.92mg/g, the measured content range is 1.10-4.00mg/g, the SD (standard deviation) is 1.12, the allowable range of the protocatechuic acid content is 1.35-2.50mg/g according to the average value of 70-130%, the allowable range of the protocatechuic acid content of the standard decoction is 0.80-5.28 mg/g according to the low limit of minus SD and the high limit of plus 3 SD. The average protocatechuic acid transfer rate is 64.02%, the transfer rate range is 37.8-87.8%, the SD is 19.43, according to technical requirements for quality control and standard formulation of Chinese medicinal granules, the allowable range of the protocatechuic acid content transfer rate is calculated according to 70-130% of the transfer rate average value, and the allowable range of the protocatechuic acid content transfer rate is 44.81-83.23%. The allowable range of the protocatechuic acid content transfer rate is 25.16-102.88% calculated according to +/-2 SD. According to the data, the determination limit of the protocatechuic acid content of the standard decoction is drawn as follows: 0.80 mg/g-5.20 mg/g, and the allowable range of the content transfer rate is 25.0-100.0%. The results show that the protocatechuic acid content and the transfer rate thereof in the standard decoction of a plurality of batches are in an allowable range, so the invention can provide reference basis for the quality standard research of the rhizoma cibotii formula granules.

Those skilled in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to those examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.