Detection method of traditional Chinese medicine preparation for liver and gallbladder damp-heat accumulation

Abstract

The invention discloses a detection method of a traditional Chinese medicine preparation for liver and gallbladder damp-heat accumulation, the traditional Chinese medicine preparation is prepared from 200 parts by weight of glechoma longituba, 200 parts by weight of dock root, 200 parts by weight of saxifraga stolonifera, 200 parts by weight of sabia parviflora, 200 parts by weight of phoenix-tail fern, 200 parts by weight of scutellaria baicalensis, 200 parts by weight of golden cypress and 200 parts by weight of andrographis paniculata. The detection method is characterized by comprising the steps of thin-layer identification and content determination of components, wherein thin-layer chromatography identification is carried out on glechoma longituba, rumex madaio and andrographis paniculata, and content determination is carried out by using a high performance liquid chromatography to determine the content of effective components of glechoma longituba, rumex madaio and scutellaria baicalensis in the preparation. The detection method is simple, stable and reliable, and effectively ensures the product quality.

Description

TECHNICAL FIELD

[0001] The present application relates to traditional Chinese medicine preparation detection technology, in particular to a detection method for traditional Chinese medicine preparation for liver and gallbladder damp-heat accumulation. BACKGROUND

[0002] Liver and gallbladder damp-heat accumulation belongs to the category of hypochondriac pain and jaundice in traditional Chinese medicine theory, and the pathogenesis is that damp-heat evil accumulates in liver and gallbladder, obstructing the movement of qi, and further causing cholecystitis, cholelithiasis and other diseases. The classics such as Treatise on Cold Damage Diseases point out that the treatment should be centered on clearing heat and dampness, soothing liver and promoting bile.

[0003] The prevalence of cholelithiasis in Guizhou region is significantly higher than the national average due to the humid and hot climate and high-fat dietary habits. The report on epidemiology of digestive system diseases in Guizhou Province shows that the prevalence of cholelithiasis in the region is about 18%, and the recurrence rate of cholangitis is as high as 35%, which has given rise to the demand for local medicine. And most of the patients with postcholecystectomy syndrome are accompanied by damp-heat constitution (such as bitter mouth, yellow and greasy tongue fur), and there are problems such as high recurrence rate and insufficient rehabilitation intervention in western medicine treatment. And the existing traditional Chinese medicines lack the characteristics of ethnic medicines and evidence-based evidence for postoperative syndrome. Relying on the theory of Miao medicine and modern science and technology, Daliankang forms a whole course of intervention scheme of "clearing heat and dampness + promoting bile and dissolving stones + postoperative repair" through characteristic prescription, precise quality control and clinical research, and provides a differentiated treatment option for liver and gallbladder damp-heat type diseases.

[0004] Daliankang Capsule (State Food and Drug Administration Drug Code Z20026124) is composed of eight kinds of medicines such as Lianqiancao and Tuodahuang, and has the effects of clearing heat and dampness, removing stones and relieving pain, but its quality control relies on traditional methods, and the prescription composition is complex, which is difficult to accurately identify multiple components and quantitative analysis. Therefore, it is of great significance to establish thin layer chromatography identification and high performance liquid chromatography content determination method for key medicinal materials such as Lianqiancao and Huangqin to ensure the efficacy of the preparation. SUMMARY

[0005] The purpose of the present application is to solve the above problems, and provide a detection method for traditional Chinese medicine preparation for liver and gallbladder damp-heat accumulation, which has the characteristics of simplicity, convenience, stability and reliability, and can effectively detect multiple effective components such as Lianqiancao, Tuodahuang, Chuanxinlian, Huangqin and the like, and ensure product quality.

[0006] The above technical problems of the present application are mainly solved by the following technical scheme: a detection method for traditional Chinese medicine preparation for liver and gallbladder damp-heat accumulation, the traditional Chinese medicine preparation is prepared by compounding Lianqiancao, Tuodahuang, Hu'ercao, Xiaohuaqingfengteng, Fengweicao, Huangqin, Huangbai and Chuanxinlian in a ratio of 200 parts by weight; the detection method comprises thin layer identification and content determination; wherein Lianqiancao, Tuodahuang and Chuanxinlian are identified by thin layer chromatography, and the content of effective components in the preparation is determined by high performance liquid chromatography.

[0007] The identification method of the preceding Chinese medicine preparation for liver and gallbladder damp-heat accumulation is as follows: take 2.0 g of the content of the product, add 20 mL of methanol, and ultrasonically treat for 30 minutes; filter and concentrate to 4 mL as a test sample; take 0.2 g of the control medicinal material of Glechoma longituda L., and prepare in the same way as the test sample to obtain a control medicinal material solution; and take ursolic acid control substance, add methanol to prepare a solution containing 1.0 mg of ursolic acid per 1 mL as a control substance solution.

[0008] According to the thin layer chromatography test, 2-8 μL of the test sample solution, 2 μL of the control medicinal material solution, and 1 μL of the control substance solution are spotted on the same silica gel thin layer plate, and developed upward with cyclohexane: ethyl acetate: glacial acetic acid (10:3:0.5) as the developing agent. After being taken out, dried, and sprayed with 10% sulfuric acid ethanol solution, it is observed under visible light; the test sample chromatogram shows the same color purple spots at the positions corresponding to the control medicinal material and control substance chromatogram.

[0009] The identification method of the preceding Chinese medicine preparation for liver and gallbladder damp-heat accumulation is as follows: take 0.25 g of the content of the product, add 10 mL of methanol, and reflux in a water bath for 30 minutes; filter and concentrate to 4 mL to obtain a test sample solution; take 0.2 g of the control medicinal material of Rumex nepalensis Spreng., and prepare in the same way as the test sample to obtain a control medicinal material solution; and take physcion control substance, add methanol to prepare a solution containing 1 mg per 1 mL as a control substance solution.

[0010] According to the thin layer chromatography test, 2-8 μL of the test sample solution, 2-4 μL of the control medicinal material solution and the control substance solution are spotted on the same silica gel thin layer plate, and developed upward with petroleum ether: freshly prepared methyl formate: formic acid (15:5:1) with a boiling range of 60-90°C as the developing agent. After being taken out, dried, and placed under a 254 nm ultraviolet lamp, it is observed; the test sample chromatogram shows the same color yellow fluorescent spots at the positions corresponding to the control substance and control medicinal material chromatogram.

[0011] The identification method of the preceding Chinese medicine preparation for liver and gallbladder damp-heat accumulation is as follows: take 4.0 g of the content of the product, add 30 mL of ethanol, and ultrasonically treat for 20 minutes; filter and concentrate to 4 mL as a test sample solution; take 0.2 g of the control medicinal material of Andrographis paniculata (Burm.f.) Nees, and prepare in the same way as the test sample to obtain a control medicinal material solution; and take dehydrated andrographolide control substance, add ethanol to prepare a solution containing 1 mg per 1 mL as a control substance solution. Chromatography, 4-10 μL of the test solution, 5 μL of the drug solution and 1 μL of the reference solution are applied to the same silica gel thin layer plate, and developed with petroleum ether-ethyl acetate-ethanol (4:2:1) as the developing agent at 60-90 ℃. After being removed and dried, the plate is examined under UV light at 254 nm. The test solution should show the same color fluorescent spots as the reference solution and the drug solution at the corresponding positions.

[0012] In the foregoing detection method of the traditional Chinese medicine preparation for liver and gallbladder damp-heat accumulation, the content determination method of Herba Reineckiae and Radix et Rhizoma Rumicis: is determined by high performance liquid chromatography.

[0013] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel is used as the filler, acetonitrile is used as the mobile phase (A) and 0.1% phosphoric acid is used as the mobile phase (B) for gradient elution, the column temperature is 35 ℃, the flow rate is 1.0 mL / min, and the detection wavelength is 0-25 min (330 nm) and 25-60 min (254 nm); the theoretical plate number calculated based on the rosmarinic acid and chrysophanol peaks should both be not less than 3000.

[0014] Preparation of the reference solution: appropriate amounts of the chrysophanol reference and the rosmarinic acid reference are precisely weighed and dissolved in methanol to prepare a mixed solution containing 0.5 mg of chrysophanol and 0.4 mg of rosmarinic acid per 1 mL.

[0015] Preparation of the test solution: the content of the product is finely ground, 1.0 g is precisely weighed, placed in a conical flask with a stopper, 25 mL of methanol is precisely added, the weight is determined, heated to reflux for 1 h, allowed to cool, the weight is determined again, the lost weight is made up with methanol, shaken well, filtered, and the filtrate is obtained.

[0016] Determination method: 10 μL of the reference solution and the test solution are precisely taken respectively, injected into the liquid chromatograph, and determined.

[0017] Each tablet of the product contains not less than 86.0 μg of rosmarinic acid (C 18 H 16 O8) and not less than 0.5 mg of chrysophanol (C 15 H 10 O4). The total weight of each tablet of the product is 0.5 g.

[0018] In the foregoing detection method of the traditional Chinese medicine preparation for liver and gallbladder damp-heat accumulation, the gradient elution is as follows: 0-20 minutes, 25% A-75% B; 20-25 minutes, 75% A-25% B; and 25-60 minutes, 80% A-20% B.

[0019] In the foregoing detection method of the traditional Chinese medicine preparation for liver and gallbladder damp-heat accumulation, the content determination method of baicalin in Scutellaria is: Determination by high performance liquid chromatography.

[0020] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler; acetonitrile-0.1% phosphoric acid (20:80) as the mobile phase; isocratic elution; column temperature 35℃, flow rate 1.0 mL / min, baicalin detection wavelength 280 nm; the theoretical plate number of baicalin peak should not be less than 3000.

[0021] Preparation of the reference solution: take the baicalin reference substance, accurately weigh, add methanol to prepare a solution containing 0.4 mg of baicalin per 1 mL.

[0022] Preparation of the test solution: take the content of the product, grind finely, accurately weigh 0.2 g, put it in a conical flask with a plug, accurately add 25 mL of 75% methanol, weigh, ultrasonic for 30 minutes, cool, weigh again, make up the weight loss with 75% methanol, shake well, filter, take the filtrate, and get it.

[0023] Determination method: accurately take 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and get it.

[0024] The above traditional Chinese medicine preparation for treating liver and gallbladder damp-heat accumulation is by 200 parts of Reineckia hex fan, 200 parts of Rheum palmatum, 200 parts of Saxifraga stolonifera, 200 parts of Sabia parviflora, 200 parts of Sarcopyramis humilis, 200 parts of Scutellaria baicalensis, 200 parts of Phellodendron amurense, and 200 parts of Andrographis paniculata, wherein the Rheum palmatum and Scutellaria baicalensis are crushed into fine powder, the rest Reineckia hex fan and the like are decocted with water twice, each time for 2 hours, the decoction is filtered, the filtrates are combined, concentrated to a relative density of 1.20-1.25 (80℃), the above fine powder and appropriate starch are added, granulated, dried, and loaded into capsules, and the traditional Chinese medicine preparation is obtained.

[0025] Compared with the prior art, the beneficial effects of the present application are: the thin layer identification of Rheum palmatum and Andrographis paniculata is newly added, the thin layer identification method of Reineckia hex fan is optimized, the accuracy is high and there is no interference; meanwhile, the content determination method of Reineckia hex fan, Rheum palmatum and Scutellaria baicalensis is newly added, the operation is simple, the cost is low, the test time is short, the separation effect is good, the recovery rate is high, the stability is high, the specificity is strong, the repeatability is good, the precision is high; the product can be effectively detected whether it is qualified, the quality is good or bad, the pharmaceutical efficacy consistency and quality stability are ensured, and the clinical medication safety and reliability are ensured. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The figure is the thin layer identification effect diagram of Reineckia hex fan with different sample application amounts.

[0027] Figure 2 The figure is the thin layer identification effect diagram of Reineckia hex fan with different developing agents and specificity.

[0028] Figure 3Different manufacturers of GF254 silica gel plate of Galium aparine L. TLC identification effect diagram.

[0029] Figure 4 Different temperature and humidity of Galium aparine L. TLC development effect diagram.

[0030] Figure 5 Different saturation time of Galium aparine L. TLC development effect diagram.

[0031] The above Figures 2 to 5 Among them, 1, 2, 3, 4 respectively represent: 1, Danyankang sample (2 μL) (batch number A20220804); 2, Galium aparine L. control medicinal materials (2 μL); 3, ursolic acid (1 μL); 4, negative control (2 μL) (batch number 20230830).

[0032] Figure 6 Different sample application amounts of Rumex madaio Yokoyama TLC development effect diagram.

[0033] Figure 7 Different developing agents and specificities of Rumex madaio Yokoyama TLC development effect diagram.

[0034] Figure 8 Different manufacturers of GF254 silica gel plate of Rumex madaio Yokoyama TLC development effect diagram.

[0035] Figure 9 Different temperature and humidity of Rumex madaio Yokoyama TLC identification effect diagram.

[0036] Figure 10 Different saturation time of Rumex madaio Yokoyama TLC identification effect diagram.

[0037] The above Figures 6 to 10 Among them, 1 to 4 respectively represent: 1, Danyankang sample (2 μl) (batch number A20220804); 2, Rumex madaio Yokoyama control medicinal materials (2 μl); 3, physcion (2 μl); 4, negative control (2 μl) (batch number 20230830).

[0038] Figure 11 Different sample application amounts of Andrographis paniculata (Burm. F.) Nees TLC identification effect diagram.

[0039] Figure 12 Different developing agents and specificities of Andrographis paniculata (Burm. F.) Nees TLC development effect diagram.

[0040] Figure 13 Different manufacturers of HSGF254 silica gel plate of Andrographis paniculata (Burm. F.) Nees TLC development effect diagram.

[0041] Figure 14 Different temperature and humidity of Andrographis paniculata (Burm. F.) Nees TLC development effect diagram.

[0042] Figure 15is the thin layer development effect diagram of Andrographis with different saturation times.

[0043] The above Figures 11 to 15 Among them, 1-4 respectively represent: 1, Danyan Kang sample (4 μl) (batch number A20220804); 2, Andrographis control medicinal materials (5 μl); 3, Dehydrated andrographolide (1 μl); 4, negative control (4 μl) (batch number 20230830).

[0044] Figure 16 is the full wavelength scanning diagram of rosmarinic acid and chrysophanol detection.

[0045] Figure 17 is the HPLC diagram of the specificity test of baicalin content determination.

[0046] Figure 18 is the HPLC diagram of the specificity test of chrysophanol content determination.

[0047] Figure 19 is the full wavelength ultraviolet scanning diagram of baicalin.

[0048] Figure 20 is the HPLC diagram of the specificity test of baicalin content determination. DETAILED DESCRIPTION

[0049] The technical solutions of the present application will be further specifically described below by examples, and in combination with the accompanying drawings.

[0050] As Figure 1 shown, the detection method of the traditional Chinese medicine preparation for liver and gallbladder dampness and heat accumulation of the present embodiment is prepared by compounding Rubia cordifolia, Rheum palmatum, Saxifraga stolonifera, Sabia parvifolia, Geranium carolinianum, Scutellaria baicalensis, Phellodendron amurense, Andrographis panniculata in a ratio of 200 parts by weight, and the above eight raw materials are prepared; the detection method comprises thin layer identification and content determination; wherein Rubia cordifolia, Rheum palmatum and Andrographis panniculata are identified by thin layer chromatography, and the content of the effective components of Rubia cordifolia, Rheum palmatum and Scutellaria baicalensis in the preparation is determined by high performance liquid chromatography.

[0051] The identification method of Rubia cordifolia is as follows: 2.0 g of the content of the product is added to 20 mL of methanol, and ultrasonic treatment is performed for 30 minutes. Filter and concentrate to 4 mL as the test sample. Another 0.2 g of Rubia cordifolia control medicinal material is prepared in the same way as the control medicinal material solution. Then, 1.0 mg of ursolic acid is precisely weighed and dissolved in methanol to prepare a solution containing 1.0 mg of ursolic acid per 1 mL as the control solution.

[0052] Test by thin-layer chromatography (General Rule 0502). Take 2-8 μL of the test solution, 2 μL of the control drug solution and 1 μL of the control solution, and apply them separately to the same GF254 silica gel thin-layer plate. Develop with cyclohexane: ethyl acetate: glacial acetic acid = 10:3:0.5 as the developing agent. Take out, dry, spray with 10% sulfuric acid ethanol solution, and examine under visible light (daylight). In the test sample chromatogram, the same color purple spots appear at the corresponding positions of the control drug and control substance chromatograms. See Figures 1 to 5 .

[0053] II. The identification method of Radix Rumicis Cripi is as follows: take 0.25 g of the content of the product, add 10 mL of methanol, and reflux in water bath for 30 minutes. Filter, and concentrate to 4 mL to obtain the test solution. Take 0.2 g of the control drug of Radix Rumicis Cripi, and prepare in the same way to obtain the control drug solution. Take emodin methyl ether control substance, and add methanol to prepare a solution containing 1 mg per 1 mL as the control substance solution.

[0054] Test by thin-layer chromatography (General Rule 0502). Take 2-8 μL of the test solution, 2-4 μL of the control drug solution and 2-4 μL of the control substance solution, and apply them separately to the same GF254 silica gel thin-layer plate. Develop with petroleum ether: freshly prepared methyl formate: formic acid = 15:5:1 as the developing agent. Take out, dry, and examine under ultraviolet lamp (366 nm). In the test sample chromatogram, the same color yellow fluorescent spots appear at the corresponding positions of the control substance and control drug chromatograms. See Figures 6 to 10 .

[0055] III. The identification method of Andrographis Herba is as follows: take 4.0 g of the content of the product, add 30 mL of ethanol, and ultrasonicate for 20 minutes. Filter, and concentrate to 4 mL to obtain the test solution. Take 0.2 g of the control drug of Andrographis Herba, and prepare in the same way to obtain the control drug solution. Precisely weigh dehydroandrographolide lactone control substance, add ethanol to prepare a solution containing 1 mg per 1 mL as the control substance solution.

[0056] Test by thin-layer chromatography (General Rule 0502). Take 4-10 μL of the test solution, 5 μL of the control drug solution and 1 μL of the control substance solution, and apply them separately to the same HSGF254 silica gel thin-layer plate. Develop with petroleum ether: ethyl acetate: ethanol = 4:2:1 as the developing agent. Take out, dry, and examine under 254 nm ultraviolet lamp. In the test sample chromatogram, the same color fluorescent spots appear at the corresponding positions of the control substance and control drug chromatograms. See Figures 11 to 15 .

[0057] IV. The content determination method of Herba Galium and Radix Rumicis Cripi is as follows: determined by high performance liquid chromatography.

[0058] Determination of rosmarinic acid in Herba Rubi cordifoli and chrysophanol in Radix et Rhizoma Rumicis: refer to Figures 16 to 18 .

[0059] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler, acetonitrile as the mobile phase (A), 0.1% phosphoric acid as the mobile phase (B). Gradient elution 0-20 min, 25% A-75% B; 20-25 min, 75% A-25% B; 25-60 min, 80% A-20% B. Column temperature 35°C, flow rate 1.0 mL / min, detection wavelength 0-25 min (330 nm), 25-60 min (254 nm); the theoretical plate number should not be less than 3000 calculated by rosmarinic acid and chrysophanol peaks.

[0060] Preparation of reference solution: take chrysophanol reference substance and rosmarinic acid reference substance, accurately weigh, add methanol to prepare a mixed solution containing 0.5 mg of chrysophanol and 0.4 mg of rosmarinic acid per 1 mL, and obtain.

[0061] Preparation of test solution: take the contents of the product, grind finely, accurately weigh 1.0 g, place in a conical flask with a stopper, accurately add 25 mL of methanol, weigh, heat to reflux for 1 h, cool, re-weigh, make up the weight loss with methanol, shake well, filter, and take the filtrate, and obtain.

[0062] Determination method: accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

[0063] Each tablet of the product contains not less than 86.0 μg of rosmarinic acid (C 18 H 16 O8) and not less than 0.5 mg of chrysophanol (C 15 H 10 O4). The total weight of each tablet of the product is 0.5 g.

[0064] Five, the content determination method of baicalin in Scutellaria is determined by high performance liquid chromatography.

[0065] Determination of baicalin in Scutellaria: refer to Figure 19 , Figure 20 .

[0066] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler, acetonitrile-0.1% phosphoric acid (20:80), isocratic elution, column temperature 35°C, flow rate 1.0 mL / min, baicalin detection wavelength 280 nm, the theoretical plate number should not be less than 3000 calculated by baicalin peak.

[0067] Preparation of reference solution: accurately weigh a certain amount of baicalin reference substance, add methanol to prepare a solution containing 0.4 mg of baicalin per 1 mL.

[0068] Preparation of test solution: Take the contents of the product, finely grind, accurately weigh 0.2 g, add 25 mL of 75% methanol into a conical flask with a stopper, weigh the weight, ultrasonic for 30 minutes, cool, re-weigh, make up the weight loss with 75% methanol, shake well, filter, take the filtrate, and obtain.

[0069] Determination method: accurately pipette 10 μL of the control solution and the test solution respectively, inject into the liquid chromatograph, determine, and obtain.

[0070] Six, the preparation method of the pharmaceutical preparation in this embodiment is as follows: According to weight parts, take 200 parts of Herba Galium, 200 parts of Radix Rumicis, 200 parts of Herba Saxifragae, 200 parts of Herba Sabiaeflorae, 200 parts of Herba Sedi, 200 parts of Radix Scrophulariae, 200 parts of Cortex Phellodendri, and 200 parts of Herba Andrographitis. Grind Radix Rumicis and Radix Scrophulariae into fine powder, and add the rest of Herba Galium and six other ingredients to water to decoct twice, 2 hours each time. Filter the decoction, combine the filtrate, concentrate to relative density of 1.20-1.25 (80°C), add the fine powder and appropriate amount of starch, granulate, dry, and fill into capsules, and obtain.

[0071] In order to ensure that the detection method of this embodiment is scientific, reasonable and feasible, the detection method of this embodiment is further studied and investigated.

[0072] (I) Identification of Herba Galium Herba Galium is the dry aboveground part of Herba Galium verum L. Glechoma longituba (Nakai) Kupr. It is recorded in the 2020 edition of Chinese Pharmacopoeia (Volume I). In this experiment, Herba Galium was selected as the control drug, and ursolic acid was selected as the control. The characteristic components of Herba Galium in Danyan Kang were extracted by ultrasonic extraction method, and the thin layer identification of Herba Galium was carried out. The influence of specificity, different thin layer plates, low temperature, high humidity, low humidity and other durability on the thin layer chromatography of Herba Galium in the preparation was investigated. The test results show that: using Herba Galium as the control drug, ursolic acid as the control, cyclohexane: ethyl acetate: glacial acetic acid (10:3:0.5) as the developing system, the thin layer identification of Herba Galium has obvious characteristics and strong specificity, which can be used as the thin layer identification method of Herba Galium in Danyan Kang capsules, so it is included in the detection project of this invention.

[0073] (1) Instruments, reagents and samples Instrument selection: KS-7200DE type liquid crystal ultrasonic cleaner

Kunshan XXX ultrasonic instrument Co., Ltd.

Shanghai XXX experimental equipment Co., Ltd.

Li X enterprise Co., Ltd.

Wuyi XX electronic Co., Ltd.

XXX-XXX instrument (Shanghai) Co., Ltd.

[0074] Reagents: ethyl acetate (AR)

Xilong XX Co., Ltd., batch number: 231016

Xilong XX Co., Ltd., batch number: 230510

Xilong XX Co., Ltd., batch number: 20220105

Xilong XX Co., Ltd., batch number: 20201124

Chengdu XX Chemical Co., Ltd., batch number: 2023061203

Yantai XX silica gel development Co., Ltd., batch number: 20241112, 200×50 mm; 20240122, 100×200 mm

Qingdao XX Chemical Co., Ltd., batch number: 20190318, 100×200 mm; Qingdao XX silica gel dryer Co., Ltd., batch number: 20240401, 100×200 mm

Chengdu XXX Biotechnology Co., Ltd., batch number: C30H4803

[0075] Garden balsam control drug material (batch number: Z8E6NGF2) sample: test product batch number (A20220804), negative control product batch number (20230830) were provided by the applicant.

[0076] Garden balsam control drug material sample: test product, negative control product.

[0077] (2) Preparation of test product solution: take the content of the product 2.0 g, add methanol 20 mL, ultrasonic treatment for 30 minutes. Filter and concentrate to about 4 mL as test product solution.

[0078] (3) Preparation of negative sample: according to the prescription proportion and preparation process, negative sample without garden balsam medicinal material was prepared, and test product solution was prepared according to the same method, as negative sample solution.

[0079] (4) Preparation of control drug material solution: take garden balsam control drug material 0.2 g, prepare according to the same method of test product solution, as control drug material solution.

[0080] (5) Preparation of control product solution: accurately weigh ursolic acid control product, add methanol to prepare a solution containing 1.0 mg ursolic acid per 1 mL, as control product solution.

[0081] (6) Developing system: cyclohexane: ethyl acetate: glacial acetic acid (10:3:0.5).

[0082] (7) Chromogenic agent: 10% sulfuric acid ethanol solution.

[0083] (8) Methodology verification: Spotting amount investigation: the above-mentioned solution of ursolic acid reference substance, Dianyankang sample, and Herba Galiums sinense reference material were respectively taken in different spotting amounts, and the spotting amount investigation experiment was carried out according to the above method. The results are shown in Figure 1 .

[0084] As can be seen from Figure 1 , each spotting amount is clear and obvious, and 2-8 μl of the test sample solution, 2 μl of the reference material solution, and 1 μl of the reference substance solution are selected.

[0085] Developing agent and specificity investigation: Dianyankang sample (2 μL), Herba Galiums sinense reference material (2 μL), ursolic acid reference substance (1 μL), and negative control (2 μL) were taken, and the developing conditions of the developing agent cyclohexane: ethyl acetate: glacial acetic acid (10:3:0.5), cyclohexane: ethyl acetate: formic acid (9:2:0.2), n-butanol: glacial acetic acid (3:1:0.5), and cyclohexane: ethyl acetate (9:4) were respectively investigated according to the above method. The results are shown in Figure 2 .

[0086] As can be seen from Figure 2 , the spots are clear when the developing agent is cyclohexane: ethyl acetate: glacial acetic acid (10:3:0.5), the Rf value is moderate, and therefore this developing condition is selected as the subsequent investigation. The negative control has no interference and strong specificity.

[0087] Different manufacturers' thin layer plates investigation: Dianyankang sample (2 μL), Herba Galiums sinense reference material (2 μL), ursolic acid (1 μL), and negative control (2 μL) were taken, and different brands and specifications of thin layer plates were investigated (Yantai XX: HSGF254 silica gel plate, Qingdao XX Chemical: GF254 silica gel plate, Qingdao XX silica gel: GF254 silica gel plate) with cyclohexane: ethyl acetate: glacial acetic acid (10:3:0.5) as the developing condition. The results are shown in Figure 3 .

[0088] As can be seen from Figure 3 , the spots of GF254 silica gel plates of Qingdao XX Chemical and Qingdao XX silica gel and HSGF254 silica gel plate of Yantai XX are clear, indicating that the developing condition has good applicability and good durability under different manufacturers' thin layer plates.

[0089] Different temperature and humidity investigation: different temperature and humidity (10% RH, 45% RH, and 70% RH, 4°C and 25°C) were respectively taken as the variable factors, and the experiment was carried out according to the above identification method. The results are shown inFigure 4 .

[0090] From Figure 4 It can be seen that under different humidity and temperature conditions, the spot separation effect is good and each spot is still clear, indicating that the development condition has good durability.

[0091] Saturation time investigation: under unsaturated, saturated for 15 minutes and 30 minutes, the above identification method was used for experiment. The results are shown in Figure 5 .

[0092] From Figure 5 It can be seen that the length of the pre-saturation time does not affect the sample spots. Therefore, no pre-saturation time is selected.

[0093] (II) Identification of Radix Rumicis Cripii The experiment selected Radix Rumicis Cripii as the control medicinal material, emodin methyl ether as the control, extracted the characteristic components of Radix Rumicis Cripii in Danyan Kang by water bath reflux extraction method, identified the Radix Rumicis Cripii by thin layer, and investigated the influence of special property, different thin layer plates, low temperature, high humidity, low humidity and other durability on the thin layer chromatography of the medicinal material of Lianqiancao in the preparation. The test results show that: using Radix Rumicis Cripii as the control medicinal material, emodin methyl ether as the control, petroleum ether (60-90℃): methyl formate: formic acid (15:5:1) as the developing system, the thin layer identification characteristics of Radix Rumicis Cripii are obvious, and the special property is strong, which can be used as the thin layer identification method of Radix Rumicis Cripii in Danyan Kang capsules, so it is included in the detection items of the application.

[0094] (1) Instruments, reagents and samples DHG-9240A electric heating constant temperature blast drying oven

Shanghai XXX Experimental Equipment Co., Ltd.

XX Enterprise Co., Ltd.

Tianjin XXX Experimental Analysis Instrument Manufacturing Plant

Wuyi XX Electronic Co., Ltd.

XXX-XXX Instrument (Shanghai) Co., Ltd.

XX Scientific Co., Ltd., batch number: 230911

Shanghai XX Chemical Technology Co., Ltd., batch number: RH581526

XX Scientific Co., Ltd., batch number: 20230311

batch number DHJM20170418 purchased from Nanjing XX Bioengineering Co., Ltd.

batch number: 6KLD-9GHA purchased from China XX Institute of Drug Identification

Yantai XX Silica Gel Development Co., Ltd., batch number: 20241112, 200×50 mm; 20240122, 100×200 mm

Qingdao XX Chemical Co., Ltd., batch number: 20190318, 100×200 mm

Qingdao XX Silica Gel Drier Co., Ltd., batch number: 20240401, 100×200 mm

[0095] (2) Preparation of test sample solution: 0.25 g of the contents was taken, 10 mL of methanol was added, and it was refluxed in a water bath for 30 minutes, filtered, and concentrated to 4 mL to obtain the test sample solution.

[0096] (3) Preparation of negative sample: according to the prescription proportion and preparation process, a negative sample containing no radix et rhizoma rhei officinalis was prepared, and was prepared as a negative sample solution according to the same method as the test sample solution.

[0097] (4) Preparation of control drug material solution: 0.2 g of the control drug material of radix et rhizoma rhei officinalis was taken and prepared according to the same method as the test sample solution as the control drug material solution.

[0098] (5) Preparation of control sample solution: rheum emodin methyl ether control sample was accurately weighed, and methanol was added to prepare a solution containing 1 mg per 1 mL as the control sample solution.

[0099] (6) Developing system: petroleum ether (60-90℃): methyl formate: formic acid (15:5:1) as developing agent.

[0100] (7) Methodology verification: Different sample amounts of the above-mentioned solution of rhein methyl ether reference standard, cholecystitis-relieving sample, and rhubarb reference medicinal material solution were sampled and tested according to the above method to investigate the sample amount. The results are shown in the figure. Figure 6 .

[0101] Depend on Figure 6 As can be seen, the sample amounts of each spot are clear and obvious. After comprehensive consideration, the sample amount of the test sample solution is selected to be 2~8 μl, and the sample amounts of the control medicinal material solution and the control solution are 2~4 μl each.

[0102] Developing solvent and specificity investigation: Two μL of the cholecystitis-relieving sample, two μL of *Rheum palmatum* (reference material), one μL of rhein methyl ether, and one 2 μL of negative control were taken and their development in different solvents were investigated using the method described above. The solvents tested were petroleum ether (60–90℃): methyl formate: formic acid (15:5:1), chloroform: methanol (9:1), petroleum ether (60–90℃): ethyl acetate: formic acid (15:5:1), and petroleum ether (60–90℃): ethyl acetate: acetic acid (15:5:1). Results are shown below. Figure 7 .

[0103] Depend on Figure 7 It is evident that the spots are clear and the Rf value is moderate when the developing solvent is petroleum ether (60–90℃): methyl formate: formic acid (15:5:1). Therefore, this developing condition was chosen for subsequent investigation. At the corresponding positions of the reference material, both the reference material and the test sample showed yellow spots, while the negative sample showed no yellow spots, indicating strong specificity.

[0104] Thin-layer plate tests from different manufacturers: Two samples of Cholecystitis Relief (2 μL), Rheum palmatum (2 μL) as a control, emodin methyl ether (2 μL), and a negative control (2 μL) were taken. Thin-layer plates of different brands and specifications (Yantai XX-HSGF254 silicone plate, Qingdao XX-GF254 silicone plate, Qingdao XX-GF254 silicone plate) were tested using the method described above. Results are shown below. Figure 8 .

[0105] Depend on Figure 8 As can be seen, the spots on the Qingdao XX, XX-GF254 silicone plates and the Yantai XX-HSGF254 silicone plates are all clear, indicating that the development conditions are well applicable.

[0106] Investigations under different temperatures and humidity levels: Experiments were conducted using the above identification method, taking different temperatures and humidity levels (10%RH, 45%RH, and 70%RH, 4℃ and 25℃) as varying factors. Results are shown below. Figure 9 .

[0107] Depend on Figure 9 It is evident that the spot separation effect is good and each spot remains clear under different humidity and temperature conditions, indicating that the development conditions are durable.

[0108] Saturation time investigation: The conditions of unsaturated, saturated for 15 minutes, and 30 minutes were investigated respectively, following the identification method described above. Results are shown below. Figure 10 .

[0109] Depend on Figure 10 It is evident that the length of the presaturation time does not affect the sample spots. Therefore, a presaturation time that is not required is chosen.

[0110] (III) Identification of Andrographis paniculata This product is Andrographis paniculata, a plant belonging to the Acanthaceae family. Andrographis paniculata (Burm. f.) Nees The dried aerial parts of Andrographis paniculata. Included in the 2020 edition of the Chinese Pharmacopoeia (Part I). This experiment selected Andrographis paniculata reference material as an indicator component for testing. Thin-layer chromatography (TLC) identification was performed using Andrographis paniculata reference material as a control, and the effects of specificity, different TLC plates, different temperatures, and different humidity on the TLC of Andrographis paniculata in Cholecystitis Capsules were investigated. The experimental results showed that using Andrographis paniculata reference material as a control, and petroleum ether (60–90℃): ethyl acetate: ethanol (4:2:1) as the developing system, the TLC identification characteristics of Andrographis paniculata were obvious and the specificity was strong. Therefore, it can be used as a TLC identification method for Andrographis paniculata in Cholecystitis Capsules, and is therefore included in the detection items of this invention.

[0111] (1) Instruments, reagents and samples KS-7200DE type liquid crystal ultrasonic cleaning machine

Kunshan XXX Ultrasonic Instrument Co., Ltd.

Shanghai XXX Experimental Equipment Co., Ltd.

XX Enterprise Co., Ltd.

Wuyi XX Electronic Co., Ltd.

XXX-XXX Instrument (Shanghai) Co., Ltd.

XX Scientific Inc., batch number: 230911

XX Scientific Inc., batch number: 231016

XX Scientific Inc., batch number: 230510

Yantai XX Silica Gel Development Co., Ltd., batch number: 20241112, 200×50 mm; 20240122, 100×200 mm

Qingdao XX Chemical Co., Ltd., batch number: 20190318, 100×200 mm

Qingdao XX Silica Gel Drier Co., Ltd., batch number: 20240401, 100×200 mm

batch number: P17J10F80032 purchased from Shanghai XX Biological Technology Co., Ltd.

[0112] (2) Preparation of test sample solution: 4.0 g of the content was taken, 30 mL of ethanol was added, and ultrasonic was performed for 20 minutes. After filtration, it was concentrated to about 4 mL as the test sample solution.

[0113] (3) Preparation of negative sample: according to the prescription proportion and preparation process, a negative sample without Andrographis paniculata medicinal material was prepared, and was prepared as a negative sample solution according to the same method as the test sample solution.

[0114] (4) Preparation of control drug material solution: 0.2 g of Andrographis paniculata control drug material was taken and prepared according to the same method as the test sample solution as the control drug material solution.

[0115] (5) Preparation of control sample solution: dehydroandrographolide control sample was accurately weighed, and a solution containing 1.0 mg per 1 mL was prepared by adding ethanol as the control sample solution.

[0116] (6) Developing system: petroleum ether (60-90℃) ∶ ethyl acetate ∶ ethanol (4∶2∶1).

[0117] (7) Methodology verification Spotting quantity investigation: respectively take the above-mentioned solution dehydrated andrographolide (1 μL, 2 μL, 3 μL, 4 μL), Dianyankang sample (4 μL, 6 μL, 8 μL, 10 μL), Andrographis paniculata control drug (2 μL, 3 μL, 4 μL, 5 μL), and spot on HSGF245 silica gel plate, the developing condition is petroleum ether (60-90 ℃) : ethyl acetate: ethanol (4:2:1), and observe at 254 nm. The results are shown in Figure 11 .

[0118] From Figure 11 It can be seen that each spotting quantity is clear and obvious, and the dehydrated andrographolide spotting quantity is selected as 1 μL, the Andrographis paniculata control drug is selected as 5 μL, and the Dianyankang capsule sample is selected as 4-10 μL.

[0119] Developing agent and specificity investigation: take Dianyankang sample (4 μL), Andrographis paniculata control drug (4 μL), dehydrated andrographolide (1 μL), and negative control (4 μL), and respectively investigate four different developing systems: petroleum ether (60-90 ℃) : ethyl acetate: ethanol (4:2:1), petroleum ether (60-90 ℃) : ethyl acetate (1:1), chloroform: ethyl acetate: methanol (4:3:0.4), petroleum ether (60-90 ℃) : ethyl acetate: methanol (4:2:1) according to the above method. See Figure 12 .

[0120] From Figure 12 It can be seen that the spot is clear in petroleum ether (60-90 ℃) : ethyl acetate: ethanol (4:2:1), so this developing condition is selected as subsequent investigation. The control drug and the test sample show black spots on the corresponding position of the control, and the negative sample has no black spot, and the specificity is strong.

[0121] Different manufacturers' thin layer plate test: take Dianyankang sample (4 μL), Andrographis paniculata control drug (4 μL), dehydrated andrographolide (1 μL), and negative control (4 μL), and respectively investigate different brands and specifications of thin layer plates (Yantai XX-HSGF254 silica gel plate, Qingdao XX-GF254 silica gel plate, Qingdao XX-GF254 silica gel plate) according to the above method. See Figure 13 .

[0122] From Figure 13 It can be seen that the spots of the two Qingdao XX and XX-GF254 silica gel plates and the Yantai XX-HSGF254 silica gel plate are clear, indicating that the developing condition is suitable. The developing agent of the Yantai XX-HSGF254 silica gel plate does not upwardly bleed, so this silica gel plate is selected for subsequent investigation.

[0123] Different temperature and different humidity investigation: Different temperature and different humidity (10% RH, 45% RH and 70% RH, 4℃ and 25℃) as the variable factors, the above identification method was carried out. The results are shown in Figure 14 .

[0124] From Figure 14 It can be seen that under different humidity and different temperature conditions, the spot separation effect is good and each spot is still clear, indicating that the development condition has good durability.

[0125] Saturation time investigation: Unsaturated, saturated for 15 minutes and 30 minutes were investigated respectively, and the above identification method was carried out. The results are shown in Figure 15 .

[0126] From Figure 15 It can be seen that the length of pre-saturation time does not affect the sample spots. Therefore, no pre-saturation time is selected.

[0127] (Four) Investigation on the content determination method of Galium verum and Rumex nepalensis Spreng. Rosmarinic acid and chrysophanol are the effective components of Galium verum and Rumex nepalensis Spreng, so rosmarinic acid and chrysophanol are selected as the index components of the internal quality of the preparation. Methanol is used for sample pretreatment by heating and reflux, and high performance liquid chromatography is used for sample determination. The results show that the method is simple in operation, low in cost and short in test time, and has the advantages of good separation effect, sensitivity, accuracy, etc.

[0128] (1) Instruments, reagents and samples Wan Yi high performance liquid chromatograph (including P3210Q pump, UV3210 detector, automatic sampler, Smart Lab CDS 2.0 data processing software); chromatographic column: ZORBAX SB C18 (4.6x150 mm, 5 μm); HH-6S type water bath pot

Tianjin XXX experimental analysis instrument manufacturing plant

Wuyi XX electronic Co., Ltd.

XXX-XXX instrument (Shanghai) Co., Ltd.

Anhui XX high-purity solvent Co., Ltd., batch number: 24036107

Anhui XX high-purity solvent Co., Ltd., batch number: 24036107

Shanghai XXX biochemical technology Co., Ltd., batch number: E23231386

batch numbers are A12HB191467 and JB241724 respectively, both purchased from Shanghai XX biological technology Co., Ltd.

[0129] (2) Chromatographic conditions and system suitability test Take octadecylsilane bonded silica gel as the filler; take acetonitrile as the mobile phase (A) and 0.1% phosphoric acid as the mobile phase (B), gradient elution (0-25 minutes, 25% A-75% B; 20-25 minutes, 75% A-25% B; 25-60 minutes, 80% A-20% B), column temperature 35℃, flow rate 1.0 mL / min, detection wavelength 0-25 min (330 nm), 25-60 min (254 nm). The theoretical plate number should not be less than 3000 calculated by rosmarinic acid and chrysophanol peaks.

[0130] (3) Preparation of reference substance solution: accurately weigh chrysophanol reference substance and rosmarinic acid reference substance, add methanol to prepare a solution containing 0.5 mg of chrysophanol and 0.4 mg of rosmarinic acid per 1 mL; accurately take 5 mL of the above reference substance solution respectively, mix well, and the mixed reference substance solution is obtained.

[0131] (4) Preparation of test solution: accurately weigh 1.0 g of Danyan Kang powder, accurately add 25 mL of methanol into a conical flask with a stopper, accurately weigh, heat and reflux for 1 h, cool, re-weigh, make up the weight loss with methanol, shake well, filter, and the filtrate is obtained.

[0132] (5) Preparation of negative control solution: According to the prescription ratio and preparation process, a negative sample containing no Herba Siegesbeckiae and Radix et Rhizoma Rumicis Cripi was prepared, and the negative sample solution was prepared by the same method as the test sample solution.

[0133] (6) Methodology verification a. Determination of detection wavelength: In this study, a high performance liquid chromatograph with a UV-visible spectrophotometric detector was used for full wavelength scanning. According to the results of the atlas, the detection wavelength of rosmarinic acid was 330 nm, and the detection wavelength of chrysophanol was 254 nm. See Figure 16 .

[0134] As can be seen from Figure 16 , the detection wavelength of rosmarinic acid is tentatively set at 330 nm, and the detection wavelength of chrysophanol is 254 nm.

[0135] b. Investigation of mobile phase: According to the above chromatographic conditions and sample treatment method, the different proportions of different mobile phases and water phases were investigated, and the results showed that the addition of an appropriate amount of acid in the mobile phase could improve the tailing phenomenon of the peak, and different concentrations of phosphoric acid had little effect on the tailing factor, therefore the following gradient was selected as the final elution condition: the mobile phase was acetonitrile (A) -0.1% phosphoric acid (B); gradient elution (0-20 minutes, 25% A; 20-25 minutes, 75% A; 25-60 minutes, 80% A), detection wavelength 0-25 min (330 nm), 25-60 min (254 nm).

[0136] c. Investigation of extraction method: According to the above chromatographic conditions and sample treatment method, the ultrasonic and reflux extraction methods were investigated respectively. The results showed that the peak areas of rosmarinic acid and chrysophanol extracted by reflux were higher than those extracted by ultrasonic, therefore the extraction method was selected as reflux.

[0137] d. Investigation of extraction solvent: According to the above chromatographic conditions and sample treatment method, the peak areas of rosmarinic acid and chrysophanol were compared when 100% methanol, 75% methanol, and 50% methanol were used as extraction solvents. The results showed that the extraction effect of 100% methanol was the best, therefore 100% methanol was selected as the extraction solvent.

[0138] e. Investigation of solvent amount: According to the above chromatographic conditions and sample treatment method, the peak areas of rosmarinic acid and chrysophanol were compared when 100% methanol was 25 mL, 50 mL, and 75 mL. The results showed that the content of the extracted compounds was higher when the solvent amount was 25 mL. Therefore, 25 mL was selected as the extraction solvent amount.

[0139] f. Extraction time investigation: according to the above chromatographic conditions and sample processing method, the peak areas of rosmarinic acid and chrysophanol extracted by heating reflux for 30 minutes, 45 minutes and 60 minutes were compared, and the results showed that the content was relatively high when extracted by reflux for 60 minutes. Therefore, reflux for 60 minutes was selected as the extraction time.

[0140] g. Investigation of linearity and range: the mixed reference solution (C = 0.1115 mg / mL) was diluted by 2, 4, 8, 16, 32, 64, 128, 256 times in turn, and the mixed reference solution and each dilution were injected into the liquid chromatograph, and the peak area integral values were measured. The peak area integral values were linearly regressed, and the standard curve was drawn with the concentration of the reference as the abscissa and the measured peak area as the ordinate. The results showed that the linear range of rosmarinic acid was 0.0009-0.1115 mg / mL, the regression equation was y = 42285x + 64.441, R 2 = 0.9992, and the linear range of chrysophanol was 0.0009-0.1115 mg / mL, the regression equation was y = 54015x - 5.0802, R 2 = 1, both showing good linear relationships.

[0141] h. Precision test The same test sample solution was repeatedly injected 6 times on the same day, and the peak areas of rosmarinic acid and chrysophanol were measured according to the above chromatographic conditions. The experimental results showed that the peak area of rosmarinic acid had an RSD of 1.001%, and the peak area of chrysophanol had an RSD of 0.759%, indicating that the instrument precision was good.

[0142] i. Reproducibility test The same batch of sample was repeatedly prepared 6 times according to the above test sample solution preparation method, and then measured according to the above content determination method to determine the peak area. The peak areas of rosmarinic acid and chrysophanol were recorded, and the experimental results showed that the peak area of rosmarinic acid had an RSD of 2.2%, and the peak area of chrysophanol had an RSD of 0.7%, indicating that the method had good reproducibility.

[0143] j. Stability test The same test sample solution was precisely taken 10 μL, and was injected at 0, 2, 4, 8, 10, 12 and 24 hours, respectively. The peak areas of rosmarinic acid and chrysophanol were recorded, and the peak area integral values were measured. The RSD value of rosmarinic acid was 2.7%, and the RSD value of chrysophanol was 1.0%. It was shown that the test sample solution of rosmarinic acid and chrysophanol had good stability within 24 h.

[0144] k. Specificity test The test sample and negative control sample of Duanyan Kang capsule were added with excipients, and then the specificity test was carried out according to the above chromatographic conditions and sample treatment method, and the peak time was compared. The results are shown in Figure 17 , Figure 18 .

[0145] From Figure 17 , Figure 18 It can be seen that the rosmarinic acid peak and the chrysophanol peak are separated from other components to the baseline, and the peak shape is good; the negative sample test results show that other components have no interference with the determination of rosmarinic acid and chrysophanol.

[0146] l. Sample recovery rate test Accurately weigh 0.5 g of Duanyan Kang powder, 6 portions, add the appropriate amount of reference solution, prepare 6 portions of test sample solution according to the method under the item of "preparation of test sample solution", and inject under the conditions of "chromatographic conditions and system suitability test" item, measure the recovery rate of rosmarinic acid and chrysophanol, calculate the average recovery rate and RSD%, according to the provisions of "China Pharmacopoeia" 2020 edition four chapters 9101 accuracy, the content of the component to be determined is between 0.01% and 0.1%, the recovery rate limit is between 85% and 110%, the content of the component to be determined is between 0.1% and 1%, and the recovery rate limit is between 90% and 108%.(See Table 1 below)

[0147] From the results in Table 1, the average recovery rate of rosmarinic acid was 96.57%, and the RSD was 2.265%; the average recovery rate of chrysophanol was 102.03%, and the RSD was 1.051%, indicating that the method has high accuracy.

[0148] m. Durability test According to the above chromatographic conditions and sample treatment method, the test investigated different temperatures 25℃, 30℃, 35℃, different brands of chromatographic column InerSustain ®C18, Luna ®5μm C18, ZORBAXSB C18, different high performance liquid chromatography system Wan Yi 3210, Agilent 1200, the results showed that under the conditions of different temperatures at 25℃-35℃, there was no obvious difference in the determination of the content of rosmarinic acid and chrysophanol; the chromatographic peak separation of rosmarinic acid and chrysophanol determined by different chromatographic columns was greater than 1.5, and there was no obvious difference in the content results; the content results of rosmarinic acid and chrysophanol determined by different high performance liquid chromatography systems were close, indicating that the method has good durability.

[0149] (Five) Methodology investigation of determination of baicalin content Baicalin in Huangqin has a high content in Danyan Kang Capsules, so it is selected as the index component for content limitation. The sample is treated by ultrasonic, and determined by high performance liquid chromatography. The results show that the method has the advantages of simple operation, low cost, short test time, good separation effect, sensitivity and accuracy, etc.

[0150] (1) Instruments, reagents and samples Wan Yi high performance liquid chromatograph (including P3210Q pump, UV3210 detector, automatic sampler, Smart LabCDS 2.0 data processing software); chromatographic column: ZORBAX SB C18 (4.6x150mm, 5µm); KS-7200DE type liquid crystal ultrasonic cleaner

Kunshan XXX Ultrasonic Instrument Co., Ltd.

Wuyi XX Electronic Co., Ltd.

XXX-XXX Instrument (Shanghai) Co., Ltd.

Anhui XXX Pure Solvent Co., Ltd., batch number 24036107

Anhui XXX Pure Solvent Co., Ltd., batch number 24036107

Shanghai XXX Biochemical Technology Co., Ltd., batch number E23231386

[0151] Baicalin

(batch number: JB246114) purchased from Shanghai XX Biotechnology Co., Ltd.

[0152] (2) Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler; acetonitrile-0.1% phosphoric acid (20:80) as the mobile phase; isocratic elution; column temperature 35℃, flow rate 1.0mL / min, baicalin detection wavelength 280nm. The theoretical plate number of baicalin peak should not be less than 3000.

[0153] (3) Preparation of reference solution: accurately weigh a certain amount of baicalin reference substance, add methanol to prepare a solution containing 0.4mg of baicalin per 1mL.

[0154] (4) Preparation of test solution: Take 0.2 g of Dianfankang powder, accurately weigh, place in a conical flask with a stopper, accurately add 75% methanol 25 mL, weigh, ultrasonic for 30 minutes, cool, re-weigh, make up the weight loss with 75% methanol, shake well, filter, take the filtrate, and obtain.

[0155] (5) Preparation of negative control solution: Prepare a negative sample containing no Scutellaria by the prescription proportion and preparation process, and prepare the negative sample solution by the same method as the "test solution".

[0156] (6) Methodology verification a. Determination of detection wavelength: In this study, high performance liquid chromatograph with ultraviolet-visible spectrophotometric detector was used for full wavelength scanning. According to the results of the atlas and the literature report, 280 nm was selected as the detection wavelength. See Figure 19 .

[0157] b. Investigation of mobile phase: According to the above chromatographic conditions and sample treatment method, different mobile phases and different proportions of aqueous phases were investigated. The results showed that adding an appropriate amount of acid in the mobile phase could improve the tailing phenomenon of the peak, therefore the following conditions were selected as the final elution conditions: mobile phase was acetonitrile (A) -0.1% phosphoric acid (B); isocratic elution (20% A-80% B).

[0158] c. Investigation of extraction method: According to the above chromatographic conditions and sample treatment method, ultrasonic and reflux extraction methods were investigated respectively. The results showed that the content of ultrasonic extraction was higher than that of reflux extraction, therefore ultrasonic extraction was selected.

[0159] d. Investigation of extraction solvent: According to the above chromatographic conditions and sample treatment method, the peak area of baicalin was compared when 100% methanol, 75% methanol, 50% methanol were used as extraction solvent. The results showed that the content of 75% methanol extraction was the highest, therefore this concentration was selected as the extraction solvent.

[0160] e. Investigation of solvent amount: According to the above chromatographic conditions and sample treatment method, 75% methanol 25 mL, 50 mL, 75 mL were accurately added, and the peak area of baicalin was compared. The results showed that there was no difference in content, therefore the amount of extraction solvent was selected as 25 mL.

[0161] f. Investigation of extraction time: According to the above chromatographic conditions and sample treatment method, the peak area of baicalin was compared under the conditions of ultrasonic extraction for 30 minutes, 45 minutes and 60 minutes. The results showed that the extraction of medicinal materials was more complete under the condition of ultrasonic extraction for 30 minutes, therefore the extraction time was selected as 30 minutes.

[0162] g. Linearity range investigation: Baicalin reference solution (C=0.401 mg / mL) was successively diluted 2, 4, 8, 16, 32, 64, 128, and 256 times. 10 μl of the reference standard and each dilution was precisely injected into the liquid chromatograph, and the peak area integral value was measured. Linear regression analysis was performed on the peak area integral value. A standard curve was plotted with the concentration of the reference standard on the x-axis and the measured peak area on the y-axis, yielding the regression equation: Y = 61998x - 79.272 (Y is peak area, X is concentration), R0 2 =0.997, indicating that baicalin showed a good linear relationship with the peak area integral value in the range of 0.00156–0.401 mg / mL.

[0163] h. Precision test: The same test solution was injected 6 times consecutively on the same day. The peak area of ​​baicalin was measured and recorded under the above chromatographic conditions. The experimental results showed that the peak area of ​​baicalin had an RSD of 1.5%, indicating that the instrument precision was good.

[0164] i. Repeatability test: Six samples from the same batch were prepared repeatedly using the above-described method for preparing the test solution. The content was then determined using the above-described method, and the peak area was measured and recorded. The experimental results showed that the RSD of the baicalin peak area was 0.6%, indicating good repeatability of the method.

[0165] j. Stability test: Take the same test solution and accurately pipette 10 μl of the test solution at 0, 2, 4, 8, 10, 12, and 24 hours respectively. Record the peak area of ​​baicalin and measure its peak area integral value. Calculate the RSD value of baicalin based on the average peak area. The RSD value of baicalin is 0.4%, indicating that the test solution of baicalin has good stability in 24 hours.

[0166] k. Specificity test: Following the chromatographic conditions and sample preparation methods described above, a specificity test was performed. The baicalin peak achieved baseline separation from other components, and the peak shape was good. The results of the negative sample test showed that other components did not interfere with the determination of baicalin. See results below. Figure 20 .

[0167] 1. Recovery Test: Accurately weigh 0.1 g of the contents of this product into 6 portions, add an appropriate amount of reference solution to each portion, and prepare 6 test solutions according to the method under "Preparation of Test Solution". Inject the samples according to the conditions under "Chromatographic Conditions and System Suitability Study", determine the recovery rate of baicalin, and calculate the average recovery rate and RSD%. According to the Accuracy section of General Chapter 9101 of Part IV of the 2020 Chinese Pharmacopoeia, the content of the analyte is between 1% and 10%, and the recovery limit is between 92% and 105%. The results are shown in Table 2 below.

[0168] From the results of Table 2, the average recovery rate of the sample addition was 94.01%, and the RSD was 1.845%, indicating that the method has high accuracy.

[0169] m. Durability test: According to the above chromatographic conditions and sample processing method, the test investigated different temperatures 25℃, 30℃, 35℃; different brands of chromatographic column InerSustain ®C18, Luna ®5μm C18, ZORBAX SB C18; different high performance liquid chromatography system Wan Yi 3210, Agilent 1200; the results showed that under the conditions of different temperatures at 25℃-35℃, there was no significant difference in the determination of the content of baicalin; the chromatographic peak separation of different chromatographic columns for determining baicalin was all greater than 1.5, and there was no significant difference in the content results; different high performance liquid chromatography systems for determining the content of baicalin had no significant difference, indicating that the durability of the method is good The application is used for the detection method of traditional Chinese medicine preparation for treating liver and gallbladder damp-heat accumulation, adds the thin layer identification of radix et rhizoma rhei and andrographis, optimizes the thin layer identification method of gemma marisci and adds the content determination method of gemma marisci, radix et rhizoma rhei and radix scrophulariae. The content determination method of gemma marisci, radix et rhizoma rhei and radix scrophulariae has the advantages of simple operation, low cost, short test time, good separation effect, high recovery rate, high stability, strong specificity, good repeatability, high precision, high accuracy and the like. The identification method of gemma marisci, radix et rhizoma rhei and andrographis has the advantages of high accuracy, no interference and the like. The application can effectively detect whether the product is qualified and the quality is good or bad, ensures the consistency of drug efficacy and the stability of quality, and further ensures the stability of product quality and the safety and effectiveness of clinical medication.

[0170] The above examples are illustrative of the present application and are not limiting thereof, and any equivalent changes or equivalent modifications made according to the technical ideas of the present application, without departing from the principles of the present technical solution, still belong to the protection scope of the present technical solution.

Claims

1. A detection method for traditional Chinese medicine preparation for treating liver-gallbladder dampness-heat accumulation, the traditional Chinese medicine preparation is prepared by mixing Rubia cordifolia, Rheum palmatum, Saxifraga stolonifera, Sabia parviflora, Pteridium aquilinum, Scutellaria baicalensis, Phellodendron amurense and Andrographis paniculata in a ratio of 200 parts by weight; the eight raw medicinal materials are mixed to form the traditional Chinese medicine preparation; and the detection method is characterized in that, The detection method comprises thin layer identification and content determination of components; wherein the thin layer chromatography is used for identifying Herba Galium and Radix Rumicis Chinensis, and the high performance liquid chromatography is used for determining the content of effective components of Herba Galium, Radix Rumicis Chinensis and Radix Scrophulariae in the preparation.

2. The detection method of the traditional Chinese medicine preparation for treating liver-gallbladder dampness-heat accumulation according to claim 1, characterized in that, The identification method of Herba Galium is as follows: 2.0 g of the content of the product is added with 20 mL of methanol, and ultrasonic treatment is conducted for 30 minutes; filtration is conducted, and concentration is conducted to 4 mL to obtain a test sample; 0.2 g of Herba Galium control drug is taken, and the same method is used to prepare a control drug solution; ursolic acid control sample is weighed, and methanol is used to prepare a solution containing 1.0 mg of ursolic acid per 1 mL, which is used as a control sample solution; According to the thin layer chromatography test, 2-8 μL of the test sample solution, 2 μL of the control drug solution and 1 μL of the control sample solution are taken, and are spotted on the same silica gel thin layer plate; cyclohexane: ethyl acetate: glacial acetic acid (10:3:0.5) is used as a developing agent to develop upwards, and is taken out, dried, and sprayed with 10% sulfuric acid ethanol solution to be observed under visible light; the test sample chromatogram shows the same color purple spots at the positions corresponding to the control drug and the control sample chromatogram.

3. The method for detecting a traditional Chinese medicine preparation for treating liver-gallbladder dampness-heat accumulation according to claim 1, characterized in that, The identification method of Radix Rumicis Chinensis is as follows: 0.25 g of the content of the product is added with 10 mL of methanol, and water bath reflux is conducted for 30 minutes; filtration is conducted, and concentration is conducted to 4 mL to obtain a test sample solution; 0.2 g of Radix Rumicis Chinensis control drug is taken, and the same method is used to prepare a control drug solution; emodin methyl ether control sample is weighed, and methanol is used to prepare a solution containing 1 mg per 1 mL, which is used as a control sample solution; According to the thin layer chromatography test, 2-8 μL of the test sample solution, 2-4 μL of the control drug solution and the control sample solution are taken, and are spotted on the same silica gel thin layer plate; petroleum ether with a boiling range of 60-90 ℃: freshly prepared methyl formate: formic acid (15:5:1) is used as a developing agent to develop upwards, and is taken out, dried, and observed under a UV lamp; the test sample chromatogram shows the same color yellow fluorescent spots at the positions corresponding to the control sample and the control drug chromatogram.

4. The detection method of the traditional Chinese medicine preparation for treating liver-gallbladder dampness-heat accumulation according to claim 1, characterized in that, The identification method of Herba Galium is as follows: 2.0 g of the content of the product is added with 20 mL of methanol, and ultrasonic treatment is conducted for 30 minutes; filtration is conducted, and concentration is conducted to 4 mL to obtain a test sample; 0.2 g of Herba Galium control drug is taken, and the same method is used to prepare a control drug solution; ursolic acid control sample is weighed, and methanol is used to prepare a solution containing 1.0 mg of ursolic acid per 1 mL, which is used as a control sample solution; According to the thin layer chromatography test, 2-8 μL of the test sample solution, 2-4 μL of the control drug solution and the control sample solution are taken, and are spotted on the same silica gel thin layer plate; petroleum ether with a boiling range of 60-90 ℃: freshly prepared methyl formate: formic acid (15:5:1) is used as a developing agent to develop upwards, and is taken out, dried, and observed under a UV lamp; the test sample chromatogram shows the same color yellow fluorescent spots at the positions corresponding to the control sample and the control drug chromatogram.

5. The method for detecting a traditional Chinese medicine preparation for treating liver-gallbladder dampness-heat accumulation according to claim 1, characterized in that, The content determination method of Herba Galium and Radix Rumicis Chinensis is as follows: the high performance liquid chromatography is used for determination; Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler; acetonitrile as the mobile phase (A), 0.1% phosphoric acid as the mobile phase (B), gradient elution, column temperature 35°C, flow rate 1.0 mL / min, detection wavelength 0-25 min (330 nm), 25-60 min (254 nm); the theoretical plate number should not be less than 3000 according to the rosmarinic acid, chrysophanol peak; Preparation of reference solution: take chrysophanol reference substance and rosmarinic acid reference substance, accurately weigh, add methanol to prepare a mixed solution containing 0.5 mg of chrysophanol and 0.4 mg of rosmarinic acid per 1 mL; Preparation of test solution: take the content of the product, grind finely, take 1.0 g, accurately weigh, put into a conical flask with a stopper, accurately add 25 mL of methanol, weigh, heat reflux for 1 h, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, and you get it. Determination method: accurately take 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine. Each tablet contains not less than 86.0 μg of rosmarinic acid (C 18 H 16 O8) and not less than 0.5 mg of chrysophanol (C 15 H 10 O4).

6. The detection method of a traditional Chinese medicine preparation for treating liver-gallbladder dampness-heat accumulation according to claim 5, characterized in that, The gradient elution is: 0-20 min, 25% A-75% B; 20-25 min, 75% A-25% B; 25-60 min, 80% A-20% B.

7. The method for detecting a traditional Chinese medicine preparation for treating liver-gallbladder dampness-heat accumulation according to claim 1, characterized in that, The content of baicalin in Huangqin is determined by: Determined by high performance liquid chromatography; Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler; acetonitrile-0.1% phosphoric acid (20:80); isocratic elution; column temperature 35°C, flow rate 1.0 mL / min, baicalin detection wavelength 280 nm; the theoretical plate number should not be less than 3000 according to the baicalin peak; Preparation of reference solution: take baicalin reference substance, accurately weigh, add methanol to prepare a solution containing 0.4 mg of baicalin per 1 mL; Preparation of test solution: take the content of the product, grind finely, take 0.2 g, accurately weigh, put into a conical flask with a stopper, accurately add 25 mL of 75% methanol, weigh, ultrasonic for 30 min, cool, weigh again, make up the weight loss with 75% methanol, shake well, filter, take the filtrate, and you get it. Determination method: accurately take 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

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