Quality detection method of Shuangshitonglin capsules

Abstract

The application belongs to the technical field of detection, and relates to a quality detection method of Shuishitonglin capsules, which comprises identifying Poria cocos, Acorus gramineus Soland, Qingdai, Phellodendri Amurensis Cortex, Salvia miltiorrhiza and Atractylodes lancea in the Shuishitonglin capsules by adopting thin layer chromatography; and further comprises detecting the content of berberine hydrochloride, tanshinone II A, salvianolic acid B, geniposidic acid and / or verbascoside in the Shuishitonglin capsules by adopting high performance liquid chromatography. The quality detection method of the Shuishitonglin capsules can accurately identify Poria cocos, Acorus gramineus Soland, Qingdai, Phellodendri Amurensis Cortex, Salvia miltiorrhiza and Atractylodes lancea in the Shuishitonglin capsules, and detect the content of berberine hydrochloride, tanshinone II A, salvianolic acid B, geniposidic acid and / or verbascoside contained in the Shuishitonglin capsules, and has high accuracy, good precision, high sensitivity, good reproducibility, simple operation, short time and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and relates to a quality detection method of Shishitonglin capsules. BACKGROUND

[0002] The Shishitonglin capsules are a unique variety, and the State Drug Permit Z20080028. The original name of the drug is Huazhu Tonglin Capsules. The drug has the effects of clearing heat and reducing dampness, resolving turbidity and dredging the urinary tract, and is used for chronic prostatitis belonging to damp-heat obstruction. Symptoms include burning sensation in the urethra, frequent and urgent urination, incomplete urination, dripping white after urination, wetness in the genital area, pain or discomfort in the perineum, lower abdomen and lumbosacral region, red tongue with yellow fur, and stringy or slippery pulse. Due to the complexity of traditional Chinese medicines, various production links and parameters may cause problems in the effective or index transfer rate. Therefore, for traditional Chinese medicines, multiple means need to be used for detection to ensure the reliability and stability of the detection results. At present, there is no systematic quality detection method for Shishitonglin capsules, and the existing detection means is not comprehensive enough to reflect the overall internal quality of Shishitonglin capsules, and cannot meet the quality detection requirements of Shishitonglin capsules.

[0003] To perfect the quality detection standard of Shishitonglin capsules, a suitable analysis method and evaluation strategy are needed. Generally, thin layer chromatography is a commonly used qualitative analysis method, and high performance liquid chromatography is a commonly used quantitative analysis method. The Shishitonglin capsules are made of ten kinds of traditional Chinese medicines such as Phellodendri Ammonium, and the overall composition is complex. Therefore, when using thin layer chromatography to qualitatively analyze whether the traditional Chinese medicine is missing a certain medicine, how to determine the accurate thin layer chromatography condition to exclude other interference becomes a key problem. Secondly, when using high performance liquid chromatography to analyze and evaluate the quality of Shishitonglin capsules, first of all, representative index components need to be determined, and the index components need to be accurately separated and determined. How to determine the index components and accurately detect them is a problem to be solved for constructing the quality detection standard of Shishitonglin capsules. SUMMARY

[0004] In view of the above technical problems existing in the quality detection of the existing Shishitonglin capsules, the application provides a quality detection method of Shishitonglin capsules, which can accurately qualitatively identify Fuling, Shichangpu, Qingdai, Guanhuangbai, Danshen and Cangzhu in the Shishitonglin capsules, and detect the content of berberine hydrochloride, danshenshuan II A, salvianolic acid B, geniposidic acid and / or verbascoside contained in the Shishitonglin capsules. The method has high accuracy, good precision, high sensitivity, good reproducibility, simple operation, short time and the like.

[0005] In order to achieve the above purpose, the application provides a quality detection method of Shishitonglin capsules. The quality detection method comprises using thin layer chromatography to identify Fuling, Shichangpu, Qingdai, Guanhuangbai, Danshen and Cangzhu in the Shishitonglin capsules; and / or

[0006] The content of berberine hydrochloride, tanshinone II A, salvianolic acid B, geniposidic acid and / or verbascoside in Shishitonglin Capsules is determined by high performance liquid chromatography.

[0007] The pachyma, alocasia, indigo, amur cork tree, salvia and atractylodes in Shishitonglin Capsules are identified by thin layer chromatography, including:

[0008] The Shishitonglin Capsules are shelled, and the content is extracted and treated, and the extract is collected;

[0009] The reference solution is prepared;

[0010] The extract and the reference solution are identified by first thin layer chromatography, and whether the Shishitonglin Capsules contain pachyma is determined based on the detection result; and / or

[0011] The extract and the reference solution are identified by second thin layer chromatography, and whether the Shishitonglin Capsules contain alocasia is determined based on the detection result; and / or

[0012] The extract and the reference solution are identified by third thin layer chromatography, and whether the Shishitonglin Capsules contain indigo is determined based on the detection result; and / or

[0013] The extract and the reference solution are identified by fourth thin layer chromatography, and whether the Shishitonglin Capsules contain amur cork tree is determined based on the detection result; and / or

[0014] The extract and the reference solution are identified by fifth thin layer chromatography, and whether the Shishitonglin Capsules contain salvia is determined based on the detection result; and / or

[0015] The extract and the reference solution are identified by sixth thin layer chromatography, and whether the Shishitonglin Capsules contain atractylodes is determined based on the detection result.

[0016] According to the embodiments of the present application, the identification method can further include at least one of the following additional technical features:

[0017] Further, when identifying Poria cocos, the Shuishitonglin Capsule content is extracted by: taking 2g of Shuishitonglin Capsule content, adding 20mL of ethanol, ultrasonic treatment for 30min, then filtering, evaporating the filtrate, adding 1mL of methanol to completely dissolve the residue, and collecting the extract; when identifying Acorus gramineus Soland and Atractylodes lancea, the Shuishitonglin Capsule content is extracted by: taking 2g of Shuishitonglin Capsule content, grinding, adding 15mL of water, stirring, and placing, then taking the upper layer solution, extracting with n-hexane twice, 20mL each time, combining the n-hexane liquid, evaporating in a water bath, dissolving the residue in 1mL of n-hexane, and collecting the extract; when identifying indigo, the Shuishitonglin Capsule content is extracted by: taking 1g of Shuishitonglin Capsule content, grinding, adding 5mL of chloroform, ultrasonic treatment for 15min, filtering, and collecting the filtrate to obtain the extract; when identifying Phellodendri chinensis, the Shuishitonglin Capsule content is extracted by: taking 1g of Shuishitonglin Capsule content, grinding, placing in a 50mL volumetric flask, adding 45mL of hydrochloric acid-methanol mixed solution with a volume ratio of 1:100, ultrasonic treatment for 30min, diluting to the mark with methanol, shaking, filtering, and collecting the filtrate to obtain the extract; and when identifying Salvia miltiorrhiza, the Shuishitonglin Capsule content is extracted by: taking 5g of Shuishitonglin Capsule content, grinding, adding 10mL of diethyl ether, shaking, filtering, evaporating the filtrate, dissolving the residue in 0.5mL of ethyl acetate, and collecting the extract.

[0018] Further, when identifying Poria cocos and Phellodendri chinensis, 2g of Poria cocos control medicinal material and 0.1g of Phellodendri chinensis control medicinal material are respectively taken to replace the Shuishitonglin Capsule content, and the control solution is prepared in the same way; when identifying Acorus gramineus Soland and Atractylodes lancea, 0.5g of Acorus gramineus Soland control medicinal material and 0.2g of Atractylodes lancea control medicinal material are respectively taken, 2mL of n-hexane is added, ultrasonic treatment is performed for 15min, and the supernatant is taken as the control solution; when identifying indigo, indigo and indigo blue control samples are taken, chloroform is added to prepare a solution containing 1mg per 1mL, which is used as the control solution; and when identifying Salvia miltiorrhiza, a Salvia miltiorrhiza ketone II A control sample is taken, ethyl acetate is added to prepare a solution containing 2mg per 1mL, which is used as the control solution.

[0019] Further, the first thin layer identification condition is: thin layer plate: silica gel G thin layer plate; sample volume: 5-10 μL; developing agent: toluene-ethyl acetate-formic acid with a volume ratio of 20:5:0.5; development, removal, air drying, and inspection under ultraviolet light with a wavelength of 365 nm; the second thin layer identification condition is: thin layer plate: silica gel G thin layer plate; sample volume: 5 μL; developing agent: petroleum ether-ethyl acetate with a volume ratio of 20:1; color developing agent: 5% vanillin sulfuric acid solution; and hot air blowing until the spots are clearly colored; the third thin layer identification condition is: thin layer plate: silica gel G thin layer plate; sample volume: 5 μL; developing agent: benzene-trichloromethane-acetone with a volume ratio of 5:4:1; development, removal, and air drying; the fourth thin layer identification condition is: thin layer plate: silica gel G thin layer plate; sample volume: 2 μL; developing agent: ethyl acetate-butanone-formic acid-water with a volume ratio of 10:6:1:1; ammonia vapor saturated chromatographic jar; development, removal, and inspection under ultraviolet light with a wavelength of 365 nm; the fifth thin layer identification condition is: thin layer plate: silica gel G thin layer plate; sample volume: 5 μL; developing agent: petroleum ether-ethyl acetate with a volume ratio of 8:2; development, removal, and air drying; and the sixth thin layer identification condition is: thin layer plate: silica gel G thin layer plate; sample volume: 5 μL; developing agent: petroleum ether-ethyl acetate with a volume ratio of 20:1; color developing agent: 10% sulfuric acid ethanol solution containing 5% p-dimethylaminobenzaldehyde; and heating until the spots are clearly colored; wherein the petroleum ether is petroleum ether with a temperature of 60-90 °C.

[0020] Further, the fourth thin layer identification method is specifically three kinds:

[0021] The first method is: 2 μL of the extract and the control sample solution are respectively spotted on the same silica gel G thin layer plate, ethyl acetate-butanone-formic acid-water with a volume ratio of 10:6:1:1 is used as the developing agent, the chromatographic jar is saturated with ammonia vapor, and the thin layer plate is inspected under ultraviolet light with a wavelength of 365 nm after development, removal, and air drying;

[0022] The second method is: 2 μL of the extract and the control sample solution are respectively spotted on the same silica gel G thin layer plate, ethyl acetate-butanone-formic acid-water with a volume ratio of 10:6:1:1 is used as the developing agent, the distance of development is 12 cm, the chromatographic jar is saturated with ammonia vapor immediately after development, and the thin layer plate is inspected under ultraviolet light with a wavelength of 365 nm;

[0023] The third method is: 2 μL of the extract and the control sample solution are respectively spotted on the same silica gel G thin layer plate, the thin layer plate is saturated with ammonia vapor in the chromatographic jar for 5-10 minutes, the thin layer plate is removed, and the thin layer plate is inspected under ultraviolet light with a wavelength of 365 nm after development in the chromatographic jar containing the developing agent ethyl acetate-butanone-formic acid-water with a volume ratio of 10:6:1:1.

[0024] The method for determining the content of berberine hydrochloride, tanshinone II A, salvianolic acid B, geniposidic acid and / or verbascoside in Shangshi Tonglin Capsules by using high performance liquid chromatography comprises the following steps:

[0025] The Shangshi Tonglin Capsules are shelled, and the content is subjected to methanol extraction treatment, and the extraction liquid is collected;

[0026] The extraction liquid is subjected to first high performance liquid chromatography detection, and the content of berberine hydrochloride in the Shangshi Tonglin Capsules is determined based on the detection result; and / or

[0027] The extraction liquid is subjected to second high performance liquid chromatography detection, and the content of tanshinone II A in the Shangshi Tonglin Capsules is determined based on the detection result; and / or

[0028] The extraction liquid is subjected to third high performance liquid chromatography detection, and the content of salvianolic acid B in the Shangshi Tonglin Capsules is determined based on the detection result; and / or

[0029] The extraction liquid is subjected to fourth high performance liquid chromatography detection, and the content of geniposidic acid and / or verbascoside in the Shangshi Tonglin Capsules is determined based on the detection result.

[0030] According to the embodiment of the present application, the content determination method can further comprise at least one of the following additional technical features:

[0031] Further, the first liquid chromatography detection condition is that the chromatographic column is a C18 chromatographic column; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is selected from acetonitrile, and the mobile phase B is selected from 0.02 mol / L potassium dihydrogen phosphate solution; the flow rate is 1.0 mL / min; the column temperature is 30℃; the detection wavelength is 345 nm; the injection amount is 10 μL; the second high performance liquid chromatography detection condition is that the chromatographic column is a C18 chromatographic column; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is selected from acetonitrile, and the mobile phase B is selected from 0.05% phosphoric acid aqueous solution; the flow rate is 1.0 mL / min; the column temperature is 30℃; the detection wavelength is 270 nm; the injection amount is 10 μL; the third high performance liquid chromatography detection condition is that the chromatographic column is a C18 chromatographic column; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is selected from methanol, and the mobile phase B is selected from 0.1% phosphoric acid aqueous solution; the flow rate is 1.0 mL / min; the column temperature is 35℃; the detection wavelength is 278 nm; the injection amount is 10 μL; and the fourth high performance liquid chromatography detection condition is that the chromatographic column is a C18 chromatographic column; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is selected from methanol, and the mobile phase B is selected from 0.5% acetic acid aqueous solution; the flow rate is 1.0 mL / min; the column temperature is 30℃; the detection wavelength is 254 nm; and the injection amount is 10 μL.

[0032] Further, the content of Shishitonglin Capsules is subjected to methanol extraction treatment as follows: the content of Shishitonglin Capsules is weighed into a conical flask with a stopper, methanol solution is added, the weight is determined, then ultrasonic mixing is performed for 40 min, the weight is determined again, the lost weight is made up with methanol solution, the mixture is shaken and filtered, the filtrate is collected, and the filtrate is filtered through a 0.22 μm microporous filter membrane to obtain an extract.

[0033] Further, when the content of berberine hydrochloride is determined, the methanol solution is a methanol solution containing 1% hydrochloric acid by volume; when the content of salvianolic acid B is determined, the methanol solution is a 80% methanol solution by volume; when the content of geniposidic acid and / or verbascoside is determined, the methanol solution is a 60% methanol solution by volume.

[0034] Further, when the content of berberine hydrochloride is determined, 45 mL of the 1% hydrochloric acid methanol solution is added based on 1 g of the content of Shishitonglin Capsules; when the content of tanshinone IIA is determined, 30 mL of the methanol solution is added based on 1 g of the content of Shishitonglin Capsules; when the content of salvianolic acid B is determined, 50 mL of the 80% methanol solution is added based on 0.5 g of the content of Shishitonglin Capsules; when the content of geniposidic acid and / or verbascoside is determined, 50 mL of the 60% methanol solution is added based on 1 g of the content of Shishitonglin Capsules.

[0035] Further, when the content of berberine hydrochloride is determined, isocratic elution is adopted for the first high performance liquid chromatography detection; the potassium dihydrogen phosphate solution is adjusted to pH 4 with phosphoric acid; the volume ratio of the mobile phase A to the mobile phase B is 25:75;

[0036] Further, when the content of tanshinone IIA is determined, the gradient elution mode adopted for the second high performance liquid chromatography detection is as follows:

[0037] 0-6 min, the mobile phase A is 61% by volume, and the mobile phase B is supplemented to 100% by volume;

[0038] 6-20 min, the mobile phase A is increased from 61% to 90% by volume, and the mobile phase B is supplemented to 100% by volume;

[0039] 20-20.5 min, the mobile phase A is decreased from 90% to 61% by volume, and the mobile phase B is supplemented to 100% by volume;

[0040] 20.5-25 min, the mobile phase A is 61% by volume, and the mobile phase B is supplemented to 100% by volume;

[0041] Further, when the content of salvianolic acid B is determined, the gradient elution mode adopted for the third high performance liquid chromatography detection is as follows:

[0042] 0-40min, mobile phase A from 32% to 50%, mobile phase B to 100% by volume;

[0043] 40-45min, mobile phase A from 50% to 32% by volume, mobile phase B to 100% by volume;

[0044] Further, when determining the content of geniposidic acid and / or verbascoside, the gradient elution method used in the fourth high performance liquid chromatography detection is:

[0045] 0-1min, mobile phase A is 5% by volume, mobile phase B to 100% by volume;

[0046] 1-40min, mobile phase A from 5% to 60% by volume, mobile phase B to 100% by volume;

[0047] 40-50min, mobile phase A from 60% to 5% by volume, mobile phase B to 100% by volume.

[0048] The beneficial effects of the present application are:

[0049] 1、Under the given thin layer chromatography conditions, the chromatogram of the extract of Shishitonglin capsule and the chromatogram of the control solution show the same color spots at the corresponding positions, thereby it can be accurately judged whether Shishitonglin capsule contains Fuling, Shichangpu, Qingdai, Guanhuangbai, Danshen and Cangzhu components, and the quality of Shishitonglin capsule is evaluated. Further, the present application also uses berberine hydrochloride, tanshinone II A, salvianolic acid B, geniposidic acid and / or verbascoside as index components and carries out quantitative analysis and determination by high performance liquid chromatography. Berberine hydrochloride is a characteristic component of Guanhuangbai, tanshinone II A and salvianolic acid B are characteristic components of Danshen, and geniposidic acid and / or verbascoside are characteristic components of Plantaginis Semen, which helps to determine the content of Guanhuangbai, Danshen and Plantaginis Semen from the quantitative point of view and gives the quantitative determination standard of whether the quality of Shishitonglin capsule is qualified. The present application combines qualitative analysis and quantitative analysis, and the given chromatography conditions can effectively exclude interference, have good separation effect, high accuracy, good precision, high sensitivity, good reproducibility, simple operation, short time, and realize effective detection of the quality of Shishitonglin capsule.

[0050] 2、When high performance liquid chromatography is used to detect the content of Shishitonglin capsule, methanol solution is used as the extracting agent, which is creatively obtained by the inventors through a large number of experiments, and it can fully extract berberine hydrochloride, tanshinone II A, salvianolic acid B, geniposidic acid and / or verbascoside in Shishitonglin capsule. When multiple components need to be extracted at the same time, multiple extraction solvents do not need to be used, which improves the extraction efficiency and reduces the extraction cost and time.

[0051] 3. Through extensive experimentation, the inventors discovered that the extraction methods for five herbs—Poria cocos, Acorus tatarinowii, Indigo naturalis, Phellodendron chinense, Salvia miltiorrhiza, and Atractylodes lancea—in Shuangshitonglin capsules are complex, and the quality transfer during the extraction process is highly traceable. Therefore, these five herbs were selected as qualitative identification standards. Research and analysis of Shuangshitonglin capsules revealed that berberine hydrochloride, tanshinone IIA, salvianolic acid B, genipin glycoside, and / or verbascoside are present in relatively high amounts. These are the active pharmaceutical ingredients, possessing advantages such as good reproducibility, ease of quantification, and ease of industrialization. They can better monitor the quality of Shuangshitonglin capsules and avoid the impact of unstable factors in raw materials, processes, or operations. Therefore, selecting these as quantitative evaluation indicators can effectively achieve quality testing of Shuangshitonglin capsules, offering advantages such as stability, controllability, good reproducibility, strong operability, and high accuracy. Attached Figure Description

[0052] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0053] Figure 1 The thin-layer chromatogram for identifying the Poria cocos components in Example 1 is shown below.

[0054] Figure 2 The thin-layer chromatogram for identifying the components of Acorus tatarinowii in Example 1 is shown below.

[0055] Figure 3 The thin-layer chromatogram for identifying the indigo component in Example 1 is shown below.

[0056] Figure 4 The thin-layer chromatogram of the Phellodendron amurense component in Example 1 is shown.

[0057] Figure 5 The thin-layer chromatograms of the components of Danshen in Example 1 are shown.

[0058] Figure 6 The thin-layer chromatogram for identifying the Atractylodes lancea components in Example 1 is shown below.

[0059] Figure 7 This is an HPLC elution efficiency evaluation chart for the determination of berberine hydrochloride content in Example 2;

[0060] Figure 8 This is the specificity chromatogram for the determination of berberine hydrochloride content in Example 2;

[0061] Figure 9 This is a system adaptability chromatogram for the determination of berberine hydrochloride content in Example 2;

[0062] Figure 10 Reference chromatogram for matching Mark peak in determination of berberine hydrochloride content in Example 2;

[0063] Figure 11 Fingerprint chromatogram for matching Mark peak in determination of berberine hydrochloride content in Example 2;

[0064] Figure 12 Reference chromatogram for matching all peaks in determination of berberine hydrochloride content in Example 2;

[0065] Figure 13 Fingerprint chromatogram for matching all peaks in determination of berberine hydrochloride content in Example 2;

[0066] Figure 14 Specificity chromatogram for determination of tanshinone IIA in Example 2;

[0067] Figure 15 Reference chromatogram for matching Mark peak in determination of tanshinone IIA in Example 2;

[0068] Figure 16 Fingerprint chromatogram for matching Mark peak in determination of tanshinone IIA in Example 2;

[0069] Figure 17 Reference chromatogram for matching all peaks in determination of tanshinone IIA in Example 2;

[0070] Figure 18 Fingerprint chromatogram for matching all peaks in determination of tanshinone IIA in Example 2;

[0071] Figure 19 Specificity chromatogram for determination of salvianolic acid B in Example 2;

[0072] Figure 20 Reference chromatogram for matching Mark peak in determination of salvianolic acid B in Example 2;

[0073] Figure 21 Fingerprint chromatogram for matching Mark peak in determination of salvianolic acid B in Example 2;

[0074] Figure 22 Reference chromatogram for matching all peaks in determination of salvianolic acid B in Example 2;

[0075] Figure 23 Fingerprint chromatogram for matching all peaks in determination of salvianolic acid B in Example 2;

[0076] Figure 24 Specificity chromatogram for determination of geniposidic acid and / or verbascoside in Example 2;

[0077] Figure 25 Mark peak matching control chromatogram for determination of geniposidic acid and / or verbascoside content in Example 2;

[0078] Figure 26 Mark peak matching fingerprint for determination of geniposidic acid and / or verbascoside content in Example 2;

[0079] Figure 27 Full spectrum peak matching control chromatogram for determination of geniposidic acid and / or verbascoside content in Example 2;

[0080] Figure 28 Full spectrum peak matching fingerprint for determination of geniposidic acid and / or verbascoside content in Example 2. DETAILED DESCRIPTION

[0081] The scheme of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be obtained commercially.

[0082] The instruments and reagents involved in the following examples are as follows:

[0083] Instruments

[0084] JY3002 electronic balance (Shanghai Puchun Measurement Instrument Co., Ltd.); ultrasonic cleaner PL-S40 (FCC RoSH Co.); U-3000 high performance liquid chromatograph (Thermo Fisher Scientific, USA); LC-2030C high performance liquid chromatograph (Shimadzu Corporation, Japan); Eclipse XDB-C18 chromatographic column (4.6 mm x 250 mm, 5 μm, Agilent Technologies, USA); Inertsil ODS-3 chromatographic column (4.6 mm x 250 mm, 5 μm, GL Sciences Inc., Japan).

[0085] Reagents

[0086] The contents of Shangshi Tonglin Capsules were provided by Shaanxi Momeide Blood and Pharmaceutical Co., Ltd.; Poria cocos reference material, Phellodendri Cortex reference material, Acori graminei Rhizoma reference material, Atractylodis Rhizoma reference material, indigo, indirubin reference substance, berberine hydrochloride (batch number: 11013-202015, purity: 98%), verbascoside (batch number: 111530-201914, purity: 95.2%), geniposidic acid (batch number: 111828-201805, purity: 98%) were purchased from China National Institute for Food and Drug Control; Salvianolic acid B (batch number: Y14M7H14804; purity: ≥98%), tanshinone IIA (batch number: Y16M10C88487; purity: ≥98%) were purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.; purified water was purchased from Hangzhou Wahaha Group Co., Ltd.; acetonitrile, methanol were chromatographic grade (Fisher Scientific); n-hexane, petroleum ether (60-90°C), ethyl acetate, vanillin, sulfuric acid, chloroform, benzene, acetone, hydrochloric acid, butanone, formic acid, diethyl ether, p-dimethylaminobenzaldehyde, ethanol, potassium dihydrogen phosphate, acetic acid (analytical grade), phosphoric acid (analytical grade) were purchased from Tianjin Kemeluo Chemical Reagent Co., Ltd.; silica gel G plate (Merck, Germany).

[0087] Example 1

[0088] Thin layer chromatography (TLC) was used to qualitatively analyze whether Shangshi Tonglin Capsules contained Poria cocos, Acori graminei Rhizoma, indigo, Phellodendri Cortex, Salvia miltiorrhiza and Atractylodis Rhizoma.

[0089] 1. Identification of Poria cocos

[0090] Preparation of Shangshi Tonglin Capsule extract: The shells of Shangshi Tonglin Capsules were removed, 2 g of the contents was taken, 20 mL of ethanol was added, and after ultrasonic treatment for 30 min, it was filtered. The filtrate was evaporated to dryness, 1 mL of methanol was added to completely dissolve the residue, and the extract was collected.

[0091] Preparation of reference solution: Poria cocos reference material was used to prepare the reference solution in the same way.

[0092] Preparation of negative control solution: A negative sample without Poria cocos was prepared according to the prescription of Shangshi Tonglin Capsules, and the negative control solution was prepared in the same way.

[0093] TLC conditions: According to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0502), 5-10 μL of each of the above two solutions was taken and spotted on the same silica gel G thin layer plate. Toluene-ethyl acetate-formic acid (20:5:0.5 by volume) was used as the developing agent, developed, taken out, dried, and observed under ultraviolet light (365 nm).

[0094] For example, the amount of the above two solutions taken can be 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, etc. This embodiment does not make specific limitations.

[0095] Results: In the qualitative analysis of Shishitonglin Capsules extract, the same fluorescent spots appeared in the corresponding position of the chromatogram of the control solution, and the negative control had no interference. The chromatogram is shown in Figure 1 , columns 1-3 are the chromatogram of Shishitonglin Capsules extract, column 4 is the chromatogram of Poria cocos control drug, columns 5-6 are the chromatogram of negative control.

[0096] From the above results, it can be known that under the given separation conditions, it can be accurately identified whether Shishitonglin Capsules contains Poria cocos medicinal materials.

[0097] 2, Acorus gramineus identification

[0098] Shishitonglin Capsules extract preparation: take the contents of Shishitonglin Capsules 2g, grind finely, add water 15mL, stir, place, take the upper layer solution, extract with n-hexane 2 times, 20mL each time, combine the n-hexane liquid, water bath dry, add n-hexane 1mL to dissolve, collect the extract.

[0099] Preparation of control solution: take Acorus gramineus control drug 0.5g, add 2mL n-hexane, ultrasonic treatment for 15 minutes, take the supernatant as the control solution.

[0100] TLC conditions: according to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0502), take 5μL of the above two solutions respectively, point on the same silica gel G thin layer plate, with petroleum ether (60-90℃)-ethyl acetate (volume ratio 20:1) as developing agent, develop, take out, dry. Spray with 5% vanillin sulfuric acid solution, hot air blow until the spots are clear.

[0101] Results: In the qualitative analysis of Shishitonglin Capsules extract, the same fluorescent spots appeared in the corresponding position of the chromatogram of the control solution, and the negative control had no interference. The chromatogram is shown in Figure 2 .

[0102] From the above results, it can be known that under the given separation conditions, it can be accurately identified whether Shishitonglin Capsules contains Acorus gramineus medicinal materials.

[0103] 3, Identification of indigo

[0104] Shishitonglin Capsules extract preparation: take the contents of Shishitonglin Capsules 1g, grind finely, add chloroform 5mL, ultrasonic treatment for 15 minutes, filter, collect the filtrate as the extract.

[0105] Preparation of control solution: take indigo, indigo red control, add chloroform to prepare a solution containing 1mg per 1mL, as the control solution.

[0106] TLC conditions: according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0502) test, 5 μL of the above two solutions were taken respectively and spotted on the same silica gel G thin layer plate, with volume ratio of 5:4:1 benzene-trichloromethane-acetone as developing agent, developed, taken out, and dried.

[0107] Results: The qualitative analysis of Shuishitonglin Capsules extract showed the same fluorescent spots at the positions corresponding to the control solution chromatogram. The chromatogram is shown in Figure 3 .

[0108] From the above results, it can be concluded that under the given separation conditions, it can be accurately identified whether Shuishitonglin Capsules contains Qingdai medicinal materials.

[0109] 4. Identification of Phellodendri Cortex

[0110] Shuishitonglin Capsules extract preparation: Take 1 g of Shuishitonglin Capsules content, grind finely, accurately weigh, add 45 mL of volume ratio 1:100 hydrochloric acid-methanol mixture to a 50 mL volumetric flask, ultrasonic treatment (power 200 W, frequency 40 kHz) for 30 minutes, cool, dilute to the mark with methanol, shake well, filter, and collect the filtrate as the extract.

[0111] Preparation of the control solution: Take 0.1 g of Phellodendri Cortex control drug instead of Shuishitonglin Capsules content, and prepare the control solution by the same method.

[0112] There are three specific identification methods for Phellodendri Cortex thin layer identification:

[0113] The first is: take 2 μL of the extract and the control solution respectively, and spot them on the same silica gel G thin layer plate, with volume ratio of 10:6:1:1 ethyl acetate-methyl ketone-formic acid-water as developing agent, place it in an ammonia vapor saturated chromatographic cylinder, take it out, dry, and observe under ultraviolet light 365 nm;

[0114] The second is: take 2 μL of the extract and the control solution respectively, and spot them on the same silica gel G thin layer plate, with volume ratio of 10:6:1:1 ethyl acetate-methyl ketone-formic acid-water as developing agent, develop, and develop for 12 cm. Immediately after development, place it in an ammonia vapor saturated chromatographic cylinder, fumigate for 2-3 minutes, and immediately observe under ultraviolet light 365 nm;

[0115] The third is: take 2 μL of the extract and the control solution respectively, and spot them on the same silica gel G thin layer plate, place the thin layer plate in an ammonia vapor saturated chromatographic cylinder for 5-10 minutes, take out the thin layer plate, immediately place it in a chromatographic cylinder containing volume ratio of 10:6:1:1 ethyl acetate-methyl ketone-formic acid-water developing agent, develop, take it out, dry, and observe under ultraviolet light 365 nm.

[0116] Results: In the qualitative analysis of Shuishitonglin Capsules extract, the same fluorescent spots were shown in the corresponding position of the chromatogram of the control solution, and the chromatogram is shown in Figure 4 , Figure 4 The third method is used to identify the chromatogram of Phellodendri chinensis Cortex.

[0117] From the above results, it can be known that under the given separation conditions, it can accurately identify whether Shuishitonglin Capsules contain Phellodendri chinensis Cortex.

[0118] 5. Identification of Salvia miltiorrhiza

[0119] Shuishitonglin Capsules extract preparation: Take the contents of Shuishitonglin Capsules 5g, grind finely, add 10mL of ethyl ether, shake, filter, evaporate the filtrate, add 0.5mL of ethyl acetate to the residue to dissolve, and collect the extract.

[0120] Preparation of control solution: Take Salvia miltiorrhiza ketone II A control, add ethyl acetate to prepare a solution containing 2mg per 1mL as the control solution.

[0121] TLC conditions: According to the test of thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0502), take 5μL of the above two solutions respectively, and point them on the same silica gel G thin layer plate. With a volume ratio of 8:2 of petroleum ether (60-90℃)-ethyl acetate as the developing agent, develop, take out, and dry.

[0122] Results: In the qualitative analysis of Shuishitonglin Capsules extract, the same fluorescent spots were shown in the corresponding position of the chromatogram of the control solution, and the chromatogram is shown in Figure 5 .

[0123] From the above results, it can be known that under the given separation conditions, it can accurately identify whether Shuishitonglin Capsules contain Salvia miltiorrhiza.

[0124] 6. Identification of Atractylodes lancea

[0125] Shuishitonglin Capsules extract preparation: Take the contents of Shuishitonglin Capsules 2g, grind finely, add 15mL of water and stir, stand, take the upper layer solution, extract with n-hexane twice, 20mL each time, combine the n-hexane liquid, evaporate on a water bath, dissolve the residue with 1mL of n-hexane, and collect the extract.

[0126] Preparation of control solution: Take 0.2g of Atractylodes lancea control drug, add 2mL of n-hexane, and ultrasonic treat (power 150W) for 15 minutes. Take the supernatant as the control solution.

[0127] TLC conditions: according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0502) test, 5 μL of the above two solutions was taken and spotted on the same silica gel G thin layer plate, with a volume ratio of 20:1 of petroleum ether (60-90 ℃)-ethyl acetate as the developing agent, sprayed with 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution, heated until the spots were clearly colored.

[0128] Results: The qualitative analysis of Shuishitonglin Capsules extract showed the same fluorescent spots in the chromatogram corresponding to the position of the control solution, and the chromatogram is shown in Figure 6 .

[0129] From the above results, it can be known that under the given separation conditions, it can be accurately identified whether Shuishitonglin Capsules contains Atractylodes rhizome medicinal materials.

[0130] Example 2

[0131] The contents of berberine hydrochloride, tanshinone II A, salvianolic acid B, geniposidic acid and / or verbascoside in Shuishitonglin Capsules were quantitatively determined by high performance liquid chromatography (HPLC).

[0132] 1. Determination of the content of berberine hydrochloride

[0133] Preparation of the first Shuishitonglin Capsules extract: 2 g of Shuishitonglin Capsules content was taken and placed in a conical flask with a stopper. 45 mL of methanol containing 1% hydrochloric acid was added, and the weight was determined. After ultrasonic treatment for 45 min, the weight was determined again. The lost weight was made up with 1% hydrochloric acid methanol solution, shaken well, filtered, and the filtrate was taken and filtered through a 0.22 μm microporous filter for standby.

[0134] Preparation of the first control solution: an appropriate amount of berberine hydrochloride control was accurately weighed and placed in a 45 mL volumetric flask. It was dissolved with 1% hydrochloric acid methanol solution to prepare a first control solution with a concentration of 0.4606 mg / mL, which was ready for use.

[0135] Preparation of the first negative control solution: 160.07 g of Danshen, 80.04 g of Yuansu, 80.09 g of Atractylodes, 160.15 g of Baijiangcua, 160.09 g of Fenbixie, 160.06 g of Cheqianzi, 80.20 g of Fuling, 95.04 g of Huashi, and 25.04 g of Qingdai were prepared according to the process of Shuishitonglin Capsules to prepare a negative sample without Huangbai. According to the preparation method of the first Shuishitonglin Capsules extract, the first negative control solution was prepared in the same way.

[0136] The first high performance liquid chromatography detection condition: using isocratic elution, the chromatographic column is Eclipse XDB-C18 chromatographic column; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is selected from acetonitrile, the mobile phase B is selected from 0.02 mol / L potassium dihydrogen phosphate solution; the potassium dihydrogen phosphate solution is adjusted to pH 4 with phosphoric acid; the volume ratio of the mobile phase A to the mobile phase B is 25:75; the flow rate is 1.0 mL / min; the column temperature is 30 DEG C; the detection wavelength is 345 nm.

[0137] Results: the content of berberine hydrochloride in Shishitonglin capsules is 2.2310 mg / g.

[0138] Methodology investigation:

[0139] Chromatographic method elution capacity evaluation: in order to ensure that the first Shishitonglin capsule extract is completely out of peak in the established chromatographic method, the experiment is prolonged to 30 min based on the effective component peak time, and the first Shishitonglin capsule extract is observed. The results show that the chromatogram baseline is flat between 17-30 min under 345 nm wavelength, and there is no obvious peak. It is proved that the first Shishitonglin capsule extract is completely out of peak in the established chromatographic method.

[0140] Specificity test: the first Shishitonglin capsule extract, the first control solution and the first negative control solution are determined under the first high performance liquid chromatography detection condition, as shown in Figure 8 The chromatogram of the first Shishitonglin capsule extract is shown in A, the chromatogram of the first control solution is shown in B, and the chromatogram of the first negative control solution is shown in C. Under this chromatographic condition, the first negative control solution has no interference at the berberine hydrochloride peak, and the berberine hydrochloride component peak in the first Shishitonglin capsule extract is well separated from the adjacent peak.

[0141] Linear test: an appropriate amount of the first control solution is diluted 1 times, 2 times, 4 times, 5 times, 8 times, 10 times and 25 times with methanol to prepare different concentrations of the first control solution. According to the first high performance liquid chromatography detection condition, the peak area of each characteristic component is determined. The standard curve Y=675.14X-1.2219, r=0.9999 is drawn with the control product concentration (μg / mL) as the abscissa and the peak area as the ordinate. The results show that the linear relationship of berberine hydrochloride is good.

[0142] In order to ensure the test rigor, as shown in Figure 9 The peak of berberine hydrochloride is identified as the standard peak 1, and the peak of unknown component A with good peak shape and large peak area is selected as the auxiliary verification peak to evaluate the instrument precision, the repeatability and stability of the first Shishitonglin capsule extract.

[0143] Precision test: take the same first Shitonglin Capsules extract, according to the first high performance liquid chromatography detection conditions, 6 needles were continuously sampled, and the chromatogram was recorded. With berberine hydrochloride as reference peak 1, the RSD values of relative retention time and relative peak area of common peaks at 345 nm wavelength were calculated, and the results were shown in Table 1. The results showed that the RSD values of relative retention time were less than 0.2%, and the RSD values of relative peak area were less than 0.3%, indicating that the precision of the analytical instrument was good.

[0144] Table 1 Precision relative retention time test results

[0145]

[0146] Repeatability test: according to the preparation method of the first Shitonglin Capsules extract, 6 first Shitonglin Capsules extracts were prepared, and 6 times were continuously determined according to the first high performance liquid chromatography detection conditions. The chromatogram was recorded. With berberine hydrochloride as reference peak 1, the RSD values of relative retention time and relative peak area of common peaks at 345 nm wavelength were calculated, and the results were shown in Table 2. The results showed that the RSD values of relative retention time of each common peak were less than 0.5%, and the RSD values of relative peak area were less than 3.5%, indicating that the repeatability of the first Shitonglin Capsules extract was good.

[0147] Table 2 Repeatability relative retention time test results

[0148]

[0149] Take the same first Shitonglin Capsules extract, according to the first high performance liquid chromatography detection conditions, respectively, at 0, 2, 4, 6, 8, 10, 12, 24 h after the preparation of the first Shitonglin Capsules extract, sample test, record the chromatogram. With berberine hydrochloride as reference peak 1, the RSD values of relative retention time and relative peak area of common peaks at 345 nm wavelength were calculated, and the results were shown in Table 3. The results showed that the RSD values of relative retention time of each common peak were less than 0.5%, and the RSD values of relative peak area were less than 2.5%, indicating that the first Shitonglin Capsules extract had good stability within 24 h.

[0150] Table 3 Stability relative retention time test results

[0151]

[0152]

[0153] Fingerprint establishment: Take 10 batches of Shuishitonglin Capsules, remove the shells and take the contents, labeled as S1-S10, respectively. Prepare 10 batches of the first Shuishitonglin Capsule extract liquid according to the first Shuishitonglin Capsule extract liquid preparation method, inject for detection according to the first high-performance liquid chromatography detection condition, and record the chromatogram. Use the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" for data analysis, take S1 as the reference spectrum, set the time window width to 0.1 min, use the median method for multi-point correction and full-spectrum peak matching, generate the Shuishitonglin Capsule fingerprint spectrum and the control fingerprint spectrum, and perform similarity analysis.

[0154] Mark peak matching: Take the characteristic component berberine hydrochloride as the Mark peak to establish the fingerprint spectrum, record the Mark peak matching control spectrum ( Figure 10 ), and the Mark peak matching fingerprint spectrum ( Figure 11 ).

[0155] Full-spectrum peak matching: Perform fingerprint spectrum analysis on 10 batches of Shuishitonglin Capsules, establish the full-spectrum peak matching fingerprint spectrum, record the full-spectrum peak matching control spectrum ( Figure 12 ), the full-spectrum peak matching fingerprint spectrum ( Figure 13 ), and the similarity (Table 4).

[0156] Table 4 Similarity of full-spectrum peak matching fingerprint spectrum of Shuishitonglin Capsules

[0157]

[0158]

[0159] 2. Determination of tanshinone IIA content

[0160] Preparation of the second Shuishitonglin Capsule extract liquid: Take 1 g of the contents of Shuishitonglin Capsules, add 30 mL of methanol to a conical flask with a stopper, weigh and record the weight, ultrasonic for 40 min, weigh and record the weight again, make up the weight loss with methanol, shake well, filter, take the filtrate, pass through a 0.22 μm microporous filter membrane, and reserve for use.

[0161] Preparation of the second control solution: Take an appropriate amount of tanshinone IIA control, prepare a control solution with a concentration of 1.035 mg / g using methanol, and reserve for use.

[0162] Preparation of the second negative control solution: Take 80.07 g of Cortex Phellodendri, 80.04 g of Rhizoma Alpiniae Officinarum, 80.09 g of Radix Atractylodis, 160.15 g of Herba Patriniae, 160.09 g of Rhizoma Dioscoreae, 160.06 g of Semen Plantaginis, 80.20 g of Poria cocos, 95.04 g of Gypsum Fibrosum, and 25.04 g of Indigo Naturalis, prepare a negative sample without Salvia miltiorrhiza according to the Shuishitonglin Capsule preparation method, and prepare a negative control solution according to the Shuishitonglin Capsule extract liquid preparation method.

[0163] The second high performance liquid chromatography detection condition: the chromatographic column is C18 chromatographic column; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is selected from acetonitrile, the mobile phase B is selected from 0.05% phosphoric acid aqueous solution; the flow rate is 1.0 mL / min; the column temperature is 30 DEG C; the detection wavelength is 270 nm; the injection amount is 10 μL; gradient elution:

[0164] 0-6min, mobile phase A is 61% by volume, and mobile phase B is supplemented to 100% by volume;

[0165] 6-20min, mobile phase A is increased from 61% by volume to 90% by volume, and mobile phase B is supplemented to 100% by volume;

[0166] 20-20.5min, mobile phase A is decreased from 90% by volume to 61% by volume, and mobile phase B is supplemented to 100% by volume;

[0167] 20.5-25min, mobile phase A is 61% by volume, and mobile phase B is supplemented to 100% by volume;

[0168] Results: the content of tanshinone IIA in Shishitonglin capsules is 1.41 mg / g.

[0169] Methodology investigation:

[0170] Specificity test: the second Shishitonglin capsule extract, the second control solution and the second negative control solution are respectively determined under the second high performance liquid chromatography detection condition, as shown in the following figure: Figure 14 Wherein A is the second Shishitonglin capsule extract chromatogram, B is the second control solution chromatogram, and C is the second negative control solution chromatogram. Under this chromatographic condition, the second negative control solution has no interference at the peak of tanshinone IIA, and the peak of tanshinone IIA in the second Shishitonglin capsule extract is well separated from the adjacent peak.

[0171] Linear test: an appropriate amount of the second control solution is diluted 1 times, 2 times, 4 times, 5 times, 8 times, 10 times, 16 times, 20 times and 50 times with methanol to prepare different concentrations of the second control solution. According to the second high performance liquid chromatography detection condition, the sample is injected, and the peak area of each characteristic component is determined. The standard curve Y=30000000X-279550, r=0.9996 is drawn with the control product concentration (μg / mL) as the abscissa and the peak area as the ordinate. The results show that the linear relationship of tanshinone IIA is good.

[0172] Precision test: the same second Shishitonglin capsule extract was taken, 6 injections were continuously injected according to the second high performance liquid chromatography detection condition, the chromatogram was recorded, the relative retention time and the relative peak area of the common peak at 270 nm wavelength were calculated with the reference peak of tanshinone II A, and the results were shown in Table 5. The results showed that the RSD value of the relative retention time was less than 0.1%, and the RSD value of the relative peak area was less than 0.4%, indicating that the precision of the analytical instrument was good.

[0173] Table 5 Precision relative retention time and relative peak area test results

[0174]

[0175] Repeatability test: the same batch of Shishitonglin capsules was taken, 6 second Shishitonglin capsule extracts were prepared according to the second Shishitonglin capsule extract preparation method, 6 injections were continuously determined according to the second high performance liquid chromatography detection condition, the chromatogram was recorded, the relative retention time and the relative peak area of the common peak at 270 nm wavelength were calculated with the reference peak of tanshinone II A, and the results were shown in Table 6. The results showed that the RSD value of the relative retention time was less than 0.1%, and the RSD value of the relative peak area was less than 4.0%, indicating that the repeatability of the second Shishitonglin capsule extract was good.

[0176] Table 6 Repeatability relative retention time and relative peak area test results

[0177]

[0178] Stability test: the same second Shishitonglin capsule extract was taken, and the sample was tested at 0, 2, 4, 6, 8, 10, 12 and 24 h after the preparation of the second Shishitonglin capsule extract according to the second high performance liquid chromatography detection condition, the chromatogram was recorded, the relative retention time and the relative peak area of the common peak at 270 nm wavelength were calculated with the reference peak of tanshinone II A, and the results were shown in Table 7. The results showed that the RSD value of each relative retention time was less than 0.1%, and the RSD value of the relative peak area was less than 0.8%, indicating that the second Shishitonglin capsule extract had good stability within 24 h.

[0179] Table 7 Stability relative retention time test results

[0180]

[0181] Fingerprint establishment: Take 10 batches of Shuishitonglin capsules, remove the shell and mark as S1-S10. Prepare 10 batches of second Shuishitonglin capsule extract liquid according to the second Shuishitonglin capsule extract liquid preparation method, inject and detect according to the second high performance liquid chromatography detection condition, and record the chromatogram. Use "Traditional Chinese Medicine Chromatography Fingerprint Similarity Evaluation System (2012 version)" for data analysis, take Danshentong II A as the reference spectrum, set the time window width to 0.1 min, use the median method, perform multi-point correction and full spectrum peak matching, generate Shuishitonglin capsule fingerprint and control fingerprint, and perform similarity analysis.

[0182] Mark peak matching: Take the characteristic component Danshentong II A as the Mark peak to establish the fingerprint, record the Mark peak matching control spectrum ( Figure 15 ), and the Mark peak matching fingerprint ( Figure 16 ).

[0183] Full spectrum peak matching: Perform fingerprint analysis on 10 batches of Shuishitonglin capsules, establish full spectrum peak matching fingerprint, record the full spectrum peak matching control spectrum ( Figure 17 ), full spectrum peak matching fingerprint ( Figure 18 ), and similarity (Table 8).

[0184] Table 8 Similarity of full spectrum peak matching fingerprint of Shuishitonglin capsule finished product powder

[0185]

[0186]

[0187] 3. Determination of the content of salvianolic acid B

[0188] Preparation of the third Shuishitonglin capsule extract liquid: Take 0.5 g of Shuishitonglin capsule content, add 50 mL of 80% methanol into a conical flask with a plug, weigh, ultrasonic for 40 min, weigh again, supplement the weight loss with 80% methanol, shake well, filter, take the filtrate, pass through a 0.22 μm microporous filter membrane, and reserve.

[0189] Preparation of the third control solution: Take an appropriate amount of salvianolic acid B control, prepare a control solution with a concentration of 1.13876 mg / g in 80% methanol, and reserve.

[0190] Third negative control solution preparation: Take 80.07 g of Cortex Phellodendri, 80.04 g of Rhizoma Acori Tatarinowii, 80.09 g of Radix Glycyrrhizae, 160.15 g of Herba Patriniae, 160.09 g of Rhizoma Alpiniae Oxophyllae, 160.06 g of Semen Plantaginis, 80.20 g of Poria Cocos, 95.04 g of Gypsum Fibrosum, and 25.04 g of Prussian Blue, and prepare a negative sample without Salvia miltiorrhiza according to the preparation method of Shuangshi Tonglin Capsules. According to the third Shuangshi Tonglin Capsule extraction liquid preparation method, the third negative control solution is prepared in the same way.

[0191] Third high performance liquid chromatography detection condition: the chromatographic column is a C18 chromatographic column; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is selected from methanol, and the mobile phase B is selected from 0.1% phosphoric acid aqueous solution; the flow rate is 1.0 mL / min; the column temperature is 35°C; the detection wavelength is 278 nm; the injection amount is 10 μL; and gradient elution is as follows:

[0192] 0-40 min, mobile phase A is increased from 32% to 50%, and mobile phase B is supplemented to 100%;

[0193] 40-45 min, mobile phase A is decreased from 50% to 32%, and mobile phase B is supplemented to 100%.

[0194] Result: the content of salvianolic acid B in Shuangshi Tonglin Capsules is 4.8586 mg / g.

[0195] Methodology investigation:

[0196] Specificity test: the third Shuangshi Tonglin Capsule extraction liquid, the third control solution and the third negative control solution are determined under the third high performance liquid chromatography detection condition, as shown in the following figure: Figure 19 A is the third Shuangshi Tonglin Capsule extraction liquid chromatogram, B is the third control solution chromatogram, and C is the third negative control solution chromatogram. Under the third high performance liquid chromatography detection condition, the third negative control solution has no interference at the peak of tanshinone II A, and the salvianolic acid B component peak in the third Shuangshi Tonglin Capsule extraction liquid is well separated from the adjacent peaks.

[0197] Linear test: an appropriate amount of the third control solution is diluted 1 times, 2 times, 4 times, 5 times, 8 times, 10 times, 16 times and 20 times with 80% methanol to prepare different concentrations of the third control solution. The sample is injected according to the third high performance liquid chromatography detection condition, and the peak area of each characteristic component is determined. The standard curve Y=10000000X-1000000, r=0.9995 is drawn with the control product concentration (μg / mL) as the abscissa and the peak area as the ordinate. The results show that the linear relationship of salvianolic acid B is good.

[0198] Precision test: the same third Shishitonglin capsule extract was taken, 6 injections were continuously injected according to the third high performance liquid chromatography detection condition, the chromatogram was recorded, the relative retention time and the relative peak area of the common peak at 278 nm wavelength were calculated with salvianolic acid B as the reference peak, and the results were shown in Table 9. The results showed that the RSD value of the relative retention time was less than 0.4%, and the RSD value of the relative peak area was less than 0.5%, indicating that the precision of the analytical instrument was good.

[0199] Table 9 Precision relative retention time and relative peak area test results

[0200]

[0201] Repeatability test: the same batch of Shishitonglin capsules were taken, 6 third Shishitonglin capsule extracts were prepared according to the above third Shishitonglin capsule extract preparation method, 6 times of determination were continuously determined according to the third high performance liquid chromatography detection condition, the chromatogram was recorded, the relative retention time and the relative peak area at 278 nm wavelength were calculated with salvianolic acid B as the reference peak, and the results were shown in Table 10. The results showed that the RSD value of the relative retention time was less than 0.36%, and the RSD value of the relative peak area was less than 4%, indicating that the repeatability of the third Shishitonglin capsule extract was good.

[0202] Table 10 Repeatability relative retention time and relative peak area test results

[0203]

[0204]

[0205] Stability test: the same third Shishitonglin capsule extract was taken, and the sample was tested at 0, 2, 4, 6, 8, 10, 12 and 24 h after the preparation of the third Shishitonglin capsule extract according to the third high performance liquid chromatography detection condition, the chromatogram was recorded, the relative retention time and the relative peak area of the common peak at 278 nm wavelength were calculated with salvianolic acid B as the reference peak, and the results were shown in Table 11. The results showed that the RSD value of each relative retention time was less than 1.0%, and the RSD value of the relative peak area was less than 0.7%, indicating that the test product had good stability within 24 h.

[0206] Table 11 Stability relative retention time test results

[0207]

[0208] Fingerprint establishment: 10 batches of Shuishitonglin capsules were taken, the shells were removed, and the contents were labeled as S1-S10. The third Shuishitonglin capsule extract was prepared according to the third Shuishitonglin capsule extraction method. The chromatogram was recorded by high performance liquid chromatography (HPLC) detection. The data was analyzed by "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)", with Salvianolic Acid B as the reference spectrum, time window width set to 0.1 min, median method, multi-point correction and full spectrum peak matching, Shuishitonglin capsule fingerprint and control fingerprint were generated, and similarity analysis was performed.

[0209] Mark peak matching: Salvianolic Acid B was used as Mark peak for fingerprint establishment. The control spectrum based on Mark peak matching ( Figure 20 ), and the fingerprint based on Mark peak matching ( Figure 21 ) were recorded.

[0210] Full spectrum peak matching: 10 batches of Shuishitonglin capsules were analyzed by fingerprint, and the full spectrum peak matching fingerprint was established. The full spectrum peak matching control spectrum ( Figure 22 ), full spectrum peak matching fingerprint ( Figure 23 ) and similarity (Table 12) were recorded.

[0211] Table 12 Similarity of full spectrum peak matching fingerprint of Shuishitonglin capsule

[0212]

[0213] 4. Content determination of geniposidic acid and verbascoside

[0214] Preparation of the fourth Shuishitonglin capsule extract: 1 g of Shuishitonglin capsule content was weighed into a conical flask with a stopper, 50 mL of 60% methanol was added, and the weight was determined. After ultrasonic treatment for 40 min, the weight was determined again, and the lost weight was made up with 60% methanol. Shake well, filter, take the filtrate, pass through a 0.22 μm microporous filter membrane, and reserve for use.

[0215] Preparation of the fourth control solution: an appropriate amount of geniposidic acid and verbascoside control was weighed, and 60% methanol was used to prepare a solution with a concentration of 0.6582 mg / g and 0.4048 mg / g, respectively. The fourth control solution includes geniposidic acid control solution and verbascoside control solution.

[0216] Preparation of the fourth negative control solution: Take 80.07 g of Cortex Phellodendri, 160.07 g of Radix Salviae Miltiorrhizae, 80.04 g of Rhizoma Alpiniae Officinarum, 80.09 g of Radix et Rhizoma Atractylodis, 160.15 g of Herba Patriniae, 160.09 g of Radix Alismatis, 80.20 g of Poria Cocos, 95.04 g of Talc, and 25.04 g of Prussian Blue, and prepare the negative sample without Trapa natans by the process for preparing Shuishi Tonglin Capsules. Then, according to the preparation method for the extract of Shuishi Tonglin Capsules, the fourth negative control solution is prepared by the same method.

[0217] The fourth HPLC detection condition: the chromatographic column is a C18 chromatographic column; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is selected from methanol, and the mobile phase B is selected from 0.5% acetic acid aqueous solution; the flow rate is 1.0 mL / min; the column temperature is 30°C; the detection wavelength is 254 nm; the injection volume is 10 μL; and the gradient elution mode is as follows:

[0218] 0-1 min, the mobile phase A is 5% by volume, and the mobile phase B is supplemented to 100% by volume;

[0219] 1-40 min, the mobile phase A is increased from 5% by volume to 60% by volume, and the mobile phase B is supplemented to 100% by volume;

[0220] 40-50 min, the mobile phase A is decreased from 60% by volume to 5% by volume, and the mobile phase B is supplemented to 100% by volume.

[0221] Results: the contents of geniposidic acid and verbascoside in Shuishi Tonglin Capsules are 1.48 mg / g and 0.75 mg / g, respectively.

[0222] Methodology investigation:

[0223] Specificity test: the fourth Shuishi Tonglin Capsule extract, the fourth control solution, and the fourth negative control solution are determined under the fourth HPLC detection condition, as shown in FIG. 1. Figure 24 A is the chromatogram of the fourth Shuishi Tonglin Capsule extract (No. 1 peak is the geniposidic acid component peak, and No. 2 peak is the verbascoside component peak), B is the geniposidic acid control solution, C is the verbascoside control solution, and D is the chromatogram of the fourth negative control solution. Under the fourth HPLC detection condition, the fourth negative control solution has no interference at the peaks of geniposidic acid and verbascoside, and the peaks of geniposidic acid and verbascoside in the fourth Shuishi Tonglin Capsule extract are well separated from adjacent peaks.

[0224] Linearity test: Take the fourth control solution, dilute it with 60% methanol to 1 times, 2 times, 4 times, 5 times, 8 times, 10 times, 16 times, 20 times, and 50 times, respectively, to prepare different concentrations of the fourth control solution. Inject the sample according to the fourth high performance liquid chromatography detection condition, and determine the peak area of each characteristic component. Take the control product concentration (μg / mL) as the abscissa and the peak area as the ordinate to draw the standard curve, as shown in Table 13 below. The results show that geniposidic acid and verbascoside have good linear relationship.

[0225] Table 13 Linear relationship of each component

[0226]

[0227] Precision test: Take the same fourth Shishi Tonglin Capsule extract, continuously inject 6 times according to the fourth high performance liquid chromatography detection condition, record the chromatogram, take geniposidic acid as the reference peak, and calculate the RSD values of the relative retention time and the relative peak area at 254 nm wavelength. The results are shown in Table 14. The results show that the RSD value of the relative retention time is less than 0.1%, and the RSD value of the relative peak area is less than 3.5%, indicating that the precision of the analysis instrument is good.

[0228] Table 14 Precision relative retention time and relative peak area test results

[0229]

[0230]

[0231] Repeatability test: Take the same batch of Shishi Tonglin Capsule, prepare 6 fourth Shishi Tonglin Capsule extracts according to the above fourth Shishi Tonglin Capsule extract preparation method, continuously determine 6 times according to the fourth high performance liquid chromatography detection condition, record the chromatogram, take geniposidic acid as the reference peak, and calculate the RSD values of the relative retention time and the relative peak area at 254 nm wavelength. The results are shown in Table 15. The results show that the RSD value of the relative retention time is less than 0.2%, and the RSD value of the relative peak area is less than 3.7%, indicating that the fourth Shishi Tonglin Capsule extract has good repeatability.

[0232] Table 15 Repeatability relative retention time and relative peak area test results

[0233]

[0234] Stability test: Take the same fourth Shishitonglin capsule extract, according to the fourth high performance liquid chromatography detection conditions, respectively, in the fourth Shishitonglin capsule extract preparation after 0, 2, 4, 6, 8, 10, 12, 24h sample test, record chromatogram, with geniposidic acid as reference peak, calculate the relative retention time and relative peak area of the total peak at 254nm wavelength, the results are shown in Table 16. The results show that the RSD value of each relative retention time is less than 0.1%, and the RSD value of relative peak area is less than 4.0%, indicating that the test product has good stability within 24h.

[0235] Table 16 Stability relative retention time test results

[0236]

[0237] Fingerprint establishment: Take 10 batches of Shishitonglin capsules, remove the shell and take the contents, mark as S1-S10, prepare 10 batches of fourth Shishitonglin capsule extract according to the fourth Shishitonglin capsule extract preparation method, and detect the sample according to the fourth high performance liquid chromatography detection condition, record the chromatogram. Using "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)", geniposidic acid was used as the reference spectrum, the time window width was set to 0.1min, the median method was used for multi-point correction and full spectrum peak matching, and the Shishitonglin capsule fingerprint and control fingerprint were generated, and the similarity analysis was carried out.

[0238] Mark peak matching: taking characteristic components and verbascoside as Mark peak to establish fingerprint, record Mark peak matching control spectrum ( Figure 25 ), Mark peak matching fingerprint ( Figure 26 ).

[0239] Full spectrum peak matching: 10 batches of Shishitonglin capsules were analyzed by fingerprint, and the full spectrum peak matching fingerprint was established, and the full spectrum peak matching control spectrum ( Figure 27 ), full spectrum peak matching fingerprint ( Figure 28 ) and similarity (Table 17) were recorded.

[0240] Table 17 Similarity of full spectrum peak matching fingerprint of Shishitonglin capsule

[0241]

[0242] Example 3

[0243] In the research process, the preparation method of Shishitonglin capsule extract and HPLC chromatography conditions in example 2 were also screened.

[0244] 1. Screening of berberine hydrochloride content determination conditions

[0245] 1.1 Different extraction solution dosage screening

[0246] Take 2 g of the contents of Shuangshi Tonglin Capsules, and add 15 mL, 20 mL, 30 mL, 45 mL, and 50 mL of methanol, respectively, for extraction. Ultrasonically extract for 60 min, and inject samples according to the first high-performance liquid chromatography detection condition to calculate the content of berberine hydrochloride.

[0247] Table 18 Detection results of different extraction solution dosages

[0248] Extraction solution dose Retention time Peak area Content mg / g Methanol 15ml 20.793 122.9987 1.511194906 Methanol 20ml 20.84 96.3232 1.588936442 Methanol 30ml 20.88 63.7854 1.603993932 Methanol 45ml 21.083 42.9537 1.657486825 Methanol 50ml 20.888 37.1742 1.610915043

[0249] As can be seen from Table 18, the content of berberine hydrochloride is the highest when the extraction solution dosage is 45 mL. The analysis reason is that berberine hydrochloride is not completely extracted when the extraction solution dosage is 15 mL, 20 mL, or 30 mL, and berberine hydrochloride is possibly diluted when the extraction solution dosage is 50 mL, resulting in a smaller content result. Therefore, 45 mL is selected as the extraction solution dosage.

[0250] 1.2 Different extraction solution screening

[0251] Take 2 g of the contents of Shuangshi Tonglin Capsules, and add 30 mL, 45 mL, and 50 mL of pure methanol, 1% hydrochloric acid methanol, 1.5% hydrochloric acid methanol, and 2% hydrochloric acid methanol, respectively, for ultrasonic extraction for 60 min. Inject samples according to the first high-performance liquid chromatography detection condition to calculate the content of berberine hydrochloride.

[0252] Table 19 Detection results of different extraction solutions

[0253] Extraction solution Retention time Peak area Content mg / g Methanol 30ml 20.88 63.7854 1.603993932 1% hydrochloric acid methanol 30ml 20.828 78.1152 1.94725008 1.5% hydrochloric acid methanol 30ml 20.806 80.2563 1.998538007 2.0% hydrochloric acid methanol 30ml 20.783 83.2047 2.069164005 Methanol 45ml 21.083 42.9537 1.657486825 1% hydrochloric acid methanol 45ml 21.08 56.1705 2.132379831 1.5% hydrochloric acid methanol 45ml 21.061 56.5029 2.144323299 2.0% hydrochloric acid methanol 45ml 21.053 54.9316 2.087864899 Methanol 50ml 20.888 37.1742 1.610915043 1% hydrochloric acid methanol 50ml 20.85 49.6743 2.109960875 1.5% hydrochloric acid methanol 50ml 20.815 49.2889 2.094574417 2.0% hydrochloric acid methanol 50ml 20.788 50.9326 2.160196423

[0254] As can be seen from Table 19, the above experimental results verify the conclusion that the content is the highest when the extraction solution dosage is 45 mL. At the same time, through the content investigation of adding acid, it is concluded that the content determination results of 1% hydrochloric acid methanol and 1.5% hydrochloric acid methanol are the most, and there is little difference. Considering cost saving and simple operation, 1% hydrochloric acid methanol is selected as the extraction solution.

[0255] 1.3 Sample amount screening

[0256] Take 1 g, 1.5 g, and 2 g of the contents of Shuangshi Tonglin Capsules, and add 1% hydrochloric acid methanol 45 mL, respectively, for ultrasonic extraction for 60 min. Inject samples according to the first high-performance liquid chromatography detection condition to calculate the content of berberine hydrochloride.

[0257] Table 20 Detection results of different sample amounts

[0258] Sample dose Retention time Peak area Content mg / g Shuangshi Tonglin capsule content 1g 21.035 27.6477 2.215053497 Shuangshi Tonglin capsule content 1.5g 20.951 45.3057 2.322662089 Shuangshi Tonglin capsule content 2g 21.08 56.1705 2.132379831

[0259] From Table 20, when the sampling amount of Shishitonglin Capsule content is 1 g, 1.5 g, or 2 g, the determination result of berberine hydrochloride content is very high. Considering that the sampling amount method is established or not is closely related to the sample addition recovery, i.e. accuracy experiment, three kinds of sampling amount sample addition recovery determination experiments are carried out to verify the optimal result.

[0260] 0.5 g, 0.75 g, or 1 g of Shishitonglin Capsule content is respectively taken, 0.5 times, 1 times, or 1.5 times of berberine hydrochloride is respectively added, 1% hydrochloric acid methanol 45 mL is added, ultrasonic extraction is carried out for 60 min, sample is injected according to the first high performance liquid chromatography detection condition, three groups of parallel experiments are respectively carried out, and the sample addition recovery rate is calculated.

[0261] Table 21: Different sampling amount addition recovery experiment

[0262] Different sample amounts Known content mg Added amount mg Measured content value mg Recovery rate (%) Content 0.5g, add 0.5 times hydrochloric acid berberine-1 0.8282 0.0043 0.8528 96.6123 Content 0.5g, add 0.5 times hydrochloric acid berberine-2 0.8241 0.0043 0.8528 96.1337 Content 0.5g, add 0.5 times hydrochloric acid berberine-3 0.7185 0.0043 0.8528 83.7429 Content 0.5g, add 1 times hydrochloric acid berberine-1 0.7639 0.0085 0.8528 88.5759 Content 0.5g, add 1 times hydrochloric acid berberine-2 0.7594 0.0085 0.8528 88.0391 Content 0.5g, add 1 times hydrochloric acid berberine-3 0.7594 0.0085 0.8528 88.0391 Content 0.5g, add 1.5 times hydrochloric acid berberine-1 0.7328 0.0128 0.8528 84.4309 Content 0.5g, add 1.5 times hydrochloric acid berberine-2 0.7630 0.0128 0.8528 87.9628 Content 0.5g, add 1.5 times hydrochloric acid berberine-3 0.6815 0.0128 0.8528 78.4141 RSD (%) 6.5 Content 0.75g, add 0.5 times hydrochloric acid berberine-1 1.3643 0.0056 1.1989 113.3265 Content 0.75g, add 0.5 times hydrochloric acid berberine-2 1.1155 0.0056 1.1989 92.5741 Content 0.75g, add 0.5 times hydrochloric acid berberine-3 1.6434 0.0056 1.1989 136.6038 Content 0.75g, add 1 times hydrochloric acid berberine-1 1.3757 0.0108 1.1989 113.8398 Content 0.55g, add 1 times hydrochloric acid berberine-2 1.1312 0.0108 1.1989 93.4530 Content 0.55g, add 1 times hydrochloric acid berberine-3 1.2411 0.0108 1.1989 102.6131 Content 0.75g, add 1.5 times hydrochloric acid berberine-1 0.8439 0.0163 1.1989 69.0239 Content 0.75g, add 1.5 times hydrochloric acid berberine-2 1.1136 0.0163 1.1989 91.5208 Content 0.75g, add 1.5 times hydrochloric acid berberine-3 1.1645 0.0163 1.1989 95.7608 RSD (%) 18.7 Content 1g, add 0.5 times hydrochloric acid berberine-1 1.1167 0.0056 1.0687 103.9706 Content 1g, add 0.5 times hydrochloric acid berberine-2 1.1229 0.0056 1.0687 104.5560 Content 1g, add 0.5 times hydrochloric acid berberine-3 1.1090 0.0056 1.0687 103.2555 Content 1g, add 1 times hydrochloric acid berberine-1 1.1310 0.0108 1.0687 104.8236 Content 1g, add 1 times hydrochloric acid berberine-2 1.1217 0.0108 1.0687 103.9497 Content 1g, add 1 times hydrochloric acid berberine-3 1.1021 0.0108 1.0687 102.1174 Content 1g, add 1.5 times hydrochloric acid berberine-1 1.1074 0.0160 1.0687 102.1210 Content 1g, add 1.5 times hydrochloric acid berberine-2 1.1198 0.0160 1.0687 103.2870 Content 1g, add 1.5 times hydrochloric acid berberine-3 1.1126 0.0160 1.0687 102.6085 RSD (%) 1.0

[0263] From Table 20 and Table 21, when the sampling amount of Shishitonglin Capsule content is 2 g, the sample addition recovery rate is between 95%-105%, which meets the relevant requirements. Therefore, 2 g is selected as the sampling amount of Shishitonglin Capsule content.

[0264] 1.4 Ultrasonic time screening

[0265] 2 g of Shishitonglin Capsule content is taken, 1% hydrochloric acid methanol 45 mL is added, ultrasonic extraction is carried out for 30 min, 45 min, or 60 min, sample is injected according to the first high performance liquid chromatography detection condition, and the content of berberine hydrochloride is calculated.

[0266] Table 22: Detection results of different ultrasonic times

[0267] Ultrasonic time Retention time Peak area Content mg / g Ultrasonic 30 min 21.073 57.3122 2.1734 Ultrasonic 45 min 21.051 58.9161 2.2310 Ultrasonic 60 min 21.17 58.7898 2.2265

[0268] From Table 22, when the ultrasonic time is 45 min, the berberine hydrochloride has been completely extracted, and the content is higher than that of 60 min. Therefore, 45 min is preferably selected as the ultrasonic extraction time.

[0269] 2, Tanshinone II A, Salvianolic Acid B, Geniposidic Acid and / or Verbascoside Content Determination Condition Screening

[0270] 2.1 Comparison of different extraction solvents

[0271] 1 g of Shishitonglin Capsule content is respectively taken, methanol, 80% methanol, 80% ethanol, and 60% methanol 50 mL is respectively added, the weight is determined, ultrasonic extraction is carried out for 40 min, the weight is determined again, the lost weight is made up with methanol, 80% methanol, 80% ethanol, and 60% methanol respectively, shaking is carried out, filtration is carried out, the filtrate is taken, 0.22 μm microporous filter membrane is passed, the extraction solution is respectively detected according to the second, third, and fourth high performance liquid chromatography detection conditions, and the detection results are shown in Table 23.

[0272] Table 23 detection results of different extraction solvents

[0273] Extraction solvent Tanshinone ⅡA content Salvianolic acid B content Geniposidic acid content Vervain glycoside content Methanol 1.44 mg / g 4.21 mg / g 1.27 mg / g 0.55 mg / g 80% methanol 1.234 mg / g 4.54 mg / g 1.32 mg / g 0.62 mg / g 80% ethanol 0.894 mg / g 4.65 mg / g 1.09 mg / g 0.34 mg / g 60% methanol 1.097 mg / g 4.87 mg / g 1.48 mg / g 0.75 mg / g

[0274] The above determination results show that when the content of tanshinone IIA is determined, the peak area of tanshinone IIA is the largest and the content of tanshinone IIA is the highest when methanol is selected as the extraction solvent; therefore, methanol is selected as the best extraction solvent; when the content of salvianolic acid B is determined, the peak area of salvianolic acid B is the largest and the content of salvianolic acid B is the highest when 80% methanol is selected as the extraction solvent; therefore, 80% methanol is selected as the best extraction solvent; when the content of geniposidic acid and / or verbascoside is determined, the peak area of geniposidic acid and / or verbascoside is the largest and the content of geniposidic acid and / or verbascoside is the highest when 60% methanol is selected as the extraction solvent; therefore, 60% methanol is selected as the best extraction solvent.

[0275] 2.2 Comparison of different extraction solvent dosages

[0276] 1g of the contents of Shuangshi Tonglin Capsules was respectively weighed into 12 conical flasks with stoppers, 10mL, 30mL, 50mL and 70mL of methanol solution was respectively added to the first group, 10mL, 30mL, 50mL and 70mL of 80% methanol solution was respectively added to the second group, 10mL, 30mL, 50mL and 70mL of 60% methanol solution was respectively added to the third group, the weight was determined, then ultrasonic extraction was performed for 40min, the weight was determined again, the lost weight was made up with methanol, methanol, 80% methanol and 60% methanol for the first, second and third groups respectively, and then the mixture was shaken and filtered, the filtrate was collected, 0.22μm microporous filter membrane was used for filtration, and the four groups of extraction solutions were detected according to the second, third and fourth high performance liquid chromatography detection conditions respectively, and the detection results are shown in Table 24.

[0277] Table 24 detection results of different extraction solvent dosages

[0278] Dose Tanshinone ⅡA content Salvianolic acid B content Geniposidic acid content Vervain glycoside content 10 mL 1.20 mg / g 4.1 mg / g 1.27 mg / g 0.63 mg / g 30 mL 1.41 mg / g 4.24 mg / g 1.31 mg / g 0.71 mg / g 50 mL 1.44 mg / g 4.54 mg / g 1.48 mg / g 0.75 mg / g 70 mL 1.37 mg / g 4.41 mg / g 1.45 mg / g 0.69 mg / g

[0279] The above determination results show that when the content of tanshinone IIA is determined, the peak area of tanshinone IIA is the largest and the content of tanshinone IIA is the highest when the extraction solvent dosage is 30mL; therefore, the best extraction solvent dosage is 30mL; when the content of salvianolic acid B is determined, the peak area of salvianolic acid B is the largest and the content of salvianolic acid B is the highest when the extraction solvent dosage is 50mL; therefore, the best extraction solvent dosage is 50mL; when the content of geniposidic acid and / or verbascoside is determined, the peak area of geniposidic acid and / or verbascoside is the largest and the content of geniposidic acid and / or verbascoside is the highest when the extraction solvent dosage is 50mL; therefore, the best extraction solvent dosage is 50mL.

[0280] 2.3 Comparison of different ultrasonic times

[0281] Tanshinone ⅡA content determination: 1 g of the content of Shuishitonglin Capsules was weighed into four conical flasks with stoppers, 30 mL of methanol was added, the weight was determined, and then the mixture was ultrasonically extracted for 20 min, 30 min, 40 min and 50 min, respectively. The weight was determined again, the lost weight was made up with methanol, the mixture was shaken and filtered, the filtrate was passed through a 0.22 μm microporous filter, and the extract was detected by the second high performance liquid chromatography detection condition.

[0282] Salvianolic acid B content determination: 1 g of the content of Shuishitonglin Capsules was weighed into four conical flasks with stoppers, 50 mL of 80% methanol was added, the weight was determined, and then the mixture was ultrasonically extracted for 20 min, 30 min, 40 min and 50 min, respectively. The weight was determined again, the lost weight was made up with 80% methanol, the mixture was shaken and filtered, the filtrate was passed through a 0.22 μm microporous filter, and the extract was detected by the third high performance liquid chromatography detection condition.

[0283] Geniposidic acid and verbascoside content determination: 1 g of the content of Shuishitonglin Capsules was weighed into four conical flasks with stoppers, 50 mL of 60% methanol was added, the weight was determined, and then the mixture was ultrasonically extracted for 40 min. The weight was determined again, the lost weight was made up with 60% methanol, the mixture was shaken and filtered, the filtrate was passed through a 0.22 μm microporous filter, and the extract was detected by the fourth high performance liquid chromatography detection condition.

[0284] The detection results are shown in Table 25.

[0285] Table 25 Detection results of different ultrasonic extraction times

[0286] Ultrasonic time Tanshinone ⅡA content Salvianolic acid B content Geniposidic acid content Vervain glycoside content 20 min 1.28 mg / g 2.98 mg / g 1.25 mg / g 0.41 mg / g 30 min 1.32 mg / g 3.21 mg / g 1.32 mg / g 0.53 mg / g 40 min 1.44 mg / g 3.54 mg / g 1.48 mg / g 0.75 mg / g 50 min 1.46 mg / g 3.50 mg / g 1.45 mg / g 0.86 mg / g

[0287] The above determination results show that when the ultrasonic extraction time is 20 min or 30 min, the peak area of each component to be detected is relatively small, and the content is low; when the ultrasonic extraction time is 40 min or 50 min, the peak area of each component to be detected is relatively high, and the content is high; in combination with the improvement of efficiency and the convenience of experimental operation, the content determination of tanshinone ⅡA, salvianolic acid B, geniposidic acid and / or verbascoside is 40 min as the best extraction time.

[0288] 2.4 Comparison of different mobile phases

[0289] In the content determination of berberine hydrochloride, tanshinone IIA, salvianolic acid B, geniposidic acid and / or verbascoside, the present application also investigates different systems of mobile phase, such as methanol-water solution, acetonitrile-water solution, methanol-0.05% acetic acid aqueous solution, acetonitrile-0.1% acetic acid aqueous solution, methanol-0.1% phosphoric acid aqueous solution, acetonitrile-0.05% phosphoric acid aqueous solution, acetonitrile-0.02 mol / L potassium dihydrogen phosphate. The theoretical plate number of the measured components of berberine hydrochloride, tanshinone IIA, salvianolic acid B, geniposidic acid and / or verbascoside in Shitonglin Capsules is used as an index, and the results are shown in Table 26.

[0290] Table 26 detection results of different mobile phases (theoretical plate number)

[0291] Mobile phase Berberine hydrochloride Tanshinone ⅡA Salvianolic acid B Geniposidic acid Vervain glycoside Methanol-water / 5632 5462 / 10012 Acetonitrile-water 3395 8463 8652 / 10254 Methanol-0.5% acetic acid water / 8465 27104 32335 49573 Acetonitrile-0.1% acetic acid water / 8562 29504 28212 56214 Methanol-0.1% phosphoric acid water 35682 87541 39070 / 24625 Acetonitrile-0.05% phosphoric acid water 43918 168700 38312 / 21493 Acetonitrile-0.02 mol / L potassium dihydrogen phosphate 58628 126541 37946 / 78325

[0292] The above determination results show that in the content determination of berberine hydrochloride, when methanol-water, methanol-0.5% acetic acid water and acetonitrile-0.1% acetic acid water are used as the mobile phase, no berberine hydrochloride component peak is detected, when acetonitrile-0.02 mol / L potassium dihydrogen phosphate is used as the mobile phase, the theoretical plate number is the highest and the separation degree is the best, therefore, acetonitrile-0.02 mol / L potassium dihydrogen phosphate is selected as the best mobile phase. In the content determination of tanshinone IIA, when acetonitrile-0.05% phosphoric acid water is used as the mobile phase, the theoretical plate number is the highest and the separation degree is the best, therefore, acetonitrile-0.05% phosphoric acid water is selected as the best mobile phase. In the content determination of salvianolic acid B, when methanol-0.1% phosphoric acid water is used as the mobile phase, the theoretical plate number is the highest and the separation degree is the best, therefore, methanol-0.1% phosphoric acid water is selected as the best mobile phase. In the content determination of geniposidic acid and verbascoside, when methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid water, acetonitrile-0.05% phosphoric acid water and acetonitrile-0.02 mol / L potassium dihydrogen phosphate are used as the mobile phase, no geniposidic acid is detected, when methanol-0.5% acetic acid water is used as the mobile phase, the theoretical plate number is the highest and the separation degree is the best, in the content determination of verbascoside, when methanol-0.5% acetic acid water and acetonitrile-0.1% acetic acid water are used as the mobile phase, the theoretical plate number is higher and the separation degree is better, and considering the economy and operability of the experiment, methanol-0.5% acetic acid water is selected as the best mobile phase.

[0293] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0294] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A quality detection method of Shuangshitonglin capsules, characterized in that, The quality detection method adopts high performance liquid chromatography to determine the content of geniposidic acid and / or verbascoside in Shangshitonglin Capsules; The content of Shangshitonglin Capsules is weighed into a conical flask with a stopper, methanol solution is added, the weight is determined, ultrasonic mixing is carried out for 40-45 min, the weight is determined again, methanol solution is added to make up for the weight loss, it is shaken uniformly, filtered, the filtrate is collected, filtered through a 0.22 μm microporous filter membrane, and the extract is obtained; The extract is detected by the fourth high performance liquid chromatography, and the content of geniposidic acid and / or verbascoside in the Shangshitonglin Capsules is determined based on the detection result; The fourth high performance liquid chromatography detection conditions are as follows: the chromatographic column is a C18 chromatographic column; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is selected from methanol, and the mobile phase B is selected from 0.5% acetic acid aqueous solution; the flow rate is 1.0 mL / min; the column temperature is 30 DEG C; the detection wavelength is 254 nm; and the injection amount is 10 μL; When the content of geniposidic acid and / or verbascoside is determined, the gradient elution mode used by the fourth high performance liquid chromatography detection is as follows: 0-1 min, the mobile phase A is 5% by volume, and the mobile phase B is supplemented to 100% by volume; 1-40 min, the mobile phase A is increased from 5% by volume to 60% by volume, and the mobile phase B is supplemented to 100% by volume; 40-50 min, the mobile phase A is decreased from 60% by volume to 5% by volume, and the mobile phase B is supplemented to 100% by volume.

2. The method for detecting the quality of Shishitong capsules according to claim 1, characterized in that, The quality detection method further includes identifying Fuling, Shichangpu, Qingdai, Guanhuangbai, Danshen and Cangzhu in Shangshitonglin Capsules by thin layer chromatography, including: The Shangshitonglin Capsules are shelled, the content is taken and extracted, and the extract is collected; A reference solution is prepared; The extract and the reference solution are identified by the first thin layer chromatography, and whether the Shangshitonglin Capsules contain Fuling is determined based on the detection result; and / or The extract and the reference solution are identified by the second thin layer chromatography, and whether the Shangshitonglin Capsules contain Shichangpu is determined based on the detection result; and / or The extract and the reference solution are identified by the third thin layer chromatography, and whether the Shangshitonglin Capsules contain Qingdai is determined based on the detection result; and / or The extract and the reference solution are identified by the fourth thin layer chromatography, and whether the Shangshitonglin Capsules contain Guanhuangbai is determined based on the detection result; and / or The extract and the reference solution are identified by the fifth thin layer chromatography, and whether the Shangshitonglin Capsules contain Danshen is determined based on the detection result; and / or The extract and the reference solution are identified by the sixth thin layer chromatography, and whether the Shangshitonglin Capsules contain Cangzhu is determined based on the detection result; When Fuling is identified, the content of Shangshitonglin Capsules is extracted as follows: 2 g of the content of Shangshitonglin Capsules is taken, 20 mL of ethanol is added, ultrasonic treatment is carried out for 30 min, and then filtration is carried out, the filtrate is evaporated to dryness, the residue is dissolved in 1 mL of methanol, and the extract is collected. When identifying Acori graminei Rhizoma and Atractylodis Rhizoma, the content of Shuishitonglin Capsules is extracted as follows: 2 g of the content of Shuishitonglin Capsules is finely ground, stirred with 15 mL of water, and left to stand. The supernatant is collected and extracted with n-hexane twice, 20 mL each time. The n-hexane solution is combined and evaporated on a water bath. The residue is dissolved in 1 mL of n-hexane, and the extract is collected. When identifying Indigo Naturalis, the content of Shuishitonglin Capsules is extracted as follows: 1 g of the content of Shuishitonglin Capsules is finely ground, stirred with 5 mL of chloroform, and ultrasonically treated for 15 minutes. The filtrate is collected as the extract. When identifying Phellodendri Chinensis Cortex, the content of Shuishitonglin Capsules is extracted as follows: 1 g of the content of Shuishitonglin Capsules is finely ground, placed in a 50 mL volumetric flask, and added with 45 mL of a mixture of hydrochloric acid and methanol (1:100 by volume). The mixture is ultrasonically treated for 30 minutes, diluted with methanol to the calibration mark, shaken, and filtered. The filtrate is collected as the extract. When identifying Salviae Miltiorrhizae Radix et Rhizoma, the content of Shuishitonglin Capsules is extracted as follows: 5 g of the content of Shuishitonglin Capsules is finely ground, stirred with 10 mL of diethyl ether, and filtered. The filtrate is evaporated, and the residue is dissolved in 0.5 mL of ethyl acetate. The extract is collected.

3. The quality detection method of Shuishitonglin Capsules according to claim 2, characterized in that, When identifying Acori graminei Rhizoma and Atractylodis Rhizoma, 2 g of Acori graminei Rhizoma and 0.1 g of Atractylodis Rhizoma are respectively used to replace the content of Shuishitonglin Capsules to prepare the reference solution. When identifying Acori graminei Rhizoma and Atractylodis Rhizoma, 0.5 g of Acori graminei Rhizoma and 0.2 g of Atractylodis Rhizoma are respectively added with 2 mL of n-hexane, ultrasonically treated for 15 minutes, and the supernatant is collected as the reference solution. When identifying Indigo Naturalis, 1 mg of indigo and indigotin is respectively dissolved in 1 mL of chloroform to prepare the reference solution. When identifying Salviae Miltiorrhizae Radix et Rhizoma, 2 mg of tanshinone II A is dissolved in 1 mL of ethyl acetate to prepare the reference solution.

4. The quality detection method of Shuishitonglin Capsules according to claim 2, characterized in that, The first thin layer chromatography identification is performed under the following conditions: thin layer plate: silica gel G thin layer plate; sample application amount: 5-10 μL; developing agent: toluene-ethyl acetate-formic acid (20:5:0.5 by volume). After development, the thin layer plate is taken out, dried, and observed under ultraviolet light at 365 nm. The second thin layer chromatography identification is performed under the following conditions: thin layer plate: silica gel G thin layer plate; sample application amount: 5 μL; developing agent: petroleum ether-ethyl acetate (20:1 by volume), development, taking out, drying, and spraying with 5% vanillin sulfuric acid solution. The third thin layer chromatography identification is performed under the following conditions: thin layer plate: silica gel G thin layer plate; sample application amount: 5 μL; developing agent: benzene-trichloromethane-acetone (5:4:1 by volume), development, taking out, and drying. The fourth thin layer chromatography identification is performed under the following conditions: thin layer plate: silica gel G thin layer plate; sample application amount: 2 μL; developing agent: ethyl acetate-butanone-formic acid-water (10:6:1:1 by volume), ammonia vapor saturated chromatography cylinder; development, taking out, and observation under ultraviolet light at 365 nm. The conditions of the fifth thin layer chromatography identification are as follows: thin layer plate: silica gel G thin layer plate; sample volume: 5 μL; developing agent: petroleum ether-ethyl acetate with a volume ratio of 8:2, development, removal, and air drying; The conditions of the sixth thin layer chromatography identification are as follows: thin layer plate: silica gel G thin layer plate; sample volume: 5 μL; developing agent: petroleum ether-ethyl acetate with a volume ratio of 20:1, development, removal, air drying, spraying with 10% sulfuric acid ethanol solution containing 5% p-dimethylaminobenzaldehyde, and heating until the spots are clearly colored; The petroleum ether is 60-90 ℃ petroleum ether.

5. The method for detecting the quality of Shangshi Tonglin capsules according to claim 4, characterized in that, The specific identification method of the fourth thin layer chromatography identification is as follows: 2 μL of the extract and the reference substance solution are respectively spotted on the same silica gel G thin layer plate, ethyl acetate-methyl ketone-formic acid-water with a volume ratio of 10:6:1:1 is used as the developing agent, the thin layer plate is placed in an ammonia vapor saturated chromatographic cylinder for development, removed, air dried, and observed under a UV lamp with a wavelength of 365 nm; or 2 μL of the extract and the reference substance solution are respectively spotted on the same silica gel G thin layer plate, ethyl acetate-methyl ketone-formic acid-water with a volume ratio of 10:6:1:1 is used as the developing agent, developed with a development distance of 12 cm, immediately placed in an ammonia vapor saturated chromatographic cylinder for fumigation for 2-3 minutes, and immediately observed under a UV lamp with a wavelength of 365 nm; or 2 μL of the extract and the reference substance solution are respectively spotted on the same silica gel G thin layer plate, the thin layer plate is placed in an ammonia vapor saturated chromatographic cylinder for saturation for 5-10 minutes, removed, immediately placed in a chromatographic cylinder containing the developing agent of ethyl acetate-methyl ketone-formic acid-water with a volume ratio of 10:6:1:1, developed, removed, and air dried under a UV lamp with a wavelength of 365 nm.

6. The method for detecting the quality of Shishitong capsules according to claim 1, wherein, The quality detection method further comprises determining the contents of berberine hydrochloride, tanshinone II A, and / or salvianolic acid B in Shishitonglin Capsules by high performance liquid chromatography; the extract is detected by the first high performance liquid chromatography, and the content of berberine hydrochloride in the Shishitonglin Capsules is determined based on the detection result; and / or the extract is detected by the second high performance liquid chromatography, and the content of tanshinone II A in the Shishitonglin Capsules is determined based on the detection result; and / or the extract is detected by the third high performance liquid chromatography, and the content of salvianolic acid B in the Shishitonglin Capsules is determined based on the detection result.

7. The quality detection method of the Shishitonglin Capsules according to claim 6, wherein the conditions of the first high performance liquid chromatography detection are as follows: the chromatographic column is a C18 chromatographic column; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is selected from acetonitrile, and the mobile phase B is selected from 0.02 mol / L potassium dihydrogen phosphate solution; the flow rate is 1.0 mL / min; the column temperature is 30 ℃; the detection wavelength is 345 nm; and the injection volume is 10 μL; the conditions of the second high performance liquid chromatography detection are as follows: the chromatographic column is a C18 chromatographic column; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is selected from acetonitrile, and the mobile phase B is selected from 0.05% phosphoric acid aqueous solution; the flow rate is 1.0 mL / min; the column temperature is 30 ℃; the detection wavelength is 270 nm; and the injection volume is 10 μL; The third high performance liquid chromatography detection condition is: the chromatographic column is C18 chromatographic column; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is selected from methanol, the mobile phase B is selected from 0.1% phosphoric acid aqueous solution; the flow rate is 1.0 mL / min; the column temperature is 35 DEG C; the detection wavelength is 278 nm; the injection amount is 10 mu L.

8. The quality detection method of Shishitonglin capsules according to claim 7, wherein, when determining the content of berberine hydrochloride, the methanol solution is a methanol solution containing 1% hydrochloric acid by volume; When determining the content of salvianolic acid B, the methanol solution is 80% methanol solution by volume; When determining the content of geniposidic acid and / or verbascoside, the methanol solution is 60% methanol solution by volume.

9. The quality detection method of Shishitonglin capsules according to claim 8, wherein, when determining the content of berberine hydrochloride, the amount of 1% hydrochloric acid methanol solution added is 45 mL based on 2 g of Shishitonglin capsule content; When determining the content of tanshinone IIA, the amount of methanol solution added is 30 mL based on 1 g of Shishitonglin capsule content; When determining the content of salvianolic acid B, the amount of 80% methanol solution added is 50 mL based on 0.5 g of Shishitonglin capsule content, When determining the content of geniposidic acid and / or verbascoside, the amount of 60% methanol solution added is 50 mL based on 1 g of Shishitonglin capsule content.

10. The quality detection method of Shishitonglin capsules according to claim 7, wherein, when determining the content of berberine hydrochloride, the first high performance liquid chromatography detection adopts isocratic elution; the potassium dihydrogen phosphate solution is adjusted to pH 4 with phosphoric acid; the volume ratio of the mobile phase A to the mobile phase B is 25:75; When determining the content of tanshinone IIA, the second high performance liquid chromatography detection adopts gradient elution mode as follows: 0-6 min, the mobile phase A is 61% by volume, and the mobile phase B is supplemented to 100% by volume; 6-20 min, the mobile phase A is increased from 61% to 90% by volume, and the mobile phase B is supplemented to 100% by volume; 20-20.5 min, the mobile phase A is decreased from 90% to 61% by volume, and the mobile phase B is supplemented to 100% by volume; 20.5-25 min, the mobile phase A is 61% by volume, and the mobile phase B is supplemented to 100% by volume; When determining the content of salvianolic acid B, the third high performance liquid chromatography detection adopts gradient elution mode as follows: 0-40 min, the mobile phase A is increased from 32% to 50% by volume, and the mobile phase B is supplemented to 100% by volume; 40-45 min, the mobile phase A is decreased from 50% to 32% by volume, and the mobile phase B is supplemented to 100% by volume. ​ ​ ​

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