CN115015418B

CN115015418B - Quality detection method of Japanese ardisia herb decoction

Info

Publication number: CN115015418B
Application number: CN202210615938.9A
Authority: CN
Inventors: 何述金; 周代俊; 熊龙富; 杨志辉; 何承东; 朱美成; 黄黎明; 田仁成
Original assignee: Changsha Xinlin Pharmaceutical Co ltd; HUNAN XINHUI PHARMACEUTICAL CO Ltd
Current assignee: Changsha Xinlin Pharmaceutical Co ltd; HUNAN XINHUI PHARMACEUTICAL CO Ltd
Priority date: 2022-06-01
Filing date: 2022-06-01
Publication date: 2023-12-15
Anticipated expiration: 2042-06-01
Also published as: CN115015418A

Abstract

The invention provides a quality detection method of Japanese ardisia herb decoction, which comprises the steps of determining the properties of Japanese ardisia herb decoction, the extract yield of dry extract, thin-layer identification, extract, characteristic spectrum and bergenin content, limiting the content of Japanese ardisia herb decoction to 32.0-66.0mg of bergenin in each 1g, wherein the extract yield of dry extract is determined by adopting a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and bergenin content are all determined by liquid chromatography. The quality detection method of the Japanese ardisia herb decoction provided by the invention evaluates the quality of the Japanese ardisia herb decoction through multi-aspect measurement, lays a solid foundation for stable quality of products, can establish feasible quality standards of the Japanese ardisia herb decoction, and realizes effective control of the quality of the Japanese ardisia herb standard decoction.

Description

Quality detection method of Japanese ardisia herb decoction

Technical Field

The invention relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a quality detection method of Japanese ardisia herb tea soup.

Background

The modern medicine needs to have three characteristics of stability, uniformity, safety and effectiveness, and the Chinese patent medicine is difficult to compare with western medicines in the aspects, so that the detection is more needed by adopting various means, and the reliability and the stability of the detection result are ensured. Herba Ardisiae Japonicae is whole plant of Ardisia japonica of Ardisiaceae, and is prepared by digging in summer and autumn when stems and leaves are exuberant, removing silt, drying, and has the functions of eliminating phlegm, relieving cough, promoting diuresis, and promoting blood circulation. Can be used for treating cough, bloody sputum, chronic bronchitis, jaundice due to damp-heat, and traumatic injury. At present, a systematic quality detection method is not formed on the Japanese ardisia herb tablet decoction, and the quality control requirement of the traditional Chinese medicine formula particles cannot be met because the Japanese ardisia herb decoction is not comprehensive enough to be detected by adopting the existing detection means. Therefore, it is necessary to establish a quality detection method of the Japanese ardisia herb decoction for controlling the quality of medicinal materials.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provide a quality detection method of the Japanese ardisia herb decoction, so as to better control the quality of the Japanese ardisia herb decoction, characterize the quality of medicines and improve the stability of the medicines.

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

the invention provides a quality detection method of Japanese ardisia herb soup, which comprises the following detection methods,

determining the properties of the Japanese ardisia herb decoction, the dry extract extraction rate, the thin-layer identification, the extract, the characteristic spectrum and the bergenin content, and limiting the standard of the Japanese ardisia herb decoction content to be 32.0-66.0mg of bergenin content per 1g, wherein the dry extract extraction rate is determined by adopting a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and bergenin content are all measured by liquid chromatography;

the determination of the characteristic spectrum by liquid chromatography comprises: performing liquid chromatograph analysis, namely taking a solution prepared from a Japanese ardisia herb reference medicinal material as a reference substance solution b, taking a solution prepared from a bergenin reference substance as a reference substance solution b, taking a solution prepared from a Japanese ardisia herb soup sample as a test substance solution b, respectively precisely sucking the reference substance solution b, the reference substance solution b and the test substance solution b, respectively injecting the reference substance solution b, the reference substance solution b and the test substance solution b into a liquid chromatograph, and measuring to obtain the Chinese ardisia herb decoction; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica column (4.6 mm. Times.250 mm,5 μm); mobile phase: using 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 of a table a;

Table a gradient elution procedure

Flow rate: 1.2mL/min; column temperature: 25 ℃; sample injection amount: 10. Mu.L; detection wavelength: 250nm.

In one embodiment, the decoction method comprises: soaking herba Ardisiae Japonicae tablet in water for 30-40min, decocting twice for 30-40min for the first time and 25-30min for the second time, separating solid from liquid, concentrating, and drying to obtain herba Ardisiae Japonicae decoction dry extract powder.

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

(1) Preparing a test sample solution a: taking 0.1g of Japanese ardisia herb decoction sample, adding 20mL of methanol, carrying out ultrasonic treatment for 30min, cooling, filtering, evaporating filtrate to dryness, and adding 1mL of methanol into residues to dissolve to obtain a sample solution a;

(2) Preparing a control medicinal material solution a: taking 1g of Japanese ardisia herb reference medicine, adding 20ml of methanol, carrying out ultrasonic treatment for 30min, cooling, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain reference medicine solution a;

(3) Thin layer chromatography analysis was performed: the thin layer chromatography conditions were as follows: silica gel G thin layer plate; sample application amount: 2uL of each of the test solution a and the control medicinal solution a; developing agent: the volume ratio is 5:4:2 in methylene chloride-ethyl acetate-methanol; color-developing agent: the volume ratio of the newly prepared solution is 1:1, and the solution is 1% ferric trichloride-1% potassium ferricyanide mixed solution, and the solution is inspected under sunlight.

In one embodiment, the hot dip method uses ethanol as a solvent and the range of the extract is determined by a hot dip method under the alcohol-soluble extract determination method.

In one embodiment, the determination of the characteristic spectrum by liquid chromatography further comprises the steps of:

(1) Preparation of reference solution b: taking 1.0g of Japanese ardisia herb reference medicine, adding 25mL of 25% methanol, carrying out ultrasonic treatment for 30min, cooling, filtering, and taking the subsequent filtrate as reference substance solution b;

(2) Preparing a reference substance solution b: taking a proper amount of bergenin reference substance, precisely weighing, adding methanol for dissolving, and preparing a reference substance solution b with the concentration of 200 ug/mL;

(3) Preparing a test sample solution b: taking 0.2g of Japanese ardisia herb soup sample, precisely weighing, placing into a conical flask with a plug, adding 25mL of precisely weighed 25% methanol, sealing, performing ultrasonic treatment for 30min, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution b.

In one embodiment, determining bergenin content using liquid chromatography comprises: performing liquid chromatograph analysis, taking a solution prepared from bergenin reference substance as a reference substance solution c, taking a solution prepared from a Japanese ardisia herb decoction sample as a test substance solution c, precisely sucking the reference substance solution c and the test substance solution c respectively, respectively injecting into the liquid chromatograph, and measuring to obtain the bergenin- -based liquid chromatography; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica column (4.6 mm. Times.250 mm,5 μm); mobile phase: eluting with methanol as mobile phase A and water as mobile phase B; flow rate: 1.2mL/min; column temperature: 25 ℃; sample injection amount: 10. Mu.L; detection wavelength: 275nm.

In one embodiment, the method for determining bergenin content by liquid chromatography further comprises the steps of:

(1) Preparing a reference substance solution: taking a proper amount of bergenin reference substance, precisely weighing, adding methanol to prepare a solution containing bergenin with the concentration of 200ug/ml, and taking the solution as reference substance solution c;

(2) Preparing a test solution: taking about 0.2g of a Japanese ardisia herb soup sample, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of methanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution c.

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

(1) The quality of the Japanese ardisia herb decoction is evaluated through research on the properties of the Japanese ardisia herb decoction, the dry extract extraction rate, thin-layer identification, extract, characteristic spectrum and bergenin content measurement and through multi-aspect measurement, a solid foundation is laid for the stable quality of products, a feasible quality standard of the Japanese ardisia herb decoction can be established, the effective control of the quality of the Japanese ardisia herb standard decoction is realized, and a chromatogram with better and clearer separation degree can be obtained by adopting the chromatographic condition of the application for liquid phase analysis.

(2) The Japanese ardisia herb tablet is decocted to prepare Japanese ardisia herb tablet decoction, and the bergenin content range of the Japanese ardisia herb standard decoction is proposed to be: 32.0-66.0 mg/g; the average bergenin transfer rate is 56.32%, and the bergenin content transfer rate range of the standard Japanese ardisia herb decoction is proposed to be: 35.3 to 77.4 percent, and the result shows that the bergenin content and the transfer rate of the bergenin in the decoction of a plurality of batches are all within the allowable range, so the invention can provide reference for the quality standard research of the Japanese ardisia herb formula particles.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIGS. 1 (a) and 1 (b) are thin layer diagrams of standard decoction of 27 batches of Japanese ardisia herb tablets in an embodiment of the invention; wherein, the group 1 of patterns is a negative control sample thin layer pattern, the group 2 and the group 17 are Japanese ardisia herb control medicinal material solution thin layer patterns, and the group 3 to 16 and the group 18 to 30 are Japanese ardisia herb tablets 27 batches of standard decoction thin layer patterns.

FIG. 2 is a graph showing the comparison of different detection wavelengths in the detection wavelength investigation of standard decoction of Japanese ardisia herb tablets according to thin layer chromatography; wherein, the detection wavelength of S1 is 270nm, the detection wavelength of S2 is 230nm, and the detection wavelength of S3 is 250nm.

FIG. 3 is a graph showing the comparison of different column temperatures in the examination of standard decoction of herba Thesii tablet by thin layer chromatography; wherein the column temperature of S1 is 30 ℃, the column temperature of S2 is 25 ℃, and the column temperature of S3 is 20 ℃.

FIG. 4 is a graph showing the comparison of different flow rates in the flow rate investigation of standard decoction of Japanese ardisia herb tablets according to thin layer chromatography; wherein the flow rate of S1 is 0.8mL/min, the flow rate of S2 is 1.0mL/min, and the flow rate of S3 is 1.2mL/min.

FIG. 5 is a graph showing the relative flow of standard decoction of herba Thesii tablet under thin layer chromatography; wherein, the mobile phase of S1 is 0.1% phosphoric acid, the mobile phase of S2 is 0.1% acetic acid, and the mobile phase of S3 is 0.1% formic acid.

FIG. 6 is a characteristic diagram of gradient 1, gradient 2 and gradient 3 in elution gradient investigation of standard decoction of herba Thesii tablet according to thin layer chromatography; wherein S1 is gradient 1, S2 is gradient 2, and S3 is gradient 3.

FIG. 7 is a comparison chart of different extraction methods in the investigation of the extraction method of the present invention; s1 is reflux extraction of a characteristic spectrum of a sample solution; s2 is ultrasonic extraction of a characteristic spectrum of the sample solution.

FIG. 8 is a graph showing the comparison of different extraction times in the extraction time investigation of the present invention; wherein S1 is a characteristic spectrum of a sample solution to be extracted for 20min by ultrasonic extraction; s2, ultrasonically extracting a characteristic spectrum of the sample solution for 30 minutes; s3, ultrasonic extraction is carried out for 40min on the characteristic spectrum of the sample solution.

FIG. 9 is a graph showing the comparison of different extraction solvents in the investigation of the extraction solvents of the present invention; wherein S1 is a characteristic spectrum of a sample solution prepared by extracting with 25% ethanol; s2 is a characteristic spectrum of a sample solution prepared by 25% methanol extraction; s3 is a characteristic spectrum of the sample solution prepared by water extraction.

FIG. 10 is a graph showing the comparison of different sample amounts in the investigation of the solvent dosage according to the present invention; wherein S1 is a characteristic spectrum of a sample solution with the solvent dosage of 15 ml; s2 is a characteristic map of a sample solution to be tested, the solvent consumption of which is 25 ml; s3 is a characteristic map of the solution of the test sample with the solvent dosage of 35 ml.

FIG. 11 is a graph of the characteristic of the mixed control solution in the identification of characteristic peaks of the characteristic graph of the present invention.

FIG. 12 is a sample solution characteristic map in characteristic map characteristic peak assignment according to the present invention.

FIG. 13 is a graph showing the comparison of gallic acid spectra in characteristic peak assignments of the characteristic spectrum of the present invention, wherein (a) is a gallic acid spectrum of a sample, and (b) is a gallic acid spectrum of a standard.

FIG. 14 is a graph showing the comparison of bergenin spectra in characteristic peak assignments of the characteristic spectrum of the present invention, wherein (a) is a bergenin spectrum of a sample, and (b) is a bergenin spectrum of a standard.

FIG. 15 is a comparison chart of quercetin spectra in characteristic spectrum characteristic peak assignment of the present invention, wherein (a) is a sample quercetin spectrum chart, and (b) is a standard quercetin spectrum chart.

FIG. 16 is a graph of blank solvent comparisons in a specificity study of the present invention; s1 is a reference substance solution characteristic map; s2 is a characteristic spectrum of the solution of the sample, and S3 is a characteristic spectrum of a blank solvent (25% methanol).

FIG. 17 is a graph of the common peak superposition characteristics for the repeatability test of the present invention; s (1) 5 is a test sample solution common peak superposition characteristic spectrum under the condition of repeatability 1; s (2) 5 is a test sample solution common peak superposition characteristic spectrum under repeatability 2; s (3) 5 is a test sample solution common peak superposition characteristic spectrum under repeatability 3; s (4) 5 is a test sample solution common peak superposition characteristic spectrum under the condition of repeatability 4; s (5) 5 is a test sample solution common peak superposition characteristic spectrum under repeatability 5; s (6) 5 is a test sample solution common peak superposition characteristic spectrum under repeatability 6.

FIG. 18 is a graph of the common peak superposition characteristics for the precision test of the present invention; s (1) 5 is a test sample solution common peak superposition characteristic spectrum under the precision of 1; s (2) 5 is a test sample solution common peak superposition characteristic spectrum under the precision of 2; s (3) 5 is a test sample solution common peak superposition characteristic spectrum under the condition of precision 3; s (4) 5 is a test sample solution common peak superposition characteristic spectrum under the condition of precision 4; s (5) 5 is a test sample solution common peak superposition characteristic spectrum under the condition of precision 5; s (6) 5 is a test sample solution common peak superposition characteristic spectrum under the condition of precision 6.

FIG. 19 is a graph of the common peak superposition characteristics for stability testing in accordance with the present invention; s (1) 5 is a test sample solution common peak superposition characteristic spectrum measured in 0 h; s (2) 5 is a test sample solution common peak superposition characteristic spectrum measured in 2 h; s (3) 5 is a test sample solution common peak superposition characteristic spectrum measured in 4 hours; s (4) 5 is a test sample solution common peak superposition characteristic spectrum measured in 8 hours; s (5) 5 is a test sample solution common peak superposition characteristic spectrum measured in 12 hours; s (6) 5 is a test sample solution common peak superposition characteristic spectrum measured at 24 hours.

FIG. 20 is a graph of common peak stacking features of column tests in different column surveys of the present invention; wherein S1 is the chromatographic column of the batch number PF-108, S2 is the chromatographic column of the batch number PF-51, and S3 is the chromatographic column of the medium-spectrum red.

FIG. 21 is a graph showing various column temperature studies in accordance with the present invention; wherein S1 is a chromatographic column with a column temperature of 23 ℃, S2 is a chromatographic column with a column temperature of 25 ℃, and S3 is a chromatographic column with a column temperature of 27 ℃.

FIG. 22 is a graph of common peak superposition characteristics for flow rate tests in different flow rate surveys of the present invention; wherein, the flow rate of S1 is 1.20min/mL, the flow rate of S2 is 1.15min/mL, and the flow rate of S3 is 1.25min/mL.

FIG. 23 is a map of bergenin control in a characteristic chromatogram determination of the present invention.

FIG. 24 is a graph of the superposition of bergenin, gallic acid, quercetin and test samples in a characteristic chromatogram assay of the present invention.

FIG. 25 is a superimposed spectrum of bergenin and a test sample in a characteristic chromatogram determination of the present invention.

FIG. 26 is a graph of a control herbal material of Japanese ardisia in a characteristic chromatogram assay of the invention.

FIG. 27 is a superimposed spectrum of 27 batches of Japanese ardisia herb decoction pieces in the determination of the characteristic chromatogram of the invention; wherein S1-S27 respectively represent superposition patterns of 1-27 batches of Japanese ardisia herb Chinese herbal pieces.

FIG. 28 shows a graph of common peaks of 27 batches of Japanese ardisia herb Chinese medicinal materials in characteristic chromatogram determination of the invention.

FIG. 29 is a superposition of standard decoction patterns of 27 batches of Japanese ardisia herb in the measurement of the characteristic chromatogram of the invention; wherein S1-S27 respectively represent 1-27 batches of standard decoction overlapping patterns of herba Ardisiae Japonicae.

FIG. 30 is a fit of 27 batches of standard decoction of Japanese ardisia herb in the determination of characteristic chromatogram of the invention.

FIG. 31 is a graph of bergenin spectra in the assay of the present invention.

FIG. 32 is a graph comparing blank solvents in a specific study of the assay methodology of the present invention; s1 is a reference substance solution characteristic map; s2 is a characteristic spectrum of the solution of the sample, and S3 is a characteristic spectrum of a blank solvent (methanol).

FIG. 33 is a graph showing the linearity of the concentration of bergenin control in a linear range assay of the present invention.

Detailed Description

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

The invention provides a quality detection method of Japanese ardisia herb decoction, comprising the following detection method, wherein the quality of Japanese ardisia herb decoction, the dry extract extraction rate, the thin-layer identification, the extract, the characteristic spectrum and the bergenin content are measured, the standard of the Japanese ardisia herb decoction content is limited to 32.0-66.0mg of bergenin in each 1g, and the dry extract extraction rate is measured by adopting a decoction method; the thin layer identification adopts thin layer chromatography for identification; the extract is measured by a hot dipping method; the characteristic spectrum and bergenin content are all determined by liquid chromatography.

In this embodiment:

preparing standard Japanese ardisia herb decoction: referring to the decoction method in the medical institution Chinese medicine decoction room management Specification (Chinese medicine administration 2009 No. 3), 15 batches of short-land tea tablets are taken, water is added until the short-land tea tablets are about 4-5cm higher than the medicinal materials, the short-land tea tablets are soaked for 30-40min and decocted for two times, the first decoction time is 30-40min, the second decoction time is 25-30min, solid-liquid separation is carried out when the short-land tea tablets are hot, the filtrates are combined, concentrated and dried, and 15 batches of short-land tea standard decoction dry paste powder are prepared.

1. Dry extract yield test

27 batches of Japanese ardisia herb decoction pieces are taken, 27 batches of standard decoction dry paste powder are prepared according to the preparation method, the paste yield of the dry extract is calculated (see table 1), and the average value, the SD value and the allowable range of the paste yield are calculated. According to the characteristics and the application of the standard decoction in the technical requirements of quality control and standard establishment of traditional Chinese medicine prescription granule of the national drug administration, the following paste yield is: extract yield and Standard Deviation (SD) were calculated from the dry extract powder. The average value plus or minus 3 times SD (or 70% -130% of the average value) is the allowable range of the paste yield. And calculating the average value of the paste rate, SD and the allowable range of the paste rate according to the paste rate data of 27 batches of standard decoction.

Table 1: paste yield

The results show that the allowable range of the paste yield of the standard decoction is 8.77-16.69% according to the SD calculation of adding or subtracting 3 times of the average value; calculated according to 70-130% of the average value, is 8.91-16.55%. By combining the two groups of calculation data, the allowable range of the plaster yield of the standard decoction is 9-16.5%.

2. Property investigation

According to the physical characteristics of 27 batches of standard decoction, the product is yellowish-brown to brown powder; light smell and slightly bitter taste.

3. Thin layer authentication

The product is a dry extract of single decoction piece Japanese ardisia herb, and is prepared by taking Japanese ardisia herb reference medicinal materials as a reference by referring to a method under the term of Japanese ardisia herb thin-layer identification in Chinese pharmacopoeia, and a thin-layer identification method is established, and 27 batches of sample tests show that spots of a test sample are clear and negative reference samples have no interference, so the method is assumed to be the product [ identification ]. The test methods and results are as follows:

3.1 test method according to thin layer chromatography (rule 0502 of four parts of Chinese pharmacopoeia 2020 edition)

Preparation of test solution: about 0.1g of the product is taken, 20ml of methanol is added, ultrasonic treatment is carried out for 30 minutes, the mixture is cooled, filtered, the filtrate is evaporated to dryness, and 1ml of methanol is added into residues to dissolve the residues to be used as a test sample solution.

Preparing a control medicinal material solution: adding methanol 20ml into herba Ardisiae Japonicae reference 1g, and making into medicinal solution.

Chromatographic conditions: the thin layer plate is a silica gel G thin layer plate; sample application amount: 2 μl of each of the test solution and the control solution; developing agent: dichloromethane-ethyl acetate-methanol (5:4:2); color-developing agent: spraying mixed solution of 1% ferric trichloride-1% potassium ferricyanide (1:1) (new formulation before use), and inspecting under sunlight.

The test was performed on 27 batches of standard decoction, and as shown in fig. 1 (a) and 1 (b), spots of the same color were developed and clear at positions corresponding to the control chromatogram in the sample chromatogram.

4. Determination of extract

The results of hot dipping method under the condition of alcohol-soluble extract assay (general rule 2201 of Chinese pharmacopoeia 2020 edition) by taking 27 batches of standard decoction and using ethanol as solvent are shown in Table 2 in detail.

Table 2: extract measurement results

The result shows that the average value of the 27 batches of standard decoction extract is 38.96 percent, and the lower limit of the allowable range (70-130 percent of average value) of the reference standard limit, namely, the 70 percent of average value is 27.27 percent; the lower limit of the allowable range of the reference standard limit (mean plus or minus 3 times SD), i.e. the mean minus 3SD, is 24.56%. By comparison, the average 70% result was both lower than the lowest detection value (31.25%) of the 27 batches of standard decoction, and was closer to the lowest detection value. Therefore, it is assumed that the alcohol-soluble extract of the product should not be less than 27.3%.

5. Feature profile testing

5.1 instruments, reagents and reagents

(1) Instrument: shimadzu high performance liquid chromatograph (LC-2030 puls, shimadzu corporation, japan); shimadzu Shim-pack GIST C18-AQ (4.6 mm. Times.250 mm,5 μm); thermostatic waterbath (HMTD-7000, yongguangming medical instruments Co., ltd., beijing); ultrasonic cleaners (KQ-300 DE, kunshan ultrasonic instruments Co., ltd.); one ten-thousandth balance (PX 224ZH, ohus instruments limited); one ten million balance (AWU 220D, japan shimadzu limited).

(2) Reagent: ethanol (Tianjin far chemical reagent Co., ltd.) and methanol (Tianjin Denko chemical reagent Co., ltd.) are chromatographically pure; the water was ultrapure water (homemade in the laboratory) and phosphoric acid (MIEuro chemical Co., ltd.).

(3) Standard substance: bergenin (lot number: 111532-201604, content: 94.1%, chinese food and drug inspection institute), gallic acid (lot number: 110831-201906, content: 91.5%, chinese food and drug inspection institute), quercetin (lot number: 111538-202007, content: 93.5%, chinese food and drug inspection institute), japanese ardisia herb reference medicine (lot number: 121213-201102, chinese food and drug inspection institute).

5.2 test methods

5.2.1 determination of chromatographic conditions

(1) Determination of the optimal absorption wavelength

The method is based on chromatographic conditions and a PAD detector, multi-wavelength scanning is carried out on the standard Japanese ardisia herb decoction sample, the scanning wavelength is 200-400nm, 3 detection wavelengths are selected for research, and the optimal absorption wavelength is determined.

Taking appropriate amount of herba Ardisiae Japonicae standard decoction fine powder (batch number: T210706), taking about 0.2g, placing into conical flask with plug, adding 25% methanol 25mL, sealing, ultrasonic treating (power 300W, frequency 40 kHz) for 30 min, cooling, shaking, filtering, and collecting filtrate.

Chromatographic conditions: octadecylsilane chemically bonded silica (4.6 mm. Times.250 mm,5 μm) (Shimadzu shimm-pack GIST C18-AQ (4.6 mm. Times.250 mm,5 μm)); gradient elution was performed as specified in table 3 with methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; the flow rate is 1.0ml per minute; the column temperature is 25 ℃; the detection wavelengths are 230 nm, 250nm and 270nm.

Table 3:

time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～40	2→15	98→85
40～75	15→50	85→50
			75～95	50→55	50→45
95～97	55→2	45→98
			97～107	2	98

The results show that the sample has stronger and more absorption between 200 nm and 300nm, and detailed figure 2 shows that when the detection wavelength is selected to be 250nm by comparing 3 detection wavelength chromatograms, the separation degree of each characteristic peak is better, the interference is smaller, and the base line is stable, so that the detection wavelength is selected to be 250 nm.

(2) Investigation of column temperature

The experiment is carried out by comparing column temperature of 20deg.C, 25deg.C and 30deg.C, and selecting proper column temperature.

Chromatographic conditions: octadecylsilane chemically bonded silica (4.6 mm. Times.250 mm,5 μm) (Shimadzu shimm-pack GIST C18-AQ (4.6 mm. Times.250 mm,5 μm)); gradient elution was performed as specified in table 4 with methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; the flow rate is 1.0ml per minute; the column temperature is 20, 25 and 30 ℃; the detection wavelength was 250nm.

Table 4:

the results show that by comparing chromatograms of 3 different column temperatures, as shown in fig. 3, the chromatographic peak information and peak shape differences of 3 mobile phases are not large, and when the temperature of 25 ℃ is selected as the column temperature, the separation degree of the selected characteristic peak is better, so that the temperature of 25 ℃ is selected as the detection column temperature.

(3) Investigation of flow Rate

The experiment selects 3 flow rates of 0.8ml/min, 1.0ml/min and 1.2ml/min for comparison, and selects a proper flow rate.

Chromatographic conditions: octadecylsilane chemically bonded silica (4.6 mm. Times.250 mm,5 μm) (Shimadzu shimm-pack GIST C18-AQ (4.6 mm. Times.250 mm,5 μm)); gradient elution was performed as specified in table 5 with methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; the flow rate is 0.8ml, 1.0ml and 1.2ml per minute; the column temperature is 25 ℃; the detection wavelength was 250nm.

Table 5:

The results show that by comparing chromatograms of 3 different flow rates, as shown in fig. 4, the chromatographic peak information and peak shape difference of 3 mobile phases are not large, and when 1.2ml/min is selected as the flow rate, the peak separation degree is better, so that 1.2ml/min is selected as the flow rate.

(4) Investigation of mobile phases

The experiment selects three mobile phases of 0.1% formic acid, 0.1% phosphoric acid and 0.1% phosphoric acid solution for comparison, and determines a proper mobile phase.

Chromatographic conditions: octadecylsilane chemically bonded silica (4.6 mm. Times.250 mm,5 μm) (Shimadzu shimm-pack GIST C18-AQ (4.6 mm. Times.250 mm,5 μm)); gradient elution was performed as specified in table 6 with methanol as mobile phase a and 0.1% formic acid, 0.1% phosphoric acid solution as mobile phase B; the flow rate is 1.2ml per minute; the column temperature is 25 ℃; the detection wavelength was 250nm.

Table 6:

The results show that by comparing the chromatograms of 3 different mobile phases, as shown in fig. 4, when 0.1% acetic acid solution is selected as the mobile phase, no obvious characteristic peak exists, and when 0.1% phosphoric acid solution is selected as the mobile phase, the peak information is complete, the separation effect is better than that of 0.1% formic acid solution, so that 0.1% phosphoric acid is selected.

(5) Gradient optimization

The experiment optimizes the elution gradient of the characteristic spectrum of the standard decoction of Japanese ardisia herb and determines the optimal gradient.

Chromatographic conditions: octadecylsilane chemically bonded silica (4.6 mm. Times.250 mm,5 μm) (Shimadzu shimm-pack GIST C18-AQ (4.6 mm. Times.250 mm,5 μm)); methanol is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specifications in tables 7, 8 and 9; the flow rate is 1.2ml per minute; the column temperature is 25 ℃; the detection wavelength was 250nm.

Table 7: gradient 1

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～20	5→15	95→85
20～85	15→55	85→45
			85～87	55→5	45→95
87～97	5	95

Table 8: gradient 2

Table 9: gradient 3

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～30	2→15	98→85
30～45	15→50	85→50
			45～75	50→55	50→45
75～77	55→2	45→98
			77～87	2	98

The result shows that the gradient 2 with more peak information, more uniform peak shape distribution and better separation degree of each peak is finally determined as the elution gradient of the standard decoction feature map of the Japanese ardisia herb by optimizing the elution gradient of the standard decoction feature map of the Japanese ardisia herb as shown in figure 6.

5.2.2 chromatographic conditions

Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica (4.6 mm. Times.250 mm,5 μm) (Shimadzu shimm-pack GIST C18-AQ (4.6 mm. Times.250 mm,5 μm)); gradient elution was performed as specified in table 10 with methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; the flow rate is 1.2ml per minute; the column temperature is 25 ℃; the detection wavelength is 250nm; the sample loading was 10. Mu.l.

Table 10:

5.3 examination of the pretreatment method of the sample solution

The method for pre-treating the sample of the characteristic spectrum of the standard decoction of Japanese ardisia herb (batch number: Y210701) is examined, and the influence of an extraction solvent, an extraction mode, an extraction time and an extraction solvent dosage (or a sampling amount) on the characteristic spectrum of the standard decoction of Japanese ardisia herb is mainly examined.

5.3.1 initial detection conditions

Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica (4.6 mm. Times.250 mm,5 μm) (Shimadzu shimm-pack GIST C18-AQ (4.6 mm. Times.250 mm,5 μm)); gradient elution was performed as specified in table 11 with methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; the flow rate is 1.2ml per minute; the column temperature is 25 ℃; the detection wavelength was 250nm.

Table 11:

Preparation of reference solution: taking 1.0g of Japanese ardisia herb reference medicine, adding 25ml of 25% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, and taking subsequent filtrate as reference medicine reference substance solution. And (3) taking a proper amount of bergenin reference substance, precisely weighing, and adding methanol to prepare solutions with 200 mug per 1ml respectively as reference substance solutions of the reference substance. The number of theoretical plates should be no less than 1500 calculated as bergenin peak.

Preparation of test solution: taking appropriate amount of herba Ardisiae Japonicae standard decoction fine powder, taking about 0.2g, placing into conical flask with plug, adding 25% methanol 25mL, sealing, performing ultrasonic treatment (power 100W, frequency 40 kHz) for 30 min, cooling, shaking, filtering, and collecting filtrate.

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

5.3.2 investigation of extraction method: the test solutions were prepared by different extraction methods, including ultrasonic extraction and reflux extraction, respectively, and were measured according to the test method 5.3.1 described above. As shown in FIG. 7, the number of main peaks is consistent, the peak shape difference of different extraction modes is not large, and the calculation result shows that the RSD value of the ratio of the total peak area to the sampling amount is 2.83% (see Table 12), which is less than 3.0%, so that the time is saved and the operation is convenient, so that the sample extraction mode is selected to be ultrasonic.

Table 12:

5.3.3 investigation of extraction time: sample solutions were prepared at different times for ultrasonic extraction, and were measured according to the test method 5.3.1 described above. The results showed that the number of main peaks was consistent and the peak shape was not much different at different extraction times (see FIG. 8), so that the ultrasonic extraction was excluded for 40 minutes. The calculation result shows that the RSD value of the ratio of the total peak area to the sampling amount is 0.38% (see Table 13), less than 3.0%, and no obvious difference exists, so that 30 minutes of extraction with larger value (total peak area/sampling amount) are selected as the extraction time.

Table 13:

5.3.4 investigation of extraction solvent: the test solutions were prepared with different extraction solvents, and were measured according to the test method 5.3.1. As shown in FIG. 9, the number of main peaks is consistent, and the peak shape difference at different extraction times is large, so that 25% ethanol is eliminated. The calculation showed that the RSD value of the ratio of the total peak area to the sampling amount was 0.31% (see table 14) and less than 3.0%, so that 25% methanol having a larger value (total peak area/sampling amount) was selected as the extraction solvent.

Table 14: investigation of results with different extraction solvents

5.3.5 solvent usage investigation: sample solutions were prepared by using different amounts of solvents (15 ml, 25ml, 35 ml), and the measurement was performed according to the 5.3.1 test method. As shown in FIG. 10, the number of main peaks is the same, and the peak shape difference is small. The calculation result showed that the RSD value of the ratio of the total peak area to the sampling amount was 1.58% (see table 15) and less than 3.0%, so that 25ml of the solvent having a larger value (total peak area/sampling amount) was selected as the extraction solvent amount.

Table 15: investigation results of different solvent usage

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In summary, the main parameters of the method for preparing the sample solution are determined as follows: taking proper amount of fine powder, taking about 0.2g, placing into a conical flask with a plug, adding 25mL of 25% methanol, sealing, performing ultrasonic treatment (power 300W, frequency 40 kHz) for 30 minutes, cooling, shaking, filtering, and collecting subsequent filtrate.

5.4 characteristic Spectrum characteristic Peak assignment

(1) Instrument, reagent and reagent

Instrument: shimadzu high performance liquid chromatograph (LC-2030 puls, shimadzu corporation, japan); shimadzu Shim-pack GIST C18-AQ (4.6 mm. Times.250 mm,5 μm); thermostatic waterbath (HMTD-7000, yongguangming medical instruments Co., ltd., beijing); ultrasonic cleaners (KQ-300 DE, kunshan ultrasonic instruments Co., ltd.); one ten-thousandth balance (PX 224ZH, ohus instruments limited); one ten million balance (AWU 220D, japan shimadzu limited).

Reagent: ethanol (Tianjin far chemical reagent Co., ltd.) and methanol (Tianjin Denko chemical reagent Co., ltd.) are chromatographically pure; the water was ultrapure water (homemade in the laboratory) and phosphoric acid (MIEuro chemical Co., ltd.).

(2) Standard substance: bergenin (lot number: 111532-202005, content: 94.4%, chinese food and drug inspection institute), gallic acid (lot number: 110831-201906, content: 91.5%, chinese food and drug inspection institute), quercetin (lot number: 111538-202007, content: 93.5%, chinese food and drug inspection institute), japanese ardisia herb reference medicinal material (lot number: 121213-201102, chinese food and drug inspection institute).

(3) Sample measurement

The detection is carried out according to the condition under the standard characteristic spectrum item of the quality standard decoction of the Japanese ardisia herb, the experimental result is shown in figures 11-15, the experimental result shows that the chromatographic peak with the consistent retention time with gallic acid, quercetin and bergenin can be found in the standard decoction characteristic spectrum of the Japanese ardisia herb, and the spectral comparison confirms that the peak 2 is gallic acid, the peak 3 is bergenin and the peak 4 is quercetin.

5.5 feature map analysis method verification

5.5.1 specificity investigation: the sample was measured under the above chromatographic conditions of 5.3.1 using 10ul of 25% methanol as a solvent. Experiments show that the blank solvent has no interference, and the method has good specificity as shown in fig. 16.

5.5.2 repeatability test: about 0.2g of Japanese ardisia herb standard decoction (batch number: Y210701) is taken, 6 parts are taken, the measurement is carried out according to the condition of 5.3.1 chromatograph, and the sample is injected for 6-needle measurement. The measurement results show that the characteristic patterns of the test samples have basically consistent peak shapes and peak numbers (see FIG. 17). The characteristic spectrum has 5 common peaks, bergenin is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The calculation showed that the relative peak area RSD value, relative retention time RSD value, were less than 3.0% and within the acceptable range (see tables 16, 17). Experiments show that the method has good reproducibility.

Table 16: relative retention time of characteristic patterns for repeatability test

Peak number	S1	S2	S3	S4	S5	S6	RSD(％)
								1	0.330	0.330	0.326	0.325	0.325	0.325	0.76
2	0.428	0.427	0.421	0.42	0.419	0.419	0.97
								3(S)	1	1	1	1	1	1	0.00
4	1.487	1.489	1.497	1.498	1.5	1.499	0.37
								5	1.604	1.607	1.616	1.617	1.62	1.619	0.41

Table 17: repetitive test of characteristic pattern relative peak area

Peak number	S1	S2	S3	S4	S5	S6	RSD(％)
								1	0.280	0.279	0.285	0.285	0.291	0.288	1.64
2	0.135	0.134	0.134	0.134	0.134	0.135	0.45
								3(S)	1.000	1.000	1.000	1.000	1.000	1.000	0.00
4	0.074	0.075	0.076	0.077	0.078	0.077	2.13
								5	0.376	0.374	0.361	0.362	0.365	0.362	1.83

5.5.3 precision test: about 0.2g of standard decoction of Japanese ardisia herb (batch number: Y210701) is taken, the measurement is carried out according to the condition of 5.3.1 chromatograph, 6-needle continuous sample injection measurement is carried out, and the measurement result shows that the peak shape and the peak number of the characteristic spectrum of the tested sample are basically consistent (see figure 18). The characteristic spectrum has 5 common peaks, bergenin is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The calculation showed that the relative peak area RSD value, relative retention time RSD value, were less than 3.0% and within the acceptable range (see tables 18, 19). Experiments show that the method has good precision.

Table 18: relative retention time of precision test characteristic spectrum

Peak number	S1	S2	S3	S4	S5	S6	RSD(％)
								1	0.326	0.324	0.325	0.326	0.328	0.329	0.57
2	0.421	0.419	0.419	0.421	0.426	0.427	0.83
								3(S)	1	1	1	1	1	1	0.00
4	1.497	1.499	1.498	1.493	1.489	1.487	0.33
								5	1.617	1.619	1.618	1.612	1.607	1.605	0.37

Table 19: relative peak area of characteristic spectrum for precision test

Peak number	S1	S2	S3	S4	S5	S6	RSD(％)
								1	0.272	0.274	0.275	0.276	0.278	0.279	0.94
2	0.131	0.133	0.133	0.133	0.131	0.134	0.96
								3(S)	1.000	1.000	1.000	1.000	1.000	1.000	0.00
4	0.078	0.076	0.076	0.076	0.074	0.074	1.95
								5	0.360	0.360	0.360	0.359	0.376	0.376	2.27

5.5.4 stability test: taking about 0.2g of Japanese ardisia herb standard decoction (batch number: Y210701), measuring according to 5.3.1 chromatographic conditions, and respectively carrying out sample injection measurement at 0h, 2h, 4h, 8h, 12h and 24h, wherein the measurement results show that the characteristic patterns of the test sample have basically consistent peak shapes and peak numbers (see figure 19). The characteristic spectrum has 5 common peaks, bergenin is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The calculation showed that the relative peak area RSD value, relative retention time RSD value, were less than 3.0% within the acceptable range (see tables 20, 21). The test shows that the test solution is stable within 24 hours.

Table 20: stability test characteristic pattern relative retention time

Peak number	0	2	4	8	12	24	RSD(％)
								1	0.326	0.325	0.325	0.325	0.327	0.324	0.29
2	0.421	0.42	0.419	0.42	0.423	0.417	0.43
								3(S)	1	1	1	1	1	1	0.00
4	1.495	1.497	1.498	1.495	1.493	1.499	0.14
								5	1.614	1.617	1.618	1.614	1.611	1.619	0.17

Table 21: stability test characteristic spectrum relative peak area

Peak number	0	2	4	8	12	24	RSD(％)
								1	0.297	0.297	0.298	0.299	0.300	0.303	0.70
2	0.148	0.148	0.137	0.146	0.148	0.149	2.82
								3(S)	1.000	1.000	1.000	1.000	1.000	1.000	0.00
4	0.077	0.075	0.075	0.076	0.079	0.074	2.22
								5	0.372	0.373	0.378	0.377	0.375	0.376	0.56

5.5.5 intermediate precision investigation

Other analysts in the same project operate on different dates and different devices, about 0.2g of the same batch of Japanese ardisia herb standard decoction (batch number: Y210701) is taken, precisely weighed, 6 parts are parallel, and test sample solutions are prepared and analyzed according to the method under the item of '5.3.1'. The bergenin is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, the RSD value is calculated, and the experimental results are shown in tables 22-25.

Table 22: intermediate precision relative retention time of Japanese ardisia herb standard decoction characteristic spectrum

Peak number	1	2	3	4	5	6	RSD(％)
								1	32.71	32.67	32.72	32.79	32.87	32.92	0.30
2	42.19	42.13	42.18	42.22	42.40	42.44	0.30
								3(S)	100.00	100.00	100.00	100.00	100.00	100.00	0.00
4	151.31	151.35	151.23	151.10	150.92	150.88	0.13
								5	163.52	163.56	163.46	163.31	163.12	163.07	0.13

Table 23: intermediate precision relative peak area of Japanese ardisia herb standard decoction characteristic spectrum

Peak number	1	2	3	4	5	6	RSD(％)
								1	28.74	28.78	28.90	28.94	29.03	29.10	0.48
2	13.93	13.93	13.95	13.98	14.06	14.10	0.51
								3(S)	100.00	100.00	100.00	100.00	100.00	100.00	0.00
4	8.35	8.45	8.48	8.46	8.40	8.30	0.84
								5	39.08	39.13	38.97	38.90	38.89	38.76	0.35

Table 24: characteristic spectrum repeatability relative retention time of Japanese ardisia herb standard decoction

Peak number	1	2	3	4	5	6	RSD(％)
								1	32.92	32.74	32.74	32.84	32.81	32.92	0.25
2	42.44	42.28	42.27	42.35	42.30	42.43	0.18
								3(S)	100.00	100.00	100.00	100.00	100.00	100.00	0.00
4	150.88	150.98	150.95	150.82	150.92	150.74	0.06
								5	163.07	163.17	163.16	163.00	163.11	162.92	0.06

Table 25: characteristic spectrum repeatability relative peak area of Japanese ardisia herb standard decoction

Peak number	1	2	3	4	5	6	RSD(％)
								1	29.10	29.35	29.73	29.54	29.66	29.33	0.80
2	14.10	14.06	14.17	14.22	14.22	14.28	0.58
								3(S)	100.00	100.00	100.00	100.00	100.00	100.00	0.00
4	8.30	8.32	8.48	8.40	8.47	7.87	2.73
								5	38.76	39.24	39.08	38.19	38.60	38.08	1.20

5.5.6 durability inspection

(1) Investigation of different chromatographic columns

A sample of about 0.2g of Japanese ardisia herb standard decoction (batch number: Y210701) was taken and assayed by the "5.3.1" test method, using 3 chromatographic columns (250 mm x4.6mm,5 μm) (Shimadzu GISTAQ-C18 (PF-108), shimadzu GIST C18 (PF-51), and Medium Spectrum Red (RD-C18), respectively, of different types. The measurement results show that the characteristic patterns of the test samples have inconsistent peak shapes and peak numbers (see figure 20). The characteristic spectrum has 5 common peaks, bergenin is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD% value is calculated. The calculation results showed that the relative retention time RSD% value, the relative peak area RSD value, were greater than 3.0% and greater than 5.0%, respectively, all within the reject ranges (see tables 26, 27). Experiments show that chromatographic columns of different models of different manufacturers have great influence on the characteristic spectrum measurement, namely, the characteristic spectrum measurement can only be carried out by using chromatographic columns of specified models.

Table 26: investigating characteristic spectrum relative retention time by different chromatographic columns

Peak number	PF-108	PF-51	Mid-spectrum red	RSD(％)
					1	0.326	0.289	0.270	9.65
2	0.420	0.359	0.383	7.93
					3(S)	1.000	1.000	1.000	0.00
4	1.496	1.618	1.528	4.09
					5	1.615	1.748	1.659	4.05

Table 27: different chromatographic columns are used for investigating the relative peak areas of characteristic patterns

Peak number	PF-108	PF-51	Mid-spectrum red	RSD(％)
					1	0.299	0.253	0.290	8.80
2	0.142	0.138	0.146	2.75
					3(S)	1.000	1.000	1.000	0.00
4	0.080	0.080	0.071	6.34
					5	0.371	0.366	0.379	1.75

(2) Investigation of different column temperatures

About 0.2g of standard decoction of Japanese ardisia herb (batch number: Y210701) was sampled and measured by the test method under item "5.3.1" at a column temperature of 23℃and 25℃and 27℃respectively. The measurement results show that the characteristic patterns of the test samples have basically consistent peak shapes and peak numbers (see FIG. 21). The characteristic spectrum has 5 common peaks, bergenin is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The calculation showed that the relative retention time RSD% value, the relative peak area RSD% value, were all less than 3.0% and within the acceptable range (see tables 28, 29). The test shows that the different column temperatures have less influence on the characteristic spectrum measurement, namely, the different column temperatures have better durability.

Table 28: different column temperatures examine the relative retention time of characteristic patterns

Peak number	23℃	25℃	27℃	RSD(％)
					1	0.334	0.323	0.318	2.52
2	0.426	0.417	0.414	1.49
					3(S)	1.000	1.000	1.000	0.00
4	1.489	1.500	1.503	0.49
					5	1.608	1.62	1.622	0.47

Table 29: different column temperatures are used for examining the relative peak areas of characteristic patterns

Peak number	23℃	25℃	27℃	RSD(％)
					1	0.282	0.277	0.277	1.18
2	0.135	0.130	0.132	1.75
					3(S)	1.000	1.000	1.000	0.00
4	0.071	0.072	0.073	1.61
					5	0.379	0.373	0.364	2.02

(3) Investigation of different flow rates

About 0.2g of the standard decoction of Japanese ardisia herb (batch number: Y210701) was sampled and measured according to the test method under item 5.3.1 at column temperatures of 1.15ml/min, 1.20ml/min and 1.25ml/min, respectively. The measurement results show that the characteristic patterns of the test samples have basically consistent peak shapes and peak numbers (see FIG. 22). The characteristic spectrum has 5 common peaks, bergenin is used as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, and the RSD value is calculated. The calculation showed that the relative retention time RSD% value, the relative peak area RSD% value, were all less than 3.0% and within the acceptable range (see tables 30, 31). The test shows that the fine flow velocity change has less influence on the characteristic spectrum measurement, namely, the durability of different flow velocities is better.

Table 30: investigation of characteristic patterns relative retention time at different flow rates

Peak number	1.15ml/min	1.20ml/min	1.25ml/min	RSD(％)
					1	0.335	0.328	0.320	2.29
2	0.431	0.425	0.415	1.91
					3(S)	1.000	1.000	1.000	0.00
4	1.485	1.490	1.501	0.55
					5	1.603	1.608	1.620	0.54

Table 31: investigation of characteristic spectrum relative peak area by different flow rates

Peak number	1.15ml/min	1.20ml/min	1.25ml/min	RSD(％)
					1	0.317	0.309	0.308	1.56
2	0.153	0.148	0.147	2.41
					3(S)	1.032	1.011	0.979	2.63
4	0.075	0.075	0.077	1.22
					5	0.374	0.376	0.373	0.36

The characteristic spectrum method meets the requirements through the verification of specificity, precision, repeatability, stability and intermediate precision and the investigation of durability, and the result shows that the established method can be well used for the characteristic spectrum measurement of the Japanese ardisia herb.

5.6 characterization analysis of Standard decoction feature atlas

5.6.1 characteristic chromatogram determination

According to the proposed characteristic spectrum analysis method, 27 batches of standard decoction of Japanese ardisia herb and 27 batches of characteristic spectrums of the traditional Chinese medicine decoction pieces used for preparation are measured, and the result shows that the standard decoction and the traditional Chinese medicine decoction pieces used for preparation have 5 common peaks in the characteristic chromatograms of the standard decoction pieces and the traditional Chinese medicine decoction pieces used for preparation, and the 5 common peaks correspond to the retention time of 5 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material, wherein the peak corresponding to the reference substance of the positioning standard bergenin is peak 3, the common peak characteristic spectrum, and the common peak characteristic spectrum is shown in figures 23-30 in detail.

5.6.2 evaluation of characteristic chromatograms relative retention time

According to the proposed characteristic spectrum analysis method, 27 batches of Japanese ardisia herb standard decoction characteristic spectrum are measured. The results show that there are 5 common peaks in the characteristic spectrum, the peak (3) corresponding to the reference peak of the positioning standard is taken as a reference peak S, and the relative retention time of the characteristic peaks peak 1, peak 2, peak 4, peak 5 and S peak is calculated, and the relative retention time and the range thereof are shown in tables 32 (1) and 32 (2).

Table 32 (1): peak relative retention time of S1-S15 standard decoction

Peak number	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10	S11	S12	S13	S14	S15
																1	0.320	0.319	0.320	0.320	0.320	0.320	0.320	0.319	0.319	0.319	0.319	0.319	0.319	0.319	0.319
2	0.401	0.400	0.400	0.401	0.400	0.400	0.400	0.400	0.400	0.400	0.400	0.401	0.400	0.400	0.400
																3(S)	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000	1.000
4	1.527	1.528	1.528	1.528	1.528	1.529	1.529	1.528	1.529	1.528	1.527	1.527	1.528	1.528	1.528
																5	1.648	1.648	1.649	1.649	1.649	1.649	1.649	1.648	1.649	1.648	1.648	1.647	1.648	1.649	1.648

Table 32 (2): peak relative retention time of S16-S27 standard decoction

In summary, the standard decoction feature spectrum measurement method established by adopting the high performance liquid chromatography is adopted, and the established method is subjected to precision, repeatability, stability verification and durability investigation according to the four-part analysis method verification guiding principle (general rule 9101) of the Chinese pharmacopoeia 2020 edition, and meets the requirements. According to the proposed characteristic spectrum analysis method, the characteristic spectrum of 27 batches of standard decoction is measured, and the result is analyzed, so that 5 common characteristic peaks are calibrated, wherein the peak 3 is bergenin. Calculating the relative retention time of another 4 characteristic peaks by taking a peak (3) corresponding to the bergenin reference as an S peak, and respectively setting the average value of the relative retention time of 27 batches of sample peaks as a specified value: 0.319 (Peak 1), 0.400 (Peak 2), 1.528 (Peak 4), 1.659 (Peak 5), and the relative retention time allowable range was assumed to be.+ -. 10% taking into consideration multi-factor errors of test operation, instrument, reagent, etc.

6. Content determination

Herba Ardisiae Japonicae is dried whole plant of Ardisia japonica (Thunb.) of Ardisia of Ardisiaceae. Produced in Hunan province, more counties such as Yiyang, anbei, taojiang, ping Jiang, etc.; other regions, such as regions in the south of the Yangtze river, are also available. The raw Dactylicapnos is mainly self-collected and carried in the Song from the trade market in a few areas. Ardisia japonica contains lachrymal I, II (Aristolol I, II), lachrymal essence (Aristin), bergenin (Bergenin), shizandra quinone (emmbelin), ardisia japonica element 1 (Bergenin ), etc. Modern pharmacological studies suggest that Ardisia Japonica extract No. 1 has antitussive effect. Therefore, the research refers to the method for measuring the content of the Japanese ardisia in the 2020 edition of Chinese pharmacopoeia, and the method for establishing the content of bergenin has important reference value for controlling the quality of Japanese ardisia standard decoction.

The bergenin reference substance (lot number: 111532-201604) is subjected to full-wavelength scanning to determine the optimal absorption wavelength, and the result shows that the detection is carried out at 275nm wavelength, the baseline is stable, the separation degree is good, no impurity peak interference exists, and therefore 275nm wavelength is selected as the detection wavelength for measuring the standard decoction content of Japanese ardisia herb.

6.1 test method

Chromatographic conditions: octadecylsilane chemically bonded silica (4.6 mm. Times.250 mm,5 μm) (Shimadzu shimm-pack GIST C18-AQ (4.6 mm. Times.250 mm,5 μm)); methanol-water (20:80) is used as a mobile phase; the flow rate is 1.2ml per minute; the column temperature is 25 ℃; the detection wavelength was 275nm. The number of theoretical plates should be no less than 1500 calculated as bergenin peak.

Preparation of reference solution: taking a proper amount of bergenin reference substance, precisely weighing, and adding methanol to prepare a solution containing 200 mug of bergenin per 1 ml.

Preparation of test solution: taking a proper amount of fine powder of the product, taking about 0.2g, precisely weighing, placing into a conical flask with a plug, precisely adding 50ml of methanol, sealing, weighing, performing ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking a subsequent filtrate.

6.2 examination of the pretreatment method of the sample solution

The extraction mode, the extraction solvent, the extraction time and the solvent amount of the content measurement of the standard decoction (batch number: Y210701) of Japanese ardisia herb are examined to determine the optimal sample pretreatment mode.

6.2.1 investigation of extraction method: sample solutions were prepared by different extraction methods, and were measured according to the test method 6.1 described above. The results showed that RSD% was less than 2% (see table 33), so the sample extraction mode was chosen to be sonication for convenience of subsequent experiments.

Table 33: comparison of different extraction methods

6.2.2 investigation of extraction time: sample solutions were prepared at different extraction times, and were measured according to the test method 6.1 described above. The results indicated that the RSD% was less than 2% and the sample was sonicated for 30 minutes with the highest content (see table 34), so the sample was selected for 30 minutes.

Table 34: comparison of different extraction times

Investigation of 6.2.3 extraction solvent: test solutions were prepared with different extraction solvents (ethanol, 80% methanol, methanol), and the test solutions were measured according to the test method 6.1 described above. The results indicated that the solvent was methanol, which was the highest (see Table 35), so the sample solvent was selected to be methanol.

Table 35: comparison of different extraction solvents

6.2.4 examination of the amount of solvent test solutions were prepared in different amounts of solvent (35 ml, 50ml, 65 ml), and were measured according to the test method 6.1 described above. The results indicated that the amount of solvent was 50ml, with the highest amount (see Table 36), so that the sample was selected to have a solvent amount of 50ml.

Table 36: comparison of different solvent amounts

In summary, the main parameters of the method for preparing the sample solution are determined as follows: taking a proper amount of fine powder of the product, taking about 0.2g, precisely weighing, placing into a conical flask with a plug, precisely adding 50ml of methanol, sealing, weighing, performing ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking a subsequent filtrate.

6.3 methodological validation of content determination

6.3.1 specificity investigation: the sample was taken and assayed with solvent methanol as described above in test 6.1, which shows that: the blank solvent was undisturbed (see figure 32) and the process was well specific.

6.3.2 repeatability test: about 0.2g of standard decoction samples in the same batch are taken, 6 parts of standard decoction samples are measured according to the test method 6.1, the average value of the bergenin content in the measured samples is 40.70mg/g, the RSD value is 0.252%, and the test shows that the method has good repeatability (see Table 37).

Table 37:

6.3.3 precision test: about 0.2g of the same batch of standard decoction sample is taken, 6 needles are continuously injected according to the test method 6.1, the peak area is measured, the RSD value of the bergenin peak area in the sample is calculated to be 0.096%, and the test shows that the instrument precision is good (see Table 38).

Table 38:

6.3.4 stability test: taking about 0.2g of a batch of standard decoction samples, injecting samples at 0h, 2h, 4h, 8h, 12h and 24h according to the test method 6.1, measuring peak areas, calculating RSD value of bergenin peak areas in the samples to be 0.573%, and testing to show that the test sample solution is stable within 24 hours (see Table 39).

Table 39:

6.3.5 linear range test: taking bergenin reference substance solution according to the concentration: 0.39757mg/ml, 0.198785mg/ml, 0.0993925mg/ml, 0.0496962mg/ml, 0.024545mg/ml, 0.004969mg/ml were determined according to the chromatographic conditions under 6.1 items.

The bergenin peak area is taken as an ordinate, the sample injection quality is taken as an abscissa, a standard curve is drawn, and the regression equation is as follows: y=13492129.9774x+9236.9974, r ² =1, bergenin was found to have a good linear relationship with its peak area in the range of 4.9 μg/ml to 397 μg/ml (see table 40 and fig. 33 for details).

Table 40: bergenin linear relation investigation result

Numbering device	Peak area	Concentration (mg/ml)
			Linearity 1	5377887	0.39757
Linearity 2	2675537	0.198785
			Linearity 3	1358495	0.0993925
Linearity 4	689020	0.0496962
			Linearity 5	343294	0.024545
Linearity 6	67019	0.004969

6.3.6 sample recovery test: samples 0.1g, 0.13g and 0.15g of the samples (bergenin content: 40.6963 mg/g) were precisely weighed three times each, 10.5ml, 7.0ml and 5.0ml of bergenin control solutions (378 ug/ml) of known concentrations were added, test solutions were prepared according to the method described in 6.1, and measured according to the chromatographic conditions described in 6.1, and the average bergenin recovery rate was calculated to be 97.50% and the RSD was calculated to be 2.85% (see table 41 for details).

Table 41: bergenin sample recovery rate test result

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6.3.7 durability inspection

(1) Investigation of different chromatographic columns

A batch of samples of about 0.2g was taken and assayed by the "6.1" test method using 3 columns (4.6 mm. Times.250 mm,5 μm) (Shimadzu GIST C18-AQ, zhong Red RD-C18, shimadzu GIST C18) of different models from different manufacturers, respectively. The calculation result shows that the RSD value of the measured content is 0.99% or less than 2.0% (see Table 42), and the test shows that the method has good durability in chromatographic columns of different models of different manufacturers.

Table 42:

(2) Investigation of different column temperatures

A batch of about 0.2g of the sample was taken and assayed by the test method under item "6.1" at a flow rate of 23℃at 25℃and at 27℃respectively. The calculation result shows that the RSD value of the measured content is 0.104% and less than 2.0% (see table 43), and the test shows that the method has good durability to small variation of column temperature.

Table 43:

(3) Investigation of different flow rates

A batch of about 0.2g of the sample was taken and assayed by the test method under item "6.1" at a flow rate of 1.0ml/min, 1.2ml/min, 1.4ml/min, respectively. The calculation shows that the RSD value of the measured content is 0.195% or less than 2.0% (see Table 44), and the test shows that the method has good durability against small variation of the flow rate.

Table 44:

(4) Investigation of different mobile phases

A batch of about 0.2g of the sample was taken and assayed by the test method under item "6.1" with methanol-water (21:79), (20:80), (19:81) respectively. The calculation result shows that the RSD value of the measured content is 0.855% and less than 3.0% (see table 45), and the test shows that the method has good durability for small fluctuation of the proportion of the flowing phase.

Table 45:

in conclusion, the whole analysis method meets the requirements through specialization, peak purity, precision, repeatability, stability, linear investigation, intermediate precision, sample recovery and durability investigation, and the established method can be well used for measuring the content of bergenin.

6.4 measurement of standard decoction, content of Chinese medicinal materials and transfer rate

The Japanese ardisia herb is primarily processed into slices through a production place, and is processed into Japanese ardisia herb decoction pieces, wherein the bergenin content of the Japanese ardisia herb decoction pieces cannot change, so that the characteristic chromatogram of the Japanese ardisia herb decoction pieces and the bergenin content refer to the medicinal material data.

6.4.1 according to the above-mentioned formulated content analysis method, 27 batches of Japanese ardisia herb standard decoction and 27 batches of Japanese ardisia herb bergenin content used for preparation thereof were measured, and the results are shown in tables 46, 47 and 48.

Table 46:27 batches of Japanese ardisia herb traditional Chinese medicine bergenin determination results

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The bergenin content in the Japanese ardisia herb decoction pieces is 9.93mg/g on average, the measured content range is 8.05 mg/g-12.64 mg/g, and the SD is 1.27; the allowable range of bergenin content is 6.95 mg/g-12.91 mg/g calculated according to 70% -130% of the average value. The allowable content range is 6.12 mg/g-13.74 mg/g calculated according to the average value-SD-average value +3SD.

Table 47: measurement result of bergenin in 27 batches of Japanese ardisia standard decoction

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The average content of bergenin in the standard decoction of the product is 44.11mg/g, the measured content range is 32.34 mg/g-65.49 mg/g, and the SD is 7.93; the allowable range of bergenin content is 30.88 mg/g-57.34 mg/g calculated according to 70% -130% of the average value. The allowable range of bergenin content is 20.32 mg/g-67.90 mg/g calculated according to the average value of-3 SD-average value of +3 SD. The highest value of the 27 batches of standard decoction is 65.49mg/g, which is not in the range (30.88 mg/g-57.34 mg/g) calculated according to 70% -130% of the average value; the minimum value of the 27 batches of standard decoction is 32.34mg/g, which is very different from the bottom limit (20.32 mg/g) of the range calculated according to the average value of-3 SD to the average value of +3SD. Therefore, according to the content range measured by 27 batches of standard decoction, the content range of bergenin of the standard decoction is more reasonable. Therefore, the bergenin content range of the standard decoction is assumed to be: 32.0 mg/g-66.0 mg/g.

6.4.1 bergenin content transfer rate: according to the detection method determined by standard decoction methodology research, 27 batches of standard decoction and traditional Chinese medicine decoction pieces for preparation are subjected to measurement, the bergenin content transfer rate is calculated, the mass transfer condition is mastered, and a basis is provided for formulating material internal control standards and the allowable range of characterization parameters thereof. The standard decoction of herba Ardisiae Japonicae tablet is prepared by decocting herba Ardisiae Japonicae tablet with water for 2 times, concentrating the filtrate, and freeze drying. The bergenin content transfer rate is shown in Table 48.

Table 48: transfer rate of bergenin content of 27 batches of Japanese ardisia herb standard decoction

From the data, the standard decoction of the short tea tablets is prepared by decocting the short tea tablets according to the scheme, the average transfer rate of bergenin of the product is 56.32%, the measured transfer rate range is 36.70% -68.63%, and the SD is 7.01. According to the technical requirements of quality control and standard formulation of traditional Chinese medicine formula particles, the allowable range of bergenin content transfer rate is 39.42-73.22% calculated according to 70-130% of the average value of the transfer rate; the average value is 35.29 to 77.35 percent calculated according to the average value of-3 SD to the average value of +3 SD. The transfer rate ranges of 27 batches of standard decoction are all in the range of average value-3 SD to average value +3SD, so the transfer rate range of bergenin content of the standard decoction is assumed to be: 35.3 to 77.4 percent.

According to the quality detection method of the Japanese ardisia herb decoction, the quality of the Japanese ardisia herb decoction is assessed through research on the properties of the Japanese ardisia herb decoction, the dry extract extraction rate, thin-layer identification, extract, characteristic spectrum and bergenin content measurement and through multi-aspect measurement, a solid foundation is laid for the stable quality of a product, a feasible quality standard of the Japanese ardisia herb decoction can be established, effective control of the quality of the Japanese ardisia herb standard decoction is realized, and a chromatogram with better and clearer separation degree can be obtained by adopting the chromatographic condition of the application for liquid phase analysis; the Japanese ardisia herb tablet is decocted to prepare Japanese ardisia herb tablet decoction, and the bergenin content range of the Japanese ardisia herb standard decoction is proposed to be: 32.0-66.0 mg/g; the average bergenin transfer rate is 56.32%, and the bergenin content transfer rate range of the standard Japanese ardisia herb decoction is proposed to be: 35.3 to 77.4 percent, and the result shows that the bergenin content and the transfer rate of the bergenin in the decoction of a plurality of batches are all within the allowable range, so the application can provide reference for the quality standard research of the Japanese ardisia herb formula particles.

Those skilled in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the application (including the claims) is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined within the idea of the application, the steps may be implemented in any order and there are many other variations of the different aspects of the application 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 which fall within the broad scope of the appended claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. of the present invention should be included in the scope of the present invention.

Claims

1. A quality detection method of Japanese ardisia herb soup is characterized by comprising the following detection methods,

the determination of the characteristic spectrum by liquid chromatography comprises: performing liquid chromatograph analysis, namely taking a solution prepared from a Japanese ardisia herb reference medicinal material as a reference substance solution b, taking a solution prepared from a bergenin reference substance as a reference substance solution b, taking a solution prepared from a Japanese ardisia herb soup sample as a test substance solution b, respectively precisely sucking the reference substance solution b, the reference substance solution b and the test substance solution b, respectively injecting the reference substance solution b, the reference substance solution b and the test substance solution b into a liquid chromatograph, and measuring to obtain the Chinese ardisia herb decoction; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica gel chromatographic column with length of 4.6mm, inner diameter of 250mm and particle diameter of 5 μm; mobile phase: using 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 of a table a;

Table a gradient elution procedure

Flow rate: 1.2mL/min; column temperature: 25 ℃; sample injection amount: 10. Mu.L; detection wavelength: 250nm;

the thin layer chromatography comprises the following steps:

s11: preparing a test sample solution a: taking 0.1g of Japanese ardisia herb decoction sample, adding 20mL of methanol, carrying out ultrasonic treatment for 30min, cooling, filtering, evaporating filtrate to dryness, and adding 1mL of methanol into residues to dissolve to obtain a sample solution a;

s12: preparing a control medicinal material solution a: taking 1g of Japanese ardisia herb reference medicine, adding 20ml of methanol, carrying out ultrasonic treatment for 30min, cooling, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain reference medicine solution a;

s13: thin layer chromatography analysis was performed: the thin layer chromatography conditions were as follows: silica gel G thin layer plate; sample application amount: 2uL of each of the test solution a and the control medicinal solution a; developing agent: the volume ratio is 5:4:2 dichloromethane, ethyl acetate, methanol solution; color-developing agent: the mixed solution of 1% ferric trichloride and 1% potassium ferricyanide which are newly prepared in the volume ratio of 1:1 before use is inspected in sunlight;

the characteristic spectrum determination by liquid chromatography further comprises the following steps:

s21: preparation of reference solution b: taking 1.0g of Japanese ardisia herb reference medicine, adding 25mL of 25% methanol, carrying out ultrasonic treatment for 30min, cooling, filtering, and taking the subsequent filtrate as reference substance solution b;

S22: preparing a reference substance solution b: taking a proper amount of bergenin reference substance, precisely weighing, adding methanol for dissolving, and preparing a reference substance solution b with the concentration of 200 ug/mL;

s23: preparing a test sample solution b: taking 0.2g of Japanese ardisia herb soup sample, precisely weighing, placing into a conical flask with a plug, adding 25mL of precisely weighed 25% methanol, sealing, performing ultrasonic treatment for 30min, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution b.

2. The method for detecting the quality of the Japanese ardisia herb decoction according to claim 1, wherein the decoction method comprises: soaking herba Ardisiae Japonicae tablet in water for 30-40min, decocting twice for 30-40min for the first time and 25-30min for the second time, separating solid from liquid, concentrating, and drying to obtain herba Ardisiae Japonicae decoction dry extract powder.

3. The quality control method of Japanese ardisia herb decoction according to claim 1, wherein the hot dipping method uses ethanol as a solvent and the range of extract is measured by the hot dipping method under the alcohol-soluble extract measurement method.

4. The method for detecting the quality of the Japanese ardisia herb decoction according to claim 1, wherein the measurement of the bergenin content by liquid chromatography comprises: performing liquid chromatograph analysis, taking a solution prepared from bergenin reference substance as a reference substance solution c, taking a solution prepared from a Japanese ardisia herb decoction sample as a test substance solution c, precisely sucking the reference substance solution c and the test substance solution c respectively, respectively injecting into the liquid chromatograph, and measuring to obtain the bergenin- -based liquid chromatography; wherein the chromatographic conditions adopted are that: octadecylsilane chemically bonded silica gel chromatographic column with length of 4.6mm, inner diameter of 250mm and particle diameter of 5 μm; mobile phase: eluting with methanol as mobile phase A and water as mobile phase B; flow rate: 1.2mL/min; column temperature: 25 ℃; sample injection amount: 10. Mu.L; detection wavelength: 275nm.

5. The method for detecting the quality of the Japanese ardisia herb soup as claimed in claim 4, wherein the measuring of the bergenin content by liquid chromatography further comprises the steps of:

s31: preparing a reference substance solution: taking a proper amount of bergenin reference substance, precisely weighing, adding methanol to prepare a solution containing bergenin with the concentration of 200ug/ml, and taking the solution as reference substance solution c;

s32: preparing a test solution: taking about 0.2g of a Japanese ardisia herb soup sample, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of methanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution c.