CN115015418A

CN115015418A - Quality detection method for Japanese ardisia herb decoction

Info

Publication number: CN115015418A
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: 2022-09-06
Anticipated expiration: 2042-06-01
Also published as: CN115015418B

Abstract

The invention provides a quality detection method of Japanese ardisia herb decoction, which comprises the steps of limiting the content of the Japanese ardisia herb decoction to 32.0-66.0mg of bergenin in each 1g of Japanese ardisia herb decoction by properties of the Japanese ardisia herb decoction, dry extract yield, thin layer identification, extract, characteristic spectrum and bergenin 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; the characteristic spectrum and the bergenin content are determined by liquid chromatography. According to the method for detecting the quality of the ardisia japonica decoction, the quality of the ardisia japonica decoction is evaluated through multi-aspect measurement, a solid foundation is laid for the stable quality of products, a feasible quality standard of the ardisia japonica decoction can be established, and the effective control of the quality of the ardisia japonica standard decoction is realized.

Description

Quality detection method for 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 method for detecting the quality of Japanese ardisia herb decoction.

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. Herba Ardisiae Japonicae is whole plant of Ardisia japonica of Ardisia of family Ardisia, collected in summer and autumn when stem and leaf are flourishing, removed of silt, and dried. Can be used for treating cough, bloody sputum, chronic bronchitis, jaundice due to damp-heat pathogen, and traumatic injury. At present, a systematic quality detection method for herba ardisiae japonicae decoction pieces is not formed, and the detection of the herba ardisiae japonicae decoction pieces by only adopting the existing detection means is not comprehensive enough and cannot meet the quality control requirement of traditional Chinese medicine formula granules. Therefore, it is necessary to establish a quality detection method of herba Ardisiae Japonicae decoction for quality control of medicinal materials.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a method for detecting the quality of ardisia japonica decoction so as to better control the quality of ardisia japonica decoction, characterize the medicine quality and improve the medicine stability.

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

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

the method comprises the steps of limiting the standard of the herba Ardisiae Japonicae decoction content to 32.0-66.0mg of bergenin per 1g by properties of the herba Ardisiae Japonicae decoction, dry extract yield, TLC identification, extract, characteristic map and bergenin content measurement, wherein the dry extract yield measurement is measured 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; measuring the characteristic spectrum and the bergenin content by liquid chromatography;

the characteristic spectrum determination by liquid chromatography comprises the following steps: performing liquid chromatograph analysis, taking the solution prepared from herba Ardisiae Japonicae reference medicinal material as reference solution b, taking the solution prepared from bergenin reference as reference solution b, taking the solution prepared from herba Ardisiae Japonicae decoction sample as test solution b, precisely sucking reference solution b, reference solution b and test solution b, respectively injecting into liquid chromatograph, and measuring; wherein, the adopted chromatographic conditions are that a chromatographic column: octadecylsilane chemically bonded silica chromatography column (4.6 mm. times.250 mm, 5 μm); mobile phase: methanol is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification of the table a;

TABLE a gradient elution procedure

Flow rate: 1.2 mL/min; column temperature: 25 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: 250 nm.

In one embodiment, the cooking method comprises: soaking herba Ardisiae Japonicae 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, concentrating, and drying to obtain herba Ardisiae Japonicae decoction dry extract powder.

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

(1) preparing a test solution a: taking 0.1g of herba Ardisiae Japonicae decoction sample, adding 20mL of methanol, performing ultrasonic treatment for 30min, cooling, filtering, evaporating filtrate, dissolving residue with 1mL of methanol to obtain sample solution a;

(2) preparing a reference medicinal material solution a: taking herba Ardisiae Japonicae reference medicinal material 1g, adding methanol 20ml, ultrasonic treating for 30min, cooling, filtering, evaporating filtrate to dryness, dissolving residue with methanol 1ml to obtain reference medicinal material solution a;

(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: 2uL of each of the test solution a and the reference medicinal material solution a; developing agent: the volume ratio is 5:4:2 dichloromethane-ethyl acetate-methanol solution; color developing agent: a new mixed solution of 1 percent ferric trichloride and 1 percent potassium ferricyanide with the volume ratio of 1:1 is prepared before use and is inspected 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 Japanese ardisia herb as a reference medicinal material, adding 25mL of 25% methanol, carrying out ultrasonic treatment for 30min, cooling, filtering, and taking a subsequent filtrate as a reference solution b;

(2) preparation of control solution b: precisely weighing appropriate amount of bergenin reference substance, and dissolving in methanol to obtain reference substance solution b with concentration of 200 ug/mL;

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

In one embodiment, the determination of the bergenin content by liquid chromatography comprises: performing liquid chromatograph analysis, taking the solution prepared from bergenin reference as reference solution c, taking the solution prepared from herba Ardisiae Japonicae decoction sample as test solution c, precisely sucking the reference solution c and the test solution c, respectively injecting into liquid chromatograph, and measuring; wherein, the adopted chromatographic conditions are that a chromatographic column: octadecylsilane chemically bonded silica chromatography column (4.6 mm. times.250 mm, 5 μm); mobile phase: taking methanol as a mobile phase A and water as a mobile phase B for elution; flow rate: 1.2 mL/min; column temperature: 25 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: 275 nm.

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

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

(2) preparing a test solution: taking about 0.2g of Japanese ardisia herb decoction sample, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of methanol, sealing the plug, weighing, ultrasonically treating for 30min, cooling, weighing again, complementing the weight loss by methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a test solution c.

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

(1) the quality of the ardisia japonica decoction is evaluated through multi-aspect measurement by researching the properties of the ardisia japonica decoction, the dry extract yield, the thin-layer identification, the extract, the characteristic map and the bergenin content measurement, a solid foundation is laid for the quality stability of products, a feasible quality standard of the ardisia japonica decoction can be established, the effective control of the quality of the ardisia japonica standard decoction is realized, and a chromatogram with better and clearer separation degree can be obtained by adopting the chromatographic condition to carry out liquid phase analysis.

(2) The Japanese ardisia herb decoction pieces are decocted to prepare the Japanese ardisia herb decoction piece, the bergenin content range of the standard Japanese ardisia herb decoction piece is: 32.0-66.0 mg/g; the average transfer rate of bergenin is 56.32%, and the transfer rate range of bergenin content of standard decoction of Japanese ardisia is: 35.3-77.4%, and the result shows that the content of bergenin and the transfer rate thereof in the decoction of a plurality of batches are within the allowable range, so the invention can provide reference basis 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 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.

FIGS. 1(a) and 1(b) are TLC charts of 27 batches of standard decoction of Japanese ardisia herb in one embodiment of the present invention; wherein, 1 group of atlas is a negative control sample thin-layer atlas, 2 groups and 17 groups of atlas are ardisia japonica control medicinal material solution thin-layer atlases, and 3-16 groups and 18-30 groups of atlas are ardisia japonica decoction piece 27 batch standard decoction thin-layer atlases.

FIG. 2 is a comparison graph of different detection wavelengths in the detection wavelength investigation of herba Ardisiae Japonicae decoction piece standard decoction by 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 250 nm.

FIG. 3 is a comparison graph of different column temperatures in column temperature investigation of herba Ardisiae Japonicae decoction piece standard decoction 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 comparison of different flow rates in thin layer chromatography of herba Ardisiae Japonicae decoction piece standard decoction; 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 1.2 mL/min.

FIG. 5 is a graph showing the relative ratio of different flows in the mobile phase examination of the Japanese ardisia herb decoction pieces by TLC; 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 the thin layer chromatography elution gradient examination of the standard decoction pieces of Japanese ardisia herb; wherein S1 is gradient 1, S2 is gradient 2, and S3 is gradient 3.

FIG. 7 is a comparison of different extraction methods in the present invention; wherein, S1 is a characteristic spectrum of the sample solution extracted by reflux; s2 is the ultrasonic extraction sample solution characteristic map.

FIG. 8 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. 9 is a comparison of different extraction solvents in the present invention; wherein S1 is a characteristic spectrum of a test solution prepared by 25% ethanol extraction; s2 is a characteristic spectrum of a test solution prepared by extracting 25% methanol; s3 is a characteristic map of the test solution prepared by water extraction.

FIG. 10 is a graph comparing different sample sizes for solvent dosage studies in accordance with the present invention; wherein S1 is a characteristic diagram of the test solution with 15ml of solvent; s2 is a characteristic map of a sample solution with 25ml of solvent; s3 is the test solution characteristic map of 35ml solvent dosage.

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

FIG. 12 is a characteristic diagram of the sample solution in the characteristic diagram peak identification of the present invention.

FIG. 13 is a comparison chart of the spectra of gallic acid in the characteristic peak identification of the present invention, wherein (a) is a spectrogram of gallic acid as a test product, and (b) is a spectrogram of gallic acid as a standard product.

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

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

FIG. 16 is a comparison of blank solvents for the specificity test of the present invention; wherein S1 is a reference substance solution characteristic map; s2 is the characteristic map of the test solution, S3 is the characteristic map of the blank solvent (25% methanol).

FIG. 17 is a common peak superposition signature for the repeatability tests of the present invention; wherein S (1)5 is a common peak superposition characteristic spectrum of the test solution under the repeatability 1; s (2)5 is a common peak superposition characteristic spectrum of the test solution under the repeatability 2; s (3)5 is a common peak superposition characteristic spectrum of the test solution under the repeatability 3; s (4)5 is a common peak superposition characteristic spectrum of the test solution under the repeatability 4; s (5)5 is a common peak superposition characteristic spectrum of the test solution under the repeatability 5; and S (6)5 is a common peak superposition characteristic spectrum of the test solution under the repeatability 6.

FIG. 18 shows a common peak overlap profile of precision tests according to the present invention; wherein, S (1)5 is a common peak superposition characteristic spectrum of the test solution under the precision 1; s (2)5 is a common peak superposition characteristic spectrum of the test solution under the precision 2; s (3)5 is a common peak superposition characteristic spectrum of the test solution under the precision 3; s (4)5 is a common peak superposition characteristic spectrum of the test solution under the precision of 4; s (5)5 is a common peak superposition characteristic spectrum of the test solution under the precision of 5; and S (6)5 is a common peak superposition characteristic spectrum of the test solution under the precision of 6.

FIG. 19 is a common peak superposition signature for the stability test of the present invention; wherein S (1)5 is a common peak superposition characteristic spectrum of the test solution measured in 0 h; s (2)5, determining a common peak superposition characteristic map of the test solution in 2 h; s (3)5 is a common peak superposition characteristic spectrum of the test sample solution measured in 4 h; s (4)5, determining a common peak superposition characteristic map of the test solution in 8 h; s (5)5, a common peak superposition characteristic map of the test solution is determined in 12 h; and S (6)5 is a common peak superposition characteristic spectrum of the test solution measured for 24 h.

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

FIG. 21 is a study of different column temperatures according to the present invention; wherein S1 is chromatographic column with column temperature of 23 ℃, S2 is chromatographic column with column temperature of 25 ℃, and S3 is chromatographic column with column temperature of 27 ℃.

FIG. 22 is a flow rate test common peak superposition signature in different flow rate surveys according to 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 1.25 min/mL.

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

FIG. 24 is a chromatogram showing the overlay of bergenin, gallic acid, quercetin and test samples.

FIG. 25 is a chromatogram showing the overlay of bergenin and test samples in the characteristic chromatogram determination of the present invention.

FIG. 26 is a graph of a herba Ardisiae Japonicae control drug in a characteristic chromatogram determination of the present invention.

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

FIG. 28 is a common peak spectrum of 27 batches of Japanese ardisia herb in the characteristic chromatogram determination of the present invention.

FIG. 29 is a chromatogram showing the overlay of the standard decoction of 27 batches of Japanese ardisia during the characteristic chromatogram determination; wherein, S1-S27 respectively represent the superposition spectrum of standard decoction of 1-27 batches of Japanese ardisia herb.

FIG. 30 is a graph of a 27 batch Standard decoction fit in a characteristic chromatogram assay of the invention.

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

FIG. 32 is a comparison of blank solvents in the content determination methodology for validation of specificity studies in accordance with the present invention; wherein S1 is a reference substance solution characteristic map; s2 is the characteristic map of the sample solution, S3 is the characteristic map of the blank solvent (methanol).

FIG. 33 is a linear plot of the various concentrations of a bergenin control in a 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 quality detection method of Japanese ardisia herb decoction, which comprises the following detection method, wherein the standard of the Japanese ardisia herb decoction content is limited to 32.0-66.0mg of bergenin in each 1g by the characteristics of the Japanese ardisia herb decoction, the yield of dry extract, the identification of a thin layer, the determination of extract, a characteristic map and the bergenin content, wherein the determination of the yield of 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 bergenin content are determined by liquid chromatography.

In this embodiment:

preparing standard decoction of Japanese ardisia herb: according to a decoction method in the management Specification of traditional Chinese medicine decoction rooms of medical institutions (No. 2009) of the State administration of traditional Chinese medicine), 15 batches of Japanese ardisia herb decoction pieces are taken and added with water until the decoction pieces submerge for about 4-5cm, the decoction pieces are soaked for 30-40min and decocted for two times, the first time of decoction is 30-40min, the second time of decoction is 25-30min, solid-liquid separation is carried out while the decoction pieces are hot, filtrates are combined, concentrated and dried, and 15 batches of Japanese ardisia herb standard decoction dry extract powder are prepared.

1. Dry extract yield test

Taking 27 batches of Japanese ardisia herb decoction pieces, preparing 27 batches of standard decoction dry extract powder according to the preparation method, calculating the dry extract yield (see table 1), and calculating the mean value, SD value and allowable range of the dry extract yield. According to the standard decoction characterization and application in the technical requirements of quality control and standard establishment of traditional Chinese medicine formula granules of the State drug administration, the plaster yield is as follows: and calculating the extract yield and Standard Deviation (SD) by using 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. From the data of the paste outlet rate of the standard decoction of 27 batches, the average value, SD and the allowable range of the paste outlet rate can be calculated.

Table 1: rate of paste discharge

The results show that the allowable range of the standard decoction cream yield is calculated by adding or subtracting 3 times SD from the mean value, and is 8.77-16.69%; calculated according to 70-130% of the average value, the average value is 8.91-16.55%. Combining two groups of calculation data, the allowable range of standard decoction cream yield is drawn up to be 9% -16.5%.

2. Trait survey

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

3. Thin layer authentication

The product is a dry extract of single-component decoction piece Japanese ardisia, the Japanese ardisia is used as a reference by referring to a method under the item of 'thin-layer identification' of Japanese ardisia in Chinese pharmacopoeia, and a thin-layer identification method of the product is established, and after 27 batches of sample tests, the spot of a test sample is clear, and a negative reference sample is free of interference, so the product is drawn as an item [ identification ]. The test methods and results are as follows:

3.1 test method thin layer chromatography (China pharmacopoeia 2020 edition four-part general rule 0502) test

Preparing a test solution: taking about 0.1g of the product, adding 20ml of methanol, performing ultrasonic treatment for 30 minutes, cooling, filtering, evaporating filtrate to dryness, and dissolving residue with 1ml of methanol to obtain a sample solution.

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

Chromatographic conditions are as follows: the thin layer plate is a silica gel G thin layer plate; sample amount of spotting: the sample solution and the reference medicinal material solution are respectively 2 mul; developing agent: dichloromethane-ethyl acetate-methanol (5:4: 2); color developing agent: spraying a mixed solution (newly prepared before use) of 1% ferric trichloride and 1% potassium ferricyanide (1:1), and inspecting in sunlight.

The 27 batches of standard decoction are tested, and as shown in fig. 1(a) and 1(b), spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatogram of the control solution, and the spots are clear.

4. Measurement of extract

Taking 27 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 the 27 batches of standard decoction extract is 38.96%, and the lower limit of the reference standard limit allowable range (70-130% of the mean value), namely 70% of the mean value, is 27.27%; the lower limit of the allowable range for the reference standard limit (mean plus or minus 3 SD), i.e., mean minus 3SD, is 24.56%. By comparison, the mean value of 70% was both lower than (31.25%) and closer to the lowest test value of the 27 standard batches of decoction. Therefore, it is determined that the alcohol soluble extract of the product is not less than 27.3%.

5. Feature map testing

5.1 instruments, reagents and reagents

(1) The instrument comprises the following steps: shimadzu high performance liquid chromatograph (LC-2030puls, Shimadzu, Japan); shimadzum-pack GIST C18-AQ (4.6 mm. times.250 mm, 5 μm); a constant temperature water bath (HMTD-7000, Yongguanming medical instruments, Inc. of Beijing); an ultrasonic cleaner (KQ-300DE, ultrasonic instruments Co., Ltd., Kunshan city); one in ten thousand balance (PX224ZH, ohauss instruments ltd); one in ten million balances (AWU220D, Shimadzu, Japan).

(2) Reagent: ethanol (Tanshizus chemical reagent, Inc. of Tianjin) and methanol (Tanshizus chemical reagent, Inc. of Tianjin) by chromatography; the water is ultrapure water (self-made in laboratory) or phosphoric acid (chemical reagent of Mimi Europe, Tianjin).

(3) And (3) standard substance: bergenin (batch No. 111532-201604, content: 94.1%, China food and drug testing institute), gallic acid (batch No. 110831-201906, content: 91.5%, China food and drug testing institute), quercitrin (batch No. 111538-202007, content: 93.5%, China food and drug testing institute), and Japanese ardisia herb control drug (batch No. 121213-201102, China food and drug testing institute).

5.2 test methods

5.2.1 determination of chromatographic conditions

(1) Determination of optimum absorption wavelength

The chromatographic condition based on the method is combined with a PAD detector to carry out multi-wavelength scanning on the standard decoction sample of the Japanese ardisia, the scanning wavelength is 200-400nm, and 3 detection wavelengths are selected for research to determine the optimal absorption wavelength.

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

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica was used as a filler (4.6 mm. times.250 mm, 5 μm) (Shimadzu-pack GIST C18-AQ (4.6 mm. times.250 mm, 5 μm)); 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; the flow rate was 1.0ml per minute; the column temperature was 25 ℃; the detection wavelength is 230, 250 and 270 nm.

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 result shows that the sample has stronger and more absorption between 200-300nm, as shown in detail in FIG. 2, when 250nm is selected as the detection wavelength by comparing the 3 detection wavelength chromatograms, the separation degree of each characteristic peak is better, the interference is smaller, the base line is stable, so 250nm is selected as the detection wavelength.

(2) Investigation of column temperature

The column temperature of 20 ℃, 25 ℃ and 30 ℃ is selected for comparison in the experiment, and the appropriate column temperature is selected.

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

Table 4:

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

(3) Investigation of flow Rate

In the experiment, 3 flow rates of 0.8ml/min, 1.0ml/min and 1.2ml/min are selected for comparison, and a proper flow rate is selected.

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica was used as a filler (4.6 mm. times.250 mm, 5 μm) (Shimadzu-pack GIST C18-AQ (4.6 mm. times.250 mm, 5 μm)); gradient elution was performed as specified in table 5 using 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 was 25 ℃; the detection wavelength was 250 nm.

Table 5:

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

(4) Investigation of mobile phase

In the experiment, three mobile phases of 0.1% formic acid, 0.1% phosphoric acid and 0.1% phosphoric acid solution are selected for comparison to determine a proper mobile phase.

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

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 distinct characteristic peak is present, when 0.1% phosphoric acid solution is selected as the mobile phase, the information of the peak is complete, the separation effect is better than that of 0.1% formic acid solution, so 0.1% phosphoric acid is selected.

(5) Gradient optimization

The elution gradient of the Japanese ardisia herb standard decoction characteristic spectrum is optimized in the experiment, and the optimal gradient is determined.

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica was used as a filler (4.6 mm. times.250 mm, 5 μm) (Shimadzu-pack GIST C18-AQ (4.6 mm. times.250 mm, 5 μm)); gradient elution was performed as specified in tables 7, 8 and 9 using methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; the flow rate was 1.2ml per minute; the column temperature was 25 ℃; the detection wavelength was 250 nm.

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 by optimizing the elution gradient of the herba ardisiae japonicae standard decoction feature map, as shown in fig. 6, 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 herba ardisiae japonicae standard decoction feature map.

5.2.2 chromatographic conditions

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

Table 10:

5.3 examination of pretreatment method of sample solution

A sample pretreatment method of the characteristic spectrum of the standard herba Ardisiae Japonicae decoction (batch number: Y210701) is examined, and the influence of an extraction solvent, an extraction mode, extraction time and the dosage (or sampling amount) of the extraction solvent on the characteristic spectrum of the standard herba Ardisiae Japonicae decoction is mainly examined.

5.3.1 initial assay conditions

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

Table 11:

Preparation of reference solutions: taking 1.0g of herba Ardisiae Japonicae as reference material, adding 25ml of 25% methanol, ultrasonic treating for 30min, cooling, and filtering to obtain filtrate as reference material solution of reference material. Taking a proper amount of bergenin reference substance, precisely weighing, and adding methanol to obtain solutions containing 200 μ g of bergenin per 1ml as reference substance solutions. The number of theoretical plates is not less than 1500 calculated according to bergenin peak.

Preparation of a test solution: taking a proper amount of herba Ardisiae Japonicae standard decoction fine powder, taking about 0.2g, placing into a conical flask with a plug, adding 25mL of 25% methanol, sealing the plug, performing ultrasonic treatment (power 100W, frequency 40kHz) for 30 minutes, cooling, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.

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

5.3.2 investigation of extraction methods: the test solutions were prepared by different extraction methods, including ultrasonic extraction and reflux extraction, and were tested according to the test method 5.3.1 above. The results show that, as shown in fig. 7, the number of main peaks is consistent, the peak shape difference is not large in different extraction modes, 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) and less than 3.0%, so that the time is saved, the operation is convenient, and the sample extraction mode is selected to be ultrasonic.

Table 12:

5.3.3 investigation of extraction time: the test solutions were prepared at different ultrasonic extraction times and tested as described above for test 5.3.1. The results show that the number of main peaks is consistent, the peak shapes are not greatly different at different extraction times (see figure 8), and ultrasonic extraction is excluded for 40 minutes. The calculation results show that the RSD value of the ratio of the total peak area to the sampling amount is 0.38% (see Table 13), and is less than 3.0%, and no obvious difference exists, so that the extraction time is 30 minutes when the extraction (total peak area/sampling amount) value is larger.

Table 13:

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

Table 14: investigation results of different extraction solvents

5.3.5 solvent dosage consideration: the test solutions were prepared with different amounts of solvent (15ml, 25ml, 35ml) and tested according to the 5.3.1 test method. The results show that different solvent amounts, as shown in fig. 10, have the same number of main peaks, and different solvent amounts have smaller peak shape differences. The calculation results showed that the RSD value of the ratio of the total peak area to the sample amount was 1.58% (see table 15) and less than 3.0%, so 25ml of the solvent having a larger value (total peak area/sample amount) was selected as the amount of the extraction solvent.

Table 15: investigation results of different solvent dosage

In summary, the main parameters for determining the preparation method of the test solution are as follows: taking a proper amount of the fine powder, taking about 0.2g, placing into a conical flask with a plug, adding 25mL of 25% methanol, sealing the plug, carrying out ultrasonic treatment (power 300W and frequency 40kHz) for 30 minutes, standing cold, shaking up, filtering, and taking a subsequent filtrate to obtain the product.

5.4 feature Pattern feature Peak identification

(1) Instruments, reagents and reagents

The instrument comprises the following steps: shimadzu high performance liquid chromatograph (LC-2030puls, Shimadzu, Japan); shim-pack GIST C18-AQ (4.6 mm. times.250 mm, 5 μm); a constant temperature water bath (HMTD-7000, Yongguang medical instruments, Inc. of Beijing); an ultrasonic cleaner (KQ-300DE, ultrasonic instruments Co., Ltd., Kunshan city); one in ten thousand balance (PX224ZH, ohauss instruments ltd); one in ten million balances (AWU220D, Shimadzu, Japan).

Reagent: ethanol (Tanshizus chemical reagent, Inc. of Tianjin) and methanol (Tanshizus chemical reagent, Inc. of Tianjin) by chromatography; the water is ultrapure water (self-made in laboratory) or phosphoric acid (chemical reagent of Mimi Europe, Tianjin).

(2) And (3) standard substance: bergenin (batch No. 111532-202005, content: 94.4%, China food and drug testing institute), gallic acid (batch No. 110831-201906, content: 91.5%, China food and drug testing institute), quercitrin (batch No. 111538-202007, content: 93.5%, China food and drug testing institute), and Japanese ardisia herb control drug (batch No. 121213-201102, China food and drug testing institute).

(3) Sample assay

The detection is carried out according to the conditions under the standard characteristic spectrum item of the quality of the ardisia japonica standard decoction, the experimental result is shown in fig. 11-15, and the experimental result shows that chromatographic peaks which are consistent with the retention time of the gallic acid, the quercitrin and the bergenin can be found in the ardisia japonica standard decoction characteristic spectrum, and the peak 2 is the gallic acid, the peak 3 is the bergenin and the peak 4 is the quercitrin through the spectral comparison confirmation.

5.5 verification of feature map analysis method

5.5.1 specialization study: the sample was measured with 10ul of 25% methanol as a solvent under the above chromatography conditions of 5.3.1. Experiments show that as shown in FIG. 16, the blank solvent is free of interference, and the method has good specificity.

5.5.2 repeatability tests: taking about 0.2g of Japanese ardisia herb standard decoction (batch number: Y210701) sample, and measuring 6 parts in total according to 5.3.1 chromatographic conditions, and carrying out sample injection and 6-needle measurement. The measurement result shows that the peak shape and the peak number of the characteristic map of the test sample are basically consistent (see figure 17). And 5 common peaks exist in the characteristic map, bergenin is taken 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 show that the relative peak area RSD value and the relative retention time RSD value are both less than 3.0 percent and are in the qualified range (see tables 16 and 17). The test shows that the method has good reproducibility.

Table 16: relative retention time of characteristic spectrum of 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: relative peak area of characteristic spectrum of repeatability test

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 Japanese ardisia standard decoction (lot number: Y210701) is taken, and is subjected to determination according to 5.3.1 chromatographic conditions, and 6-needle continuous sample injection determination shows that the characteristic spectrum peak shape and the peak number of the sample are basically consistent (see figure 18). And 5 common peaks exist in the characteristic map, bergenin is taken 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 show that the relative peak area RSD value and the relative retention time RSD value are both less than 3.0 percent and are in the qualified range (see tables 18 and 19). The test shows that the method has good precision.

Table 18: relative retention time of characteristic spectrum of precision test

Number of peak	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 of 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: about 0.2g of Japanese ardisia standard decoction (batch number: Y210701) is taken as a sample, the sample is tested according to the chromatographic condition of 5.3.1, the sample is injected for testing for 0h, 2h, 4h, 8h, 12h and 24h respectively, and the test result shows that the characteristic spectrum peak shape and the peak number of the sample are basically consistent (see figure 19). And 5 common peaks exist in the characteristic map, bergenin is taken 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 result shows that the relative peak area RSD value and the relative retention time RSD value are both less than 3.0 percent and are in a qualified range (see tables 20 and 21). The test shows that the test solution is stable within 24 hours.

Table 20: stability test feature profile 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: relative peak area of characteristic spectrum of stability test

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

The same process is carried out by other analysts on different dates and different devices, taking about 0.2g of standard decoction of Japanese ardisia herb (lot: Y210701), precisely weighing, parallel taking 6 parts, preparing test solution according to the method of '5.3.1', and analyzing. The bergenin is taken 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: middle precision relative retention time of standard decoction characteristic spectrum of Japanese ardisia herb

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: middle precision relative peak area of standard decoction characteristic spectrum of Japanese ardisia

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: herba Ardisiae Japonicae standard decoction feature chromatogram repeatability relative retention time

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 standard decoction of Japanese ardisia

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 test

(1) Investigation of different chromatography columns

About 0.2g of Japanese ardisia herb standard decoction (batch number: Y210701) is taken, and samples of 3 chromatographic columns (250mmx4.6mm, 5 μm) with different models (Shimadzu GistaQ-C18(PF-108), Shimadzu GIST C18(PF-51) and Zhongzhongchong red (RD-C18) are respectively injected and tested according to the test method under the item of '5.3.1'. The measured results show that the characteristic pattern peak shape and the peak number of the test sample are inconsistent (see figure 20). And 5 common peaks exist in the characteristic map, bergenin is taken 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 show that the relative retention time RSD% value and the relative peak area RSD value are respectively more than 3.0% and more than 5.0%, and are all in the unqualified range (see tables 26 and 27). Experiments show that chromatographic columns of different models of different manufacturers have large influence on the determination of the characteristic spectrum, namely, chromatographic columns of specified models can only be used for the determination of the characteristic spectrum.

Table 26: relative retention time of characteristic spectrum for different chromatographic column investigation

Peak number	PF-108	PF-51	Middle 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: relative peak area of characteristic spectrum for different chromatographic column investigation

Peak number	PF-108	PF-51	Middle school score 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 Japanese ardisia standard decoction (lot number: Y210701) is sampled and measured at column temperature of 23 deg.C, 25 deg.C and 27 deg.C according to the test method under item 5.3.1. The measurement result shows that the peak shape and the peak number of the characteristic spectrum of the test sample are basically consistent (see figure 21). The characteristic spectrum has 5 common peaks, bergenin is taken 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 show that the relative retention time RSD% value and the relative peak area RSD% value are both less than 3.0%, and are in an acceptable range (see tables 28 and 29). Tests show that different column temperature changes have small influence on the determination of the characteristic spectrum, namely different column temperatures have good durability.

Table 28: relative retention time of characteristic spectrum for different column temperature investigation

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: relative peak area of characteristic spectrum for different column temperature investigation

Number of peak	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 herba Ardisiae Japonicae standard decoction (lot number: Y210701) is taken, and sample injection determination is performed at column temperatures of 1.15ml/min, 1.20ml/min and 1.25ml/min according to test method under item 5.3.1. The measurement result shows that the peak shape and the peak number of the characteristic map of the test sample are basically consistent (see figure 22). And 5 common peaks exist in the characteristic map, bergenin is taken 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 show that the relative retention time RSD% value and the relative peak area RSD% value are both less than 3.0%, and are in the qualified range (see tables 30 and 31). Tests show that slight flow rate changes have small influence on characteristic map determination, namely different flow rates have good durability.

Table 30: relative retention time of characteristic spectrum for different flow velocity investigation

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: relative peak area of characteristic spectrum for different flow velocity investigation

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 is subject to specificity, precision, repeatability, stability, intermediate precision verification and durability investigation, the result meets the requirement, and the established method can be well used for measuring the characteristic spectrum of the Japanese ardisia herb.

5.6 characteristic spectrum characterization and analysis of standard decoction

5.6.1 characteristic chromatogram determination

According to the drawn characteristic spectrum analysis method, the characteristic spectrums of 27 batches of Japanese ardisia herb standard decoctions and 27 batches of Chinese medicinal decoction pieces used for preparation are determined, and the results show that 5 common peaks exist in the characteristic chromatograms of the standard decoctions and the Chinese medicinal decoction pieces used for preparation through the positioning of bergenin, quercitrin and gallic acid, and the common peaks correspond to the retention time of 5 characteristic peaks in the chromatogram of a reference substance of a reference medicinal material, wherein the peak corresponding to the reference substance of the positioning standard bergenin is peak 3, the common peak characteristic spectrum is shown in detail in figures 23-30.

5.6.2 evaluation of the relative Retention time of the characteristic chromatogram

Determining the characteristic spectrum of the 27 batches of the standard decoction of Japanese ardisia according to the established characteristic spectrum analysis method. The results showed that there were 5 common peaks in the characteristic map, and the relative retention times, their relative retention times and ranges, of characteristic peak 1, peak 2, peak 4, peak 5 and the S peak were calculated using peak (3) corresponding to the peak of the standard reference as reference peak S (see table 32(1), 32 (2)).

Table 32 (1): common 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): S16-S27 standard decoction common peak relative retention time

In conclusion, the established standard decoction characteristic spectrum determination method by adopting the high performance liquid chromatography is subjected to precision, repeatability, stability verification and durability investigation 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. According to the proposed characteristic spectrum analysis method, the characteristic spectrums of 27 batches of standard decoctions are measured, and 5 common characteristic peaks are calibrated by analyzing the result, wherein the peak 3 is bergenin. Taking the peak (3) corresponding to the bergenin reference substance as an S peak, calculating the relative retention time of other 4 characteristic peaks, and drawing up the average value of the relative retention time of the peaks of 27 batches of samples as specified values: 0.319 (peak 1), 0.400 (peak 2), 1.528 (peak 4), 1.659 (peak 5), considering the error of experiment operation, instrument, reagent and other multi-factors, the relative retention time allowed range is determined as + -10%.

6. Determination of content

The Japanese ardisia herb is dried whole plant of Ardisiajaponzca (Thunb.) Blume of Ardisiajaponzca of Ardisia of Myrsinaceae. Produced in various places of Hunan province, especially in the counties of Yang tonifying, North Ann, Taojiang, Yangjiang and the like; other provinces, such as the south of the Yangtze river, are also productive. Most of them were collected from Song, the original name of the trade market in a few regions, Taiwan Ardisia. The herba Ardisiae Japonicae contains Ardisinol I and II, Ardisin, Bergenin, embelin, and Addisol No. 1 (Bergenin). Modern pharmacological research considers that the ardisiacrispin No. 1 has the effect of relieving cough. Therefore, the research refers to a method for measuring the content of Japanese ardisia herb 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 standard decoction of the Japanese ardisia herb.

The bergenin reference substance (batch No. 111532-201604) is scanned at full wavelength to determine the good absorption wavelength, and the result shows that as shown in FIG. 31, the detection is carried out at 275nm wavelength, the baseline is stable, the separation degree is good, and no impurity peak interference exists, so the 275nm wavelength is selected as the detection wavelength for determining the standard decoction content of Japanese ardisia herb.

6.1 test methods

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica was used as a filler (4.6 mm. times.250 mm, 5 μm) (Shimadzu-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 was 1.2ml per minute; the column temperature was 25 ℃; the detection wavelength was 275 nm. The number of theoretical plates is not less than 1500 calculated according to bergenin peak.

Preparation of reference solutions: taking appropriate amount of bergenin reference substance, precisely weighing, and adding methanol to obtain solution containing bergenin 200 μ g per 1 ml.

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

6.2 examination of pretreatment method of test solution

The best sample pretreatment mode is determined by examining the extraction mode, the extraction solvent, the extraction time and the solvent amount of the content measurement of the standard decoction of Japanese ardisia (batch number: Y210701).

6.2.1 investigation of extraction methods: the test solutions were prepared by different extraction methods and tested according to the test method 6.1 described above. The results show that the RSD% is less than 2% (see Table 33), and the sample extraction mode is selected to be ultrasonic treatment for facilitating subsequent experiments.

Table 33: comparison of different extraction methods

6.2.2 investigation 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 RSD% is less than 2%, the samples were sonicated for 30 minutes, and the content was the highest (see table 34), so the samples were selected for a sonication time of 30 minutes.

Table 34: comparison of different extraction times

6.2.3 investigation of extraction solvent: the test solutions were prepared with different extraction solvents (ethanol, 80% methanol, methanol) and tested according to the test method 6.1 above. The results show that the solvent was methanol, with the highest content (see table 35), so the sample solvent was selected to be methanol.

Table 35: comparison of different extraction solvents

6.2.4 solvent amount examination test solutions were prepared with different amounts of solvent (35ml, 50ml, 65ml) and tested as described above for test 6.1. The results show that the solvent amount was 50ml, the highest content (see Table 36), and therefore the sample solvent amount was selected to be 50 ml.

Table 36: comparison of different solvent amounts

In summary, the main parameters for determining the preparation method of the test solution are as follows: taking a proper amount of the fine powder, taking about 0.2g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 300W and frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing with methanol to reduce weight loss, shaking up, filtering, and taking a subsequent filtrate to obtain the product.

6.3 assay methodology verification

6.3.1 specialization study: the sample was taken and tested with methanol as solvent according to the test method 6.1 above, which showed that: the blank solvent was not perturbed (see fig. 32), and the process was well-defined.

6.3.2 repeatability tests: about 0.2g of the same batch of standard decoction samples is taken, 6 parts in total, the average value of the bergenin content in the samples is measured to be 40.70mg/g and the RSD value is 0.252 percent according to the test method of 6.1, 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 samples are taken, sample introduction is continuously carried out for 6 needles according to the test method of 6.1, the peak area is measured, the RSD value of the peak area of the bergenin in the sample is calculated to be 0.096%, and the test shows that the precision of the instrument is good (see Table 38).

Table 38:

6.3.4 stability test: about 0.2g of a batch of standard decoction samples are taken, sample introduction is carried out for 0h, 2h, 4h, 8h, 12h and 24h respectively according to the test method of 6.1, the peak areas of the samples are measured, the RSD value of the peak area of bergenin in the samples is calculated to be 0.573%, and the test shows that the sample solution is relatively stable within 24 hours (see Table 39).

Table 39:

6.3.5 Linear Range test: taking a bergenin reference solution according to the following concentration: 0.39757mg/ml, 0.198785mg/ml, 0.0993925mg/ml, 0.0496962mg/ml, 0.024545mg/ml and 0.004969mg/ml were measured under the chromatographic conditions of 6.1.

Taking the peak area of bergenin as a vertical coordinate and the sample injection quality as a horizontal coordinate, drawing a standard curve, and performing linear regression, wherein the regression equation is as follows: 13492129.9774x +9236.9974, R ² When the peak area of bergenin is 1, it is known that bergenin has a good linear relationship with the peak area thereof in the range of 4.9 to 397 μ g/ml (see table 40 and fig. 33 for details).

Table 40: the linear relation of bergenin

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

6.3.6 sample recovery test: precisely weighing 0.1g, 0.13g and 0.15g of each sample (the bergenin content is 40.6963mg/g), adding 10.5ml, 7.0ml and 5.0ml of bergenin reference solution (378ug/ml) with known concentration, preparing test solution according to the method under item 6.1, measuring according to chromatographic conditions under item 6.1, and calculating that the average sample adding recovery rate of the bergenin is 97.50% and the RSD is 2.85% (see table 41 for details).

Table 41: test result of sample recovery rate of bergenin

6.3.7 durability examination

(1) Investigation of different chromatography columns

About 0.2g of a batch of samples was sampled and measured by 3 chromatographic columns (4.6 mm. times.250 mm, 5 μm) (Shimadzu GIST C18-AQ, Zhongpu red RD-C18, Shimadzu GIST C18, respectively) of different models from different manufacturers according to the test method under item "6.1". The calculation results show that the measured content RSD value is 0.99 percent and less than 2.0 percent (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

About 0.2g of a batch of samples was sampled and measured at a flow rate of 23 ℃, 25 ℃ and 27 ℃ according to the test method under item "6.1". The calculation results showed that the measured content RSD was 0.104% and less than 2.0% (see table 43), and the test showed that the method had good durability against small variations in column temperature.

Table 43:

(3) investigation of different flow rates

About 0.2g of a batch of samples was sampled and measured at flow rates of 1.0ml/min, 1.2ml/min and 1.4ml/min, respectively, according to the test method under item "6.1". The calculations show that the RSD value of the measured content is 0.195% and less than 2.0% (see table 44), and the tests show that the method is very robust to small variations in flow rate.

Table 44:

(4) investigation of different mobile phases

About 0.2g of a batch of samples was sampled and measured by the test method under item "6.1" using methanol-water (21: 79), (20: 80), (19: 81) respectively. The calculation results show that the measured content RSD value is 0.855% and less than 3.0% (see table 45), and the test shows that the method has good durability against small variations in the ratio of mobile phases.

Table 45:

in conclusion, the results of the whole analysis method are in accordance with the requirements through specificity, peak purity, precision, repeatability, stability, linearity inspection, intermediate precision, sample recovery and durability inspection, and the established method can be well used for measuring the content of bergenin.

6.4 Standard decoction and Chinese medicinal material content and transfer rate determination

The Japanese ardisia herb is initially processed into slices through a production place and processed into Japanese ardisia herb decoction pieces after being processed, and the bergenin content of the Japanese ardisia herb decoction pieces cannot be changed, so that the characteristic chromatogram and the bergenin content of the Japanese ardisia herb decoction pieces refer to the medicinal material data.

6.4.1 the content of bergenin in 27 batches of standard decoction of Japanese ardisia herb and 27 batches of Japanese ardisia herb decoction pieces used for preparation are determined according to the proposed content analysis method, and the results are shown in tables 46, 47 and 48.

Table 46: determination result of 27 batches of Japanese ardisia herb traditional Chinese medicine decoction pieces bergenin

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

Table 47: determination result of 27 batches of Japanese ardisia herb standard decoction bergenin

The average content of bergenin in the standard decoction of the product is 44.11mg/g, the content range is 32.34 mg/g-65.49 mg/g, and SD is 7.93; calculated according to 70-130% of the average value, the allowable range of the bergenin content is 30.88-57.34 mg/g. Calculated according to the average value of-3 SD to the average value of +3SD, the allowable range of the bergenin content is 20.32mg/g to 67.90 mg/g. The highest value of the content of the 27 batches of standard decoction is 65.49mg/g, and is not in the range (30.88 mg/g-57.34 mg/g) calculated according to 70-130% of the average value; the lowest value of the content of the 27 standard decoction batches is 32.34mg/g, and the lower limit (20.32mg/g) of the range calculated according to the mean value of-3 SD to the mean value of +3SD is greatly different. Therefore, the content range of the bergenin in the standard decoction is more reasonable according to the content range measured by 27 batches of standard decoction. Therefore, the content range of bergenin in the standard decoction is drawn up as follows: 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, the bergenin content transfer rate is calculated for the measurement results of 27 batches of standard decoction and the prepared traditional Chinese medicine decoction pieces thereof, the quality 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. Herba Ardisiae Japonicae decoction piece standard decoction is prepared by decocting herba Ardisiae Japonicae decoction piece in water for 2 times, concentrating the filtrate, and freeze drying. The bergenin content transfer rate is shown in Table 48.

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

According to the data, the ardisia japonica decoction pieces are decocted according to the scheme to prepare the ardisia japonica decoction piece standard decoction, the average transfer rate of the bergenin is 56.32%, the measured transfer rate range is 36.70% -68.63%, and the SD is 7.01. According to technical requirements for quality control and standard formulation of traditional Chinese medicine formula granules, the allowable range of the bergenin content transfer rate is 39.42-73.22% calculated according to 70-130% of the mean value of the transfer rate; calculated according to the average value of-3 SD to the average value of +3SD, the ratio is 35.29 to 77.35 percent. The transfer rate ranges of the 27 batches of standard decoction are all in the range of mean value-3 SD-mean value +3SD, so the transfer rate range of the bergenin content of the standard decoction is drawn as follows: 35.3 to 77.4 percent.

According to the method for detecting the quality of the ardisia japonica decoction, the character of the ardisia japonica decoction, the dry extract yield, the thin layer identification, the extract, the characteristic map and the bergenin content determination are researched, the quality of the ardisia japonica decoction is evaluated through multi-aspect measurement, a solid foundation is laid for the quality stability of products, a feasible quality standard of the ardisia japonica decoction can be established, the effective control of the quality of the ardisia japonica standard decoction is realized, and in addition, the chromatographic condition is adopted for liquid phase analysis, so that a chromatogram with better and clearer separation degree can be obtained; the Japanese ardisia herb decoction pieces are decocted to prepare the Japanese ardisia herb decoction piece, and the content range of bergenin in the standard Japanese ardisia herb decoction is as follows: 32.0-66.0 mg/g; the average bergenin transfer rate is 56.32%, and the bergenin content transfer rate range of the standard drawn herb tea decoction is: 35.3-77.4%, and the result shows that the bergenin content and the transfer rate thereof in the decoction of a plurality of batches are within the allowable range, so the invention can provide reference basis for the quality standard research of the Japanese ardisia herb formula particles.

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 these 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.

Claims

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

the characteristic spectrum determination by liquid chromatography comprises the following steps: performing liquid chromatograph analysis, taking the solution prepared from herba Ardisiae Japonicae reference medicinal material as reference solution b, taking the solution prepared from bergenin reference as reference solution b, taking the solution prepared from herba Ardisiae Japonicae decoction sample as test solution b, precisely sucking reference solution b, reference solution b and test solution b, respectively injecting into liquid chromatograph, and measuring; wherein, the adopted chromatographic conditions are that a chromatographic column: octadecylsilane chemically bonded silica chromatography column (4.6 mm. times.250 mm, 5 μm); 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

2. The method for detecting the quality of Japanese ardisia herb decoction of claim 1, wherein the decocting method comprises the following steps: soaking herba Ardisiae Japonicae 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, concentrating, and drying to obtain herba Ardisiae Japonicae decoction dry extract powder.

3. The method for detecting the quality of Japanese ardisia herb decoction according to claim 1, wherein the thin layer chromatography comprises the following steps:

(2) preparing a reference medicinal material solution a: taking herba Ardisiae Japonicae reference medicinal material 1g, adding methanol 20ml, ultrasonic treating for 30min, cooling, filtering, evaporating filtrate to dryness, and dissolving residue with methanol 1ml to obtain reference medicinal material solution a;

4. The method of claim 1, wherein the hot-dipping method comprises using ethanol as solvent and measuring the extract range by hot-dipping method in the section of alcohol-soluble extract measurement method.

5. The method for detecting the quality of the ardisia japonica decoction as claimed in claim 1, wherein the step of determining the characteristic map by using the liquid chromatography further comprises the following steps:

6. The method for detecting the quality of the ardisia japonica decoction as claimed in claim 1, wherein the step of determining the bergenin content by liquid chromatography comprises the following steps: performing liquid chromatograph analysis, taking the solution prepared from bergenin reference as reference solution c, taking the solution prepared from herba Ardisiae Japonicae decoction sample as test solution c, precisely sucking the reference solution c and the test solution c, respectively injecting into liquid chromatograph, and measuring; wherein, the adopted chromatographic conditions are that a chromatographic column: octadecylsilane chemically bonded silica chromatography 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: 1.2 mL/min; column temperature: 25 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: 275 nm.

7. The method for detecting the quality of Japanese ardisia herb decoction of claim 6, wherein the step of determining the content of bergenin by liquid chromatography further comprises the steps of: