Quality control method of Jingu Tongxiao pill

Abstract

The present invention relates to a quality control method for Jingutongxiao pills, belonging to the technical field of traditional Chinese medicine. The quality control method is based on the existing quality standards, by adding the thin layer identification of licorice, improving the thin layer identification of cyperus rotundus, cinnamon twig, white peony root, cyperus rotundus and gentiana, and using HPLC to simultaneously determine cryptotanshinone, tanshinone I and tanshinone II A The content of the product was optimized and the chromatographic conditions were optimized, thus achieving the technical goal of better controlling the product quality.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, relates to the quality standard of Jingu Tong Xiao Wan, and particularly relates to a quality control method of Jingu Tong Xiao Wan. Background Art

[0002] Jin Gu Tong Xiao Wan (Jingu Tong Xiao Wan) is a new Chinese medicine developed based on a representative formula from the Pingle Guo family's bone-setting practice. It contains eleven herbs: Danshen (Salvia miltiorrhiza), Millettia reticulata (Spatholobi), Cyperus rotundus (Rhizoma Cyperi), Linderae odoratum (Lingering), Cyathula officinalis (Chuanxiong), Cinnamomum cassia (Cinnamomum cassia), Clemati radix (Clematis chinensis), Gentiana macrophylla (Gentiana macrophylla), White peony root (White Peony), Rehmannia glutinosa (Rehmannia glutinosa), and Licorice root. It promotes blood circulation and qi, warms the meridians and dredges the collaterals, and relieves swelling and pain. It is used to treat knee pain, swelling, and limited mobility caused by blood stasis and cold accumulation, as well as knee osteoarthritis. It is a protected Chinese medicine, and its standard is included in Volume 28 of the "New Drug Standards." This standard was revised in 2009 and received the National Drug Standard (Revised) approval number: ZGB2009-19. Its standard number: WS3-423(Z-067)-2000(Z)-2009. It came into effect on January 10, 2010.

[0003] After years of practice, this standard still has some problems, including: ① In terms of thin-layer chromatography identification, the identification method of white peony root is not stable enough. The preparation process of the test solution includes the elution step of using a neutral alumina column. The adsorption capacity of neutral alumina of different brands varies greatly, and the elution capacity of methanol of different brands also varies. The target component is often not eluted; ② In the thin-layer chromatography identification of Cyperus rotundus, the chromatographic spots of the test sample and the control medicinal material are very weak, which affects the result judgment; ③ In the thin-layer chromatography identification of cinnamon twig, the main chromatographic spot of the test sample is weak. In addition, the information provided by the control medicinal material and the control is repeated; ④ In the thin-layer chromatography identification of Cyathula capitata, the intensity contrast of the chromatographic spots of the test sample and the control medicinal material is large; ⑤ In the thin-layer chromatography identification of Gentiana macrophylla, the Gentiana macrophylla control medicinal material is selected as the control. Since Gentiana macrophylla is a multi-source variety, the chromatographic spots of the test sample and the control medicinal material may not completely correspond; ⑥ There is no thin-layer chromatography identification of Licorice; ⑦ In terms of content determination, only Tanshinone II is used A The controllable components as indicators are quite limited. The existence of these problems is very detrimental to the quality control of the Jingutongxiaowan product. Summary of the Invention

[0004] In order to overcome the above-mentioned defects, the purpose of the present invention is to provide a quality control method for Jingu Tong Xiao Wan, which improves the thin layer identification of Cyperus rotundus, cinnamon twig, white peony root, Chuanxiong and Gentiana macrophylla, adds the thin layer identification of liquorice, and optimizes the content determination items, so that the quality control method can better control product quality.

[0005] The specific scheme adopted in the present invention is:

[0006] A quality control method for Jingutongxiao pills, including identification items and content determination items;

[0007] The identification items include thin layer chromatography identification of Rhizoma Cyperi, Ramulus Cinnamomi, Radix Paeoniae Alba, Radix Cyathulae, Gentiana and Radix Glycyrrhizae, and are as follows:

[0008] (1) The thin layer chromatography identification method of Rhizoma Cyperi: take 10 g of the product, grind finely, place in a 500 ml round bottom flask, mix with 200 ml of water and a few glass beads, mix well, connect to a volatile oil tester, add water to the top of the tester to the scale, and when the overflow goes into the flask, add 2 ml of petroleum ether with a boiling range of 60-90°C, connect a reflux condenser, heat to boiling, and maintain gentle boiling for 1 hour, cool, and take the petroleum ether layer as the test solution; take 1 g of Rhizoma Cyperi control drug, prepare a control drug solution by the same method; take α-cyperone control, add ethyl acetate to prepare a solution containing 1 mg per 1 ml as the control solution; test by thin layer chromatography, take 8 μl of the test solution, 8 μl of the control drug solution and 1 μl of the control solution, and spot them on the same silica gel GF254 thin layer plate, develop with dichloromethane-ethyl acetate-glacial acetic acid (80:1:1 by volume) as the developing agent, take out, dry, and examine under a 254 nm ultraviolet light; the test chromatogram shows the same color spots at the corresponding positions of the control drug chromatogram and the control chromatogram; spray with dinitrosyl phenylhydrazine test solution, let stand for a while, and the spots gradually change to orange red; 254 thin layer plate, develop with dichloromethane-ethyl acetate-glacial acetic acid (80:1:1 by volume) as the developing agent, take out, dry, and examine under a 254 nm ultraviolet light; the test chromatogram shows the same color spots at the corresponding positions of the control drug chromatogram and the control chromatogram; spray with dinitrosyl phenylhydrazine test solution, let stand for a while, and the spots gradually change to orange red;

[0009] (2) The thin layer chromatography identification method of Ramulus Cinnamomi: take the test solution of the identification item of Rhizoma Cyperi in (1) as the test solution; take cinnamaldehyde control, add petroleum ether with a boiling range of 60-90°C to prepare a solution containing 1 μl per 1 ml as the control solution; test by thin layer chromatography, take 4 μl of the above two solutions, spot them on the same silica gel G thin layer plate, develop with petroleum ether-ethyl acetate (17:3 by volume) with a boiling range of 60-90°C as the developing agent, take out, dry, and spray with dinitrosyl phenylhydrazine ethanol test solution; the test chromatogram shows the same color spots at the corresponding positions of the control chromatogram;

[0010] (3) Thin layer chromatography identification method of white peony root: take 3g of this product, grind it into powder, add 30ml of ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, let it cool, add 30ml of water to the residue, stir, filter, extract the filtrate twice with 20ml of n-butanol saturated with water, combine the n-butanol solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution; take another paeoniflorin reference substance, add methanol to make a solution containing 2mg per 1ml, and use it as the reference solution; according to the thin layer chromatography test, take 5μl of the test solution and 2μl of the reference solution, and spot them on the same silica gel G thin layer plate respectively, use ethyl acetate-acetone-formic acid-water with a volume ratio of 15:6:2:0.5 as the developing solvent, develop, take out, dry, spray with 5% vanillin in 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly colored; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the reference substance;

[0011] (4) Thin layer chromatography identification method of Cyathula obtusifolia: Take 3 g of this product, grind it into powder, add 40 ml of 75% ethanol, reflux extract for 1 hour, filter, evaporate the filtrate to dryness, add 10 ml of water to dissolve the residue, shake and extract with 20 ml of ether, evaporate the ether solution to dryness, add 1 ml of anhydrous ethanol to dissolve the residue, and use it as the test solution; take another 0.5 g of Cyathula obtusifolia as a control medicinal material, and prepare a control medicinal material solution in the same way; according to the thin layer chromatography test, take 4 μl of each of the above two solutions, spot them on the same silica gel G thin layer plate, use toluene-chloroform-acetone with a volume ratio of 8:4:1 as the developing solvent, develop, take out, dry, and examine under a 365 nm ultraviolet lamp; in the chromatogram of the test product, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material;

[0012] (5) Thin layer chromatography identification method of Gentiana macrophylla: Take 1 g of this product, grind it into powder, add 20 ml of methanol, ultrasonically treat it for 30 minutes, filter it, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution; take another gentiopicroside reference substance, add methanol to make a solution containing 1 mg per 1 ml, and use it as the reference solution; according to the thin layer chromatography test, take 2 μl of each of the above two solutions and spot them on the same silica gel GF 254 On a thin layer plate, develop with ethyl acetate-methanol-water in a volume ratio of 10:2:1, remove, dry, and examine under a 254nm ultraviolet lamp; in the chromatogram of the test sample, a spot of the same color will appear at the corresponding position in the chromatogram of the reference sample;

[0013] (6) Thin layer chromatography identification method of licorice: take 4g of this product, grind it, add 30ml of methanol, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, add 30ml of 5% sodium carbonate solution to dissolve the residue, wash it twice with ethyl acetate, each time 30ml, adjust the pH value of the aqueous layer to 2-3 with 10% hydrochloric acid, shake and extract it twice with ethyl acetate, each time 30ml, combine the ethyl acetate solution, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution; take another 0.2g of licorice control medicinal material, and prepare a control medicinal material solution in the same way; take the glycyrrhizin reference substance, add methanol to make it contain 0.5mg of glycyrrhizin per 1ml, and use 1ml of methanol to dissolve the residue. The solution is used as the reference solution; according to the thin layer chromatography test, 1-2 μl of each of the three solutions is taken and spotted on the same silica gel G thin layer plate, and ethyl acetate-formic acid-glacial acetic acid-water with a volume ratio of 15:1:1:2 is used as the developing solvent. The plate is developed, removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots are clearly colored, and examined under sunlight and 365nm ultraviolet light respectively; in the chromatogram of the test sample, a main spot or fluorescent spot of the same color appears at the corresponding position in the chromatogram of the reference medicinal material; in the chromatogram of the reference substance, a spot or fluorescent spot of the same color appears at the corresponding position in the chromatogram of the reference medicinal material;

[0014] Determination items: Control of cryptotanshinone, tanshinone I and tanshinone II A The total amount of the content limit is: each 1g contains cryptotanshinone, tanshinone I and tanshinone II A The total amount shall not be less than 0.42 mg.

[0015] The content determination items are determined by liquid chromatography;

[0016] The chromatographic conditions were as follows: octadecylsilane bonded silica gel as filler, acetonitrile-0.02% phosphoric acid solution as mobile phase for gradient elution, detection wavelength at 270 nm, injection volume of 10 μl; the theoretical plate number was calculated based on the tanshinone II A Peak calculation should be no less than 20,000; Preparation of reference solution: Take cryptotanshinone, tanshinone I and tanshinone II A An appropriate amount of reference substance was accurately weighed and placed in a brown volumetric flask, and methanol was added to make a solution containing cryptotanshinone and tanshinone II per 1 ml. A A mixed solution of 10 μg of tanshinone I and 5 μg of tanshinone I was obtained;

[0017] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1.0g, weigh it accurately, put it into a stoppered conical flask, add 50ml of methanol accurately, stopper it tightly, weigh it, keep it in a 60℃ water bath shaker for 1 hour, let it cool, weigh it again, make up the loss with methanol, shake it well, filter it, and take the filtrate to obtain the solution;

[0018] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatography instrument, and determine;

[0019] Calculated on the basis of dry product, each 1g of Danshen contains cryptotanshinone, tanshinone I and tanshinone II. A The total amount shall not be less than 0.42 mg.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention revises and improves the quality standard of Jingutongxiao Pills. On the basis of the existing quality standard, the thin layer identification of licorice is added, the thin layer identification of cyperus rotundus, cinnamon twig, white peony root, cyperus rotundus and gentian is improved, and the HPLC method is used to simultaneously determine cryptotanshinone, tanshinone I and tanshinone II. A The content of the glutamic acid glutamic acid was increased by 1.5%, and the chromatographic conditions were optimized. The quality standard of the present invention can better control the product quality. Through this revision, the quality standard of the glutamic acid ... pills has been greatly improved, which is of great significance for controlling product quality.

[0022] 2. In the thin layer identification of Cyperus rotundus, the test solution is prepared by extracting volatile oils to avoid interference from impurities. Using dichloromethane-ethyl acetate-glacial acetic acid (80:1:1) as the developing agent has better effects. In addition, the same test solution can be used for identification of Cinnamomum cassiae, simplifying the operation.

[0023] 3. In the thin-layer identification of white peony root, the preparation method of the test sample solution was changed, which has better reproducibility than the method using neutral alumina column elution; ethyl acetate-acetone-formic acid-water (15:6:2:0.5) was used as the developing agent, which has a fast development speed and better chromatographic separation effect of the test sample.

[0024] 4. The chromatographic results of the thin-layer chromatography (TLC) identification of Cyathula and Gentiana macrophylla were better than those of the original method. For Cyathula TLC, the sample volume was increased, the sample solution preparation method was changed, and the sample volume of the Cyathula control was reduced. For Gentiana macrophylla TLC, the gentiopicroside control was used as a control, the Gentiana macrophylla control was removed, and the sample solution preparation method and developing solvent were optimized.

[0025] 5. TLC identification of licorice is a new item. The preparation method of the test solution is to use liquiritin and other components with 7 / The -OH structure flavonoids can be dissolved in 5% sodium carbonate solution and separated and purified by alkali dissolution and acid precipitation method, which is better than the traditional n-butanol extraction. The study also found that using licorice as a control material and liquiritin as a control is more effective than using ammonium glycyrrhizate.

[0026] 6. The main effects of Jingutongxiao pills are to promote blood circulation, anti-inflammatory and relieve pain, and their effects are related to fat-soluble components represented by tanshinone. Danshen is the main drug in the formula, cryptotanshinone, tanshinone I and tanshinone II AAll of them are fat-soluble active ingredients, so measuring the total amount of the three tanshinones can better control the product quality than measuring only a single component. Referring to the chromatographic conditions of the relevant literature (Chinese Pharmacopoeia [S]. 2020 edition, Part 1.78.), and gradient optimization, the target peak can achieve baseline separation, each chromatographic parameter meets the requirements, and the negative has no interference. The preparation method of the test solution in the content determination, each parameter is also the optimal condition after repeated comparison. The present invention is controlled by tanshinone II A The content of a single ingredient was changed to control cryptotanshinone, tanshinone I and tanshinone II A The total amount of tanshinone II is limited by 1g A Not less than 0.34mg is changed to: Each 1g of Danshen contains cryptotanshinone (C 19 H 20 O3), Tanshinone Ⅰ (C 18 H 12 O3) and tanshinone II A (C 19 H 18 O3) shall not be less than 0.42 mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The following are TLC identification images of Cyperus rotundus under 254 nm (left) and visible light (right); 1 to 3 are samples (batch numbers: 201101, 201102, 201103), 4 is a Cyperus rotundus reference material, 5 is an α-cyperone reference substance, and 6 is a negative control; Figure 2 This is the TLC identification chart of cinnamon twig; among them, 1 to 3 are samples (batch numbers: 201101, 201102, 201103), 4 is the cinnamaldehyde reference substance, and 5 is the negative control;

[0028] Figure 3 This is the TLC identification chart of white peony root; 1 to 3 are samples (batch numbers: 201101, 201102, 201103), 4 is the paeoniflorin reference substance, and 5 is the negative control;

[0029] Figure 4 This is a TLC identification chart of Cyathula; 1 to 3 are samples (batch numbers: 201101, 201102, 201103), 4 is a control herb of Cyathula, and 5 is a negative control;

[0030] Figure 5 This is a TLC identification chart of Gentiana macrophylla; 1 to 3 are samples (batch numbers: 201101, 201102, 201103), 4 is a gentiopicroside reference substance, and 5 is a negative control;

[0031] Figure 6The following are TLC identification images of licorice under visible light (left) and 365 nm (right); 1 to 3 are samples (batch numbers: 201101, 201102, 201103), 4 is a licorice control medicinal material, 5 is a liquiritin reference substance, and 6 is a negative control;

[0032] Figure 7 The chromatograms of the reference substance (A), the test substance (B), and the negative control solution (C); 1 is cryptotanshinone, 2 is tanshinone I, and 3 is tanshinone II A . DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0034] Example: A quality control method for Jingutongxiao pills.

[0035] 1. Instruments and reagents

[0036] 1.1 Instrument

[0037] LC-20AD high performance liquid chromatograph (Shimadzu, Japan); AB135-S electronic analytical balance (METTLER, Switzerland); SHA-B dual-function water bath constant temperature oscillator (Hangzhou Jingfei Instrument Technology Co., Ltd.).

[0038] 1.2 Drug testing

[0039] Tanshinone II A (Batch number: 110766-202022), cryptotanshinone (Batch number: 110852-201807), tanshinone I (Batch number: 110867-201607), α-cyperone (Batch number: 110748-201312), cinnamaldehyde (Batch number: 110710-201821), paeoniflorin (Batch number: 110736-201842), gentiopicroside (Batch number: 110770-201918) and Cyperus rotundus (Batch number: 121 059-201407), Sichuan Cyathula (batch number: 121065-201707), and Licorice (batch number: 120904-202021) were all from the China Food and Drug Administration; liquiritin reference substance (batch number: MUST-19033010) was purchased from Chengdu Manster Biotechnology Co., Ltd.; Jingutongxiao Pills (batch numbers: 201101, 201102, and 201103) were produced by Henan Luozheng Pharmaceutical Co., Ltd.; Silica G and Silica GF 254 Prefabricated panels were from Yantai Chemical Industry Research Institute; acetonitrile and phosphoric acid were of chromatographic grade, and the others were of analytical grade; purified water was from Wahaha Group.

[0040] 2 Methods and Results

[0041] 2.1 Identification by thin layer chromatography

[0042] 2.1.1 Identification of Cyperus rotundus by thin layer chromatography.

[0043] The sample sampling amount and sample loading amount in the thin layer chromatography identification method of Cyperus rotundus in the current standard are small, and the sample loading amount of the control medicinal material is also small. The spots corresponding to the α-cyperone reference substance in the chromatogram of the test sample and the control medicinal material are very weak, which affects the result judgment. The thin layer chromatography identification method of Cyperus rotundus is revised in the present application. The sample sampling amount, sample preparation method, sample loading amount and developing agent are optimized, and the chromatogram effect is greatly improved. Moreover, it can be more simple and convenient to identify with a test sample solution shared with Cinnamomum cassia.

[0044] Identification method: take 10g of the product, grind finely, and put it in a 500ml round-bottom flask. Add 200ml of water and a few glass beads, mix well, and connect a volatile oil tester (d<1). Add water to the top of the tester to the scale, and when the overflow goes into the flask, add 2ml of petroleum ether (60-90℃). Connect a reflux condenser, heat to boiling, and maintain a slight boil for 1 hour. Let it cool, and take the petroleum ether layer as the test sample solution. Take 10g of a negative sample of Cyperus rotundus, and prepare a Cyperus rotundus negative sample solution in the same way. Take 1g of Cyperus rotundus control medicinal material, and prepare a control medicinal material solution in the same way. Take α-cyperone reference substance, add ethyl acetate to prepare a solution containing 1mg per 1ml, and use it as the reference substance solution. Test according to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition four general rules 0502). Take 8ul of the test sample solution, 8ul of the control medicinal material solution, 1ul of the reference substance solution and 8ul of the Cyperus rotundus negative sample solution, and point them on the same silica gel GF plate. 254 Thin layer plate, dichloromethane-ethyl acetate-glacial acetic acid (80:1:1) as developing agent, develop, take out, dry, and observe under ultraviolet light (254nm). In the test sample chromatogram, the same color spots as the control medicinal material chromatogram and the reference substance chromatogram appear at the corresponding positions; spray with dinitrosophenylhydrazine test solution, let stand for a while, and the spots gradually change to orange red. The negative sample has no interference. Figure 1 .

[0045] The present application improves the sample sampling amount, and prepares the test sample solution by extracting volatile oil, which can effectively remove the interference of other fat-soluble components compared with the present standard method. The present application compares the present standard developing agent petroleum ether (60-90℃)-ethyl acetate (15:1) and the developing agent dichloromethane-ethyl acetate-glacial acetic acid (80:1:1) in the present application. The results show that the sample chromatogram spots are better separated when using dichloromethane-ethyl acetate-glacial acetic acid (80:1:1) as the developing agent. The effects are good under ultraviolet light (254nm) and after color development.

[0046] 2.1.2 Identification of Cinnamomum cassia by thin layer chromatography.

[0047] In the current standard thin-layer chromatography identification method for cinnamon twig, the main spot on the test sample's chromatogram is weak, and the control herb's chromatogram only has a corresponding spot, cinnamaldehyde. This does not provide more valuable information than using only the cinnamaldehyde reference substance as a control. The present invention increases the sample amount, sample concentration, and sample load, and eliminates the cinnamon twig control herb as a control. The test sample's chromatographic spots are clearer and the operation is simpler.

[0048] Identification method: Take the test solution of the identification item of Cyperus rotundus as the test solution. Take another 10g of the negative sample without cinnamon twig, and prepare the cinnamon twig negative sample solution in the same way. Then take the cinnamaldehyde reference substance, add petroleum ether (60-90℃) to make a solution containing 1μl per 1ml as the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0502), 4μl of each of the above three solutions were taken and spotted on the same silica gel G thin layer plate, and petroleum ether (60-90℃)-ethyl acetate (17:3) was used as the developing agent. Develop, take out, dry, and spray with dinitrophenylhydrazine ethanol test solution. In the test sample chromatogram, spots of the same color appear at the corresponding position of the reference sample chromatogram, and there is no interference if negative. See Figure 2 .

[0049] 2.1.3 Thin layer chromatography identification of white peony root.

[0050] The current quality standards for thin-layer chromatography identification of white peony root have some problems, mainly in the preparation method of the test solution. The preparation process of the test solution includes the elution step using a neutral alumina column. Due to quality problems or quality differences of neutral alumina from different manufacturers, the adsorption capacity varies greatly, and the target component often cannot be eluted, causing problems in the test results, affecting the result judgment, and seriously troubling product quality control. To avoid this situation, the present invention improves the preparation method and developing agent of the test solution, and the results are satisfactory.

[0051] Identification method: Take 3g of this product, grind it into powder, add 30ml of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, cool, add 30ml of water to the residue, stir, filter, and extract the filtrate twice with 20ml of water-saturated n-butanol each time. Combine the n-butanol solutions, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. Prepare a negative sample solution for white peony root (3g) by the same method. Separately, prepare a paeoniflorin reference substance and add methanol to a solution containing 2mg per 1ml. This is used as the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 5 μl of the test solution, 2 μl of the reference solution and 5 μl of the white peony negative sample solution were taken and spotted on the same silica gel G thin layer plate, respectively, with ethyl acetate-acetone-formic acid-water (15:6:2:0.5) as the developing solvent, developed, taken out, dried, sprayed with 5% vanillin in 10% sulfuric acid ethanol solution, and heated at 105 ° C until the spots were clearly colored. In the chromatogram of the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the reference sample, and there was no interference with the negative. See Figure 3 .

[0052] In this study, a variety of preparation methods for test solutions were compared, including eluting with methanol on a neutral alumina column after extraction, eluting with methanol first and then with 40% methanol, as well as ultrasonic extraction with ethanol, extraction with n-butanol, and then washing with ether, ammonia test solution / 0.5% sodium hydroxide / n-butanol saturated water, etc. After comparison, it was found that washing with ammonia test solution can lose a part of paeoniflorin, and the chromatographic background is large. Using the method of the present invention, the ethanol filtrate is evaporated to dryness, cooled, and then stirred with water (without heating) and filtered, most of the impurities can be removed, the loss of paeoniflorin is small, the method is simple, and the chromatographic background is clear, so the preparation method of the test solution is finally determined to be the method proposed by the present invention. In addition, in terms of the selection of reference substances, white peony reference medicinal materials and paeoniflorin reference substances are used as reference substances. The valuable information that the two can provide is the same, so only paeoniflorin is selected as the reference substance.

[0053] In this study, several developing systems were compared with reference to relevant literature, including chloroform-n-butanol-glacial acetic acid (3:3:0.2) (current standard), chloroform-n-butanol-glacial acetic acid (13:7:2) as the lower layer, chloroform-n-butanol (1:1), ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) and (8:1:1:2), ethyl acetate-acetone-formic acid-water (10:6:2:0.5) and (15:6:2:0.5), etc. Among them, chloroform-n-butanol-glacial acetic acid (3:3:0.2), ethyl acetate-formic acid-glacial acetic acid-water (8:1:1:2) and ethyl acetate-acetone-formic acid-water (15:6:2:0.5) all performed well as developing solvents, and there was no interference in the negative results. However, using ethyl acetate-acetone-formic acid-water (15:6:2:0.5) as the developing solvent, the developing speed is fast and the separation effect of the test sample spots is good.f The value is appropriate.

[0054] 2.1.4 Thin layer chromatography identification of Cyathula.

[0055] The current quality standard for the thin-layer chromatography identification of Cyathula has some problems, mainly manifested in the fact that the chromatographic spots of the test sample are weak, while the chromatographic spots of the control herb are too strong, resulting in a large contrast between the two and affecting the result determination. The present invention increases the sample size of the test sample and reduces the sample size of the control herb, achieving an appropriate strength of the chromatographic spots of the two and satisfactory results.

[0056] Identification method: Take 3g of this product, grind it into powder, add 40ml of 75% ethanol, reflux and extract for 1 hour, filter, evaporate the filtrate to dryness, add 10ml of water to the residue to dissolve it, shake and extract with 20ml of ether, evaporate the ether solution to dryness, and add 1ml of anhydrous ethanol to dissolve the residue as the test solution. Take 3g of the negative sample lacking Cynomorium root, and prepare the Cynomorium root negative sample solution in the same way. Take another 0.5g of Cynomorium root control medicinal material, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0502), take 4μl of each of the above three solutions, and spot them on the same silica gel G thin layer plate, use toluene-chloroform-acetone (8:4:1) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm). In the chromatogram of the test product, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, and the negative is without interference. See Figure 4 .

[0057] 2.1.5 Thin layer chromatography identification of Gentiana macrophylla.

[0058] The current quality standard uses a reference herb, Gentiana macrophylla, as a control for thin-layer chromatography identification of Gentiana macrophylla. Because Gentiana macrophylla is a polygenic species, chromatographic spots of the test sample and the reference herb may not fully correspond, resulting in poor specificity and often affecting the results. To improve this situation, this draft quality standard uses a gentiopicroside reference as a control and optimizes the preparation method and developing solvent for the test sample solution, resulting in a simple method and strong specificity.

[0059] Identification method: Take 1g of this product, grind it into powder, add 20ml of methanol, ultrasonically treat it for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution. Take 3g of the negative sample lacking Gentiana macrophylla, and prepare a Gentiana macrophylla negative sample solution in the same way. Take another gentiopicroside reference substance, add methanol to make a solution containing 1mg per 1ml, and use it as the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0502), take 2μl of each of the above three solutions and spot them on the same silica gel GF 254Thin layer plate, with ethyl acetate-methanol-water (10:2:1) as developing agent, development, take out, dry, placed under UV light (254nm) inspection. The test sample chromatogram, in the corresponding position with the control sample chromatogram, showed the same color spots, negative without interference. See Figure 5 .

[0060] In this study, the preparation method of the test sample was compared, including ethanol-10% ammonia solution (50:7) immersion method (present method), ultrasonic method, methanol ultrasonic method, first extracted with ethyl acetate and then extracted with methanol ultrasonic method, etc. The results showed that the methanol ultrasonic extraction method was relatively simple.

[0061] In this study, the chromatograms of three kinds of source control medicinal materials of Gentiana macrophylla, Gentiana crassicaulis and Gentiana straminea were compared. The results showed that the chromatographic spots of the three kinds of source of Gentiana were quite different, so the control medicinal materials should not be used as control in the finished product inspection.

[0062] In this study, the use of gentiopicroside and quercitol acid control samples as control was compared. Quercitol acid cannot be displayed under UV light (254nm), so gentiopicroside control sample was selected as control in this invention.

[0063] In this study, multiple development systems were compared, including: toluene-chloroform-acetone-isopropanol (8:4:1:1) (present standard), ethyl acetate-methanol-water (10:2:1) / (14:2:1), chloroform-methanol-formic acid (50:1:0.5), lower layer of chloroform-methanol-water (30:10:3), etc. The results showed that ethyl acetate-methanol-water (10:2:1) as developing agent was the best. The lower layer of chloroform-methanol-water (30:10:3) was also very good. Because chloroform in the developing agent has certain toxicity and needs to be layered, ethyl acetate-methanol-water (10:2:1) was selected as the developing agent in this invention.

[0064] 2.1.6 TLC identification of liquorice.

[0065] There is no TLC identification of liquorice in the present quality standard. During the research of the quality control method of this product, the preparation method of the test sample solution was compared, the effects of three kinds of source control medicinal materials and glycyrrhizin, ammonium glycyrrhizinate control samples as control were compared, and the developing agent was optimized. Finally, liquorice control medicinal materials and glycyrrhizin control sample were selected as control, which was simple and specific.

[0066] Identification method: Take 4g of this product, grind it into powder, add 30ml of methanol, heat under reflux for 1 hour, filter, and evaporate the filtrate to dryness. Dissolve the residue in 30ml of 5% sodium carbonate solution. Wash twice with 30ml of ethyl acetate. Adjust the pH of the aqueous layer to 2-3 with 10% hydrochloric acid. Extract twice with 30ml of ethyl acetate by shaking. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. Prepare a licorice-negative sample solution by the same method for 4g of a negative sample lacking licorice. Prepare a control solution by the same method for 0.2g of licorice control. Prepare a 0.5mg solution per 1ml of liquiritin control solution for 0.5mg of liquiritin control solution by adding methanol. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 1 to 2 μl of each of the above four solutions were taken and spotted on the same silica gel G thin layer plate, and ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored. The plates were inspected under sunlight and ultraviolet light (365nm) respectively. In the chromatogram of the test product, the main spots or fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the control medicinal material; the spots or fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the reference product. Negative, no interference. See Figure 6 .

[0067] During this study, a corresponding study was also conducted with reference to the thin-layer identification method of liquorice in the 2020 edition of the Chinese Pharmacopoeia. The selection of the reference substance and the preparation of the test solution in this method are different from the method adopted in the present invention, wherein the reference substance selects ammonium glycyrrhizate, and the preparation method of the test solution is specifically as follows: take 4g of this product, grind it, add 40ml of ether, heat and reflux for 1 hour, filter, discard the ether solution, add 30ml of methanol to the medicinal residue, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, add 40ml of water to the residue to dissolve it, shake and extract with n-butanol 3 times, 20ml each time, merge the n-butanol solution, wash 3 times with water saturated with n-butanol, 20ml each time, evaporate the n-butanol solution to dryness, and add 1ml of methanol to the residue to dissolve it as the test solution. The results show that the interference is serious at the corresponding position of the ammonium glycyrrhizate reference substance chromatogram. Therefore, liquiritin can be used as a control, but ammonium glycyrrhizate should not be used as a control. In addition, the results showed that the chromatographic background of this test solution was large, so the preparation method of the test solution was selected according to the present invention. / The -OH structure flavonoids can be dissolved in 5% sodium carbonate solution and can be separated and purified by alkali dissolution and acid precipitation method.

[0068] Licorice comes from three sources: Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata. This study also examined the chromatographic characteristics of three reference licorice herbs. All three reference herbs were prepared using the method of the present invention. After color development, the chromatographic main spots of the three reference herbs were identical. Therefore, the present licorice identification method uses both the liquiritin reference substance and the licorice reference herbs as controls, providing more chromatographic information.

[0069] In this study, multiple development systems were compared with reference to relevant literature. In addition to the developing agent used in the present invention, toluene-ethyl acetate-formic acid (20:4:0.5), chloroform-acetone-ethyl acetate-formic acid (5:2:2:1), ethyl acetate-methanol-water (20:3:2) and ethyl acetate-acetone-water (6:6:1) were also included. The developing agent ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) used in the present invention had the best effect, with a fast development speed, a clear chromatographic background, and no negative interference.

[0070] 2.1.7 Thin layer chromatography identification study of other medicinal materials.

[0071] During the research process, referring to relevant literature, a lot of research was also conducted on the thin layer chromatography identification of Smilax glabra, Millettia reticulata, Lindera spp. and Rehmannia glutinosa in Jingu Tong Xiao Wan. The results showed serious interference and failed to achieve the ideal effect. Therefore, it was not used as a quality control method for Jingu Tong Xiao Wan.

[0072] 2.2 Content determination

[0073] 2.2.1 Chromatographic Conditions and System Suitability Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.02% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the conditions in Table 1; the column temperature was 20°C; and the flow rate was 1 ml min -1 , detection wavelength was 270nm, column temperature was 20℃, injection volume was 10μl. A The peak count should be no less than 20,000.

[0074] Table 1 HPLC gradient elution conditions

[0075]

[0076] 2.2.2 Preparation of reference solution Cryptotanshinone, Tanshinone I and Tanshinone II A An appropriate amount of reference substance was accurately weighed and placed in a brown volumetric flask, and methanol was added to make a volumetric solution containing cryptotanshinone and tanshinone II per 1 ml. A A mixed solution of 10 μg of tanshinone I and 5 μg of tanshinone I is obtained.

[0077] 2.2.3 Preparation of test solution Take an appropriate amount of the product, grind it into powder, take about 1.0 g, weigh it accurately, place it in a stoppered conical flask, accurately add 50 ml of methanol, stopper it tightly, weigh it, place it in a 60℃ water bath shaker for 1 hour, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate.

[0078] 2.2.4 Preparation of negative sample solution: Take 1.0 g of the preparation lacking Salvia miltiorrhiza, grind it into powder, and prepare the negative sample solution according to the preparation method of the test solution.

[0079] The chromatograms of the control, test and negative samples are shown in Figure 7 .

[0080] 2.2.5 Linear Relationship Investigation A series of cryptotanshinone, tanshinone I and tanshinone II were prepared A Mixed solution (concentration of 2 to 20 μg ml -1 10 μl of the mixed solution was taken separately, and the peak area was determined according to the above chromatographic conditions. The peak area integral value was used as the ordinate and the injection concentration (μg·ml -1 ) as the horizontal axis, draw the standard curve, and calculate the regression equations:

[0081] Cryptotanshinone Y = 50 209.9X–232.4, R 2 =0.999 4.

[0082] Tanshinone IY=43576.3X–4647.3, R 2 =0.999 4.

[0083] Tanshinone II A Y=57180.1X+2686.6,R 2 =0.999 6.

[0084] The results showed that cryptotanshinone, tanshinone Ⅰ, tanshinone Ⅱ A The analytical concentration is 2 to 20 μg ml -1 In the range of 1:1, there is a good linear relationship between the peak area integral value and the peak area integral value.

[0085] 2.2.6 Precision test: Accurately pipette 10 μl of the same test solution and repeat the injection 6 times. Measure the peak area and calculate the peak area of ​​cryptotanshinone, tanshinone I and tanshinone II. A The peak area RSDs were 0.47%, 0.40% and 0.25%, respectively, indicating that the precision of the instrument was good.

[0086] 2.2.7 Limit of detection and limit of quantification were determined by signal-to-noise ratio method. Cryptotanshinone, Tanshinone I, and Tanshinone II AThe detection limits were 0.301 5ng, 0.301 2ng, and 0.308 4ng, and the quantification limits were 1.005ng, 1.004ng, and 1.028ng, respectively.

[0087] 2.3.8 Stability test Accurately pipette 10 μl of the same test solution and measure the stability of cryptotanshinone, tanshinone I, and tanshinone II at 0, 2, 4, 8, 12, 18, and 24 hours. A The peak area RSDs were 0.65%, 0.30% and 0.41% respectively. The results showed that the test solution was stable within 24 hours.

[0088] 2.2.9 Repeatability Test Six samples from the same batch of test sample (batch number 210103) were extracted according to the test sample solution preparation method and analyzed according to the proposed chromatographic conditions. The average total amount of tanshinone was 0.852 mg·g -1 The RSD was 0.44%. The results showed that the method had good repeatability.

[0089] 2.2.10 Recovery of sample: Take a sample with known content (batch number 210103, 0.852 mg g -1 ) 9 parts, each about 0.50g, accurately weighed, and different concentrations of cryptotanshinone, tanshinone I, and tanshinone II were accurately added to each 3 samples. A The reference solution was prepared using the test solution preparation method. The sample solution was then assayed under the proposed chromatographic conditions, and the recoveries of the three tanshinones were calculated. The results are shown in Tables 2-4. The results showed that the recoveries of the three tanshinones were all between 95% and 105%, demonstrating that the method of the present invention has a good recovery rate.

[0090] Table 2 Cryptotanshinone recovery test results

[0091]

[0092] Table 3 Tanshinone I recovery test results

[0093]

[0094] Table 4 Tanshinone II A Recovery test results

[0095]

[0096] According to the content determination results of 20 batches of products, combined with the content regulations of Danshen medicinal materials in the 2020 edition of the "Chinese Pharmacopoeia" (Part I) and the process transfer rate, the content limit of this product is stipulated as follows: calculated on a dry basis, this product contains cryptotanshinone (C 19 H 20 O3), Tanshinone Ⅰ (C 18 H12 O3) and tanshinone II A (C 19 H 18 O3) shall not be less than 0.42 mg.

[0097] Test example: This test example optimizes the determination conditions of the HPLC method used in the content determination. The optimization process is as follows: 1. Selection of chromatographic conditions

[0098] Preparation of reference solution Cryptotanshinone, Tanshinone I and Tanshinone II A An appropriate amount of reference substance was accurately weighed and methanol was added to prepare a mixed solution containing 10 μg of each substance per 1 ml.

[0099] The test solution was prepared according to the current standard method. The product was ground into powder. 0.4 g was taken and accurately weighed. The solution was placed in a stoppered conical flask. 30 ml of methanol was accurately added and weighed. The solution was ultrasonically treated (power 600 W, frequency 40 kHz) for 45 minutes. The solution was cooled and weighed again. The lost weight was supplemented with methanol. The solution was shaken and filtered. The filtrate was obtained.

[0100] 1.1 Selection of detection wavelength

[0101] Cryptotanshinone, tanshinone I and tanshinone II A For the reference solution, 10 μl was drawn and injected into the liquid chromatograph for analysis according to the method for determining the tanshinone content in Danshen medicinal materials in Part I of the 2020 edition of the Chinese Pharmacopoeia.

[0102] Liquid chromatograph (LC-20AD, SPD-M20A diode array detector, chromatographic column: Shimadzu WondaSIL TM C18Superb 5μm 4.6×250mm).

[0103] The maximum absorption wavelength was determined by using the spectrum generated by the diode array detector. The results showed that within the wavelength range of 200-800 nm, cryptotanshinone had a maximum absorption at 263 nm, tanshinone I had a maximum absorption at 244 nm, and tanshinone II had a maximum absorption at 263 nm. A It has maximum absorption at 268nm.

[0104] 10 μl of the test solution was injected into the liquid chromatograph for analysis. The separation results of the target components in the test sample at each wavelength are shown in Table 5.

[0105] Table 5 Separation of target components in the test sample at each wavelength

[0106]

[0107] The results showed that the separation was good at the detection wavelengths of 268nm and 270nm. At the same time, referring to the detection wavelength for the determination of tanshinone content in Danshen medicinal materials in the 2020 edition of the Chinese Pharmacopoeia, 270nm was selected.

[0108] 1.2 Selection of mobile phase

[0109] Using the chromatographic conditions for the determination of tanshinone content in Danshen medicinal materials in the Part I of the 2020 edition of the Chinese Pharmacopoeia, 10 μl of the test solution was aspirated and the samples were analyzed using acetonitrile-0.01% phosphoric acid, acetonitrile-0.02% phosphoric acid, and acetonitrile-0.03% phosphoric acid as the mobile phases, respectively. The results are shown in Table 6.

[0110] Table 6 Separation of target components in the test samples under different mobile phases

[0111]

[0112] The results showed that when acetonitrile-0.01% phosphoric acid, acetonitrile-0.02% phosphoric acid and acetonitrile-0.03% phosphoric acid were used as mobile phase, the concentrations of cryptotanshinone, tanshinone I and tanshinone II in the test samples were significantly increased. A There was no significant difference in the separation degree. With reference to the mobile phase for the determination of Danshen content in the pharmacopoeia, acetonitrile-0.02% phosphoric acid was selected as the mobile phase.

[0113] 1.3 Selection of elution gradient

[0114] The tanshinone gradient elution method for the determination of the content of Danshen medicinal materials in the 2020 edition of the Chinese Pharmacopoeia was used to detect cryptotanshinone, tanshinone I and tanshinone II in the test sample. A , among which cryptotanshinone had a slightly lower separation degree, and there were many interfering peaks around the target peak. On this basis, we tried to change the gradient so that cryptotanshinone, tanshinone I and tanshinone II in the test sample A The chromatographic parameters such as retention time, separation, and tailing factor are at a good level.

[0115] In this study, several gradient elution methods were selected for comparison, as shown in Tables 7 to 9.

[0116] Table 7 Gradient elution method 1

[0117]

[0118] Table 8 Gradient elution method 2

[0119]

[0120]

[0121] Table 9 Gradient elution method 3

[0122]

[0123] The results showed that using gradient elution method 3, cryptotanshinone, tanshinone I and tanshinone II A All of them were effectively separated, and the separation degree, tailing factor, etc. met the requirements.

[0124] On the basis of these elution methods, we tried to change the initial gradient concentration, change the rate and other elution methods, but the results were not better than gradient elution method 3. Therefore, method 3 was finally selected as the gradient elution method.

[0125] 1.4 Selection of column temperature

[0126] The column temperature was set at 20, 25, 30, and 40°C, respectively. 10 μl of the test solution was taken and injected into a liquid chromatograph (LC-20AD, SPD-M20A diode array detector, chromatographic column: nacalai tesque COSMOSIL5C18-MS-Ⅱ 4.6ID×250mm) for analysis according to the proposed chromatographic conditions. The results showed that the column temperature could be between 20 and 30°C, with the best effect at 20°C.

[0127] 1.5 Determination of the number of theoretical plates

[0128] Among the three tested components, Tanshinone II A The content of tanshinone II is relatively high, so the theoretical plate number of the chromatographic conditions of this method is also based on tanshinone II. A Peak meter. In the experiment, the chromatographic conditions used were: acetonitrile-0.02% phosphoric acid as mobile phase, column temperature 20°C, elution method 3, detection wavelength 270nm, and multiple samples were analyzed on multiple chromatographic columns. Tanshinone II A When the peak theoretical plate number is more than 20,000, the target peak separation can reach more than 1.5. Therefore, the theoretical plate number of the chromatographic conditions of this method is determined to be Tanshinone II. A Peak count should be no less than 20,000.

[0129] 2. Selection of test solution preparation method

[0130] 2.1 Investigation of extraction solvent

[0131] 0.4 g of the test sample (Batch No. 210103) was finely ground and accurately weighed. The mixture was placed in a stoppered conical flask. 30 ml of methanol and 30 ml of ethanol were added and weighed. The mixture was ultrasonically treated (600 W, 40 kHz, room temperature) for 45 minutes. The mixture was allowed to cool and weighed again. The weight loss was made up with methanol or ethanol, respectively. The mixture was filtered and the filtrate was analyzed according to the proposed chromatographic conditions. The results are shown in Table 10.

[0132] Table 10 Selection of extraction solvents

[0133]

[0134] The results showed that methanol was the best solvent for extraction. Therefore, methanol was selected as the extraction solvent.

[0135] 2.2 Investigation of extraction methods

[0136] Take 0.4 g of the test sample from the same batch (batch number 210103), grind it into powder, weigh it accurately, put it into a stoppered conical flask, add 30 ml of methanol, weigh it, heat and reflux for 45 minutes, make up the lost weight with methanol, filter it, take the filtrate, and analyze it according to the proposed chromatographic conditions.

[0137] The total amount of tanshinones in the sample was determined to be 0.8943 mg / g. This indicates that the reflux method is superior to the room-temperature ultrasonic method. The optimal temperature is beneficial for the extraction of total tanshinones, and the temperature should be neither too high nor too low.

[0138] 2.3 Investigation of sample sampling volume and water bath temperature under reflux conditions

[0139] Current quality standards require a sample weight of 0.4g and 30ml of methanol, whereas common pipettes are 25 or 50ml, making extraction inconvenient. This study aimed to investigate the extraction effects of different sample weights and water bath temperatures using 50ml of methanol. The experimental conditions were set as follows:

[0140] Conditions ① 0.4 g, 50 ml methanol, heated to reflux at 70 ° C for 45 min;

[0141] Condition ② 0.6g, 50ml methanol, heated under reflux at 70℃ for 45min;

[0142] Condition ③ 0.8g, 50ml methanol, heated under reflux at 70℃ for 45min;

[0143] Conditions ④1.0g, 50ml methanol, heated under reflux at 70℃ for 45min;

[0144] Conditions ⑤ 0.8g, 50ml methanol, heated to reflux at 80℃ for 45min;

[0145] Condition ⑥ 1.0 g, 50 ml methanol, heated to reflux at 80 ° C for 45 min.

[0146] The results were analyzed according to the proposed chromatographic conditions and are shown in Table 11.

[0147] Table 11 Investigation of sample sampling volume and water bath temperature under reflux conditions

[0148]

[0149]

[0150] Results: The results obtained from several sample weighings were close, and the water bath temperature of 80℃ was slightly better than that of 70℃. Considering that the chromatographic peak area of ​​the test sample and the chromatographic peak area of ​​the reference sample were closer when the sample weight was 1.0g, the sample weight was determined to be 1.0g.

[0151] The study found that although the reflux method had better extraction efficiency than the ultrasonic method, the chromatograms of the sample obtained by the reflux method showed that the tanshinone I peak front and some tailing factors were less than 0.95. Therefore, it was necessary to explore an extraction method that could ensure both extraction efficiency and chromatographic parameters.

[0152] 2.4 Re-examination of extraction methods

[0153] Experiments with ultrasonic extraction at specific temperatures have shown that, within a certain range, increasing the temperature facilitates the extraction of total tanshinones. However, extraction temperatures between 60 and 70°C are more drastic and unstable, with the sample easily splashing out of the bottle, resulting in unreliable results. Temperatures below 50°C also result in incomplete extraction. The study found that using a water bath oscillation method is more effective, as it provides milder conditions, maintains a specific extraction temperature, minimizes sample loss, is easier to operate, and produces more stable results.

[0154] 2.5 Water bath oscillation temperature investigation

[0155] Temperature is a critical parameter for tanshinone extraction. The water bath oscillation temperature was set at 40°C, 50°C, and 60°C (higher temperatures result in a reflux-like effect), the oscillation speed was 150 rpm, and the extraction time was 60 min. The experimental results are shown in Table 12.

[0156] Table 12 Water bath oscillation temperature investigation

[0157]

[0158] The results showed that the extraction effect was better at 60℃.

[0159] 2.6 Investigation of water bath oscillation time

[0160] Under the experimental conditions of a water bath oscillation speed of 150 r / min and 60°C, the water bath oscillation time was set to 30 minutes, 45 minutes, 60 minutes, and 90 minutes, respectively, and extraction tests were carried out. The experimental results are shown in Table 13.

[0161] Table 13 Water bath oscillation time investigation

[0162]

[0163] The results showed that at 60℃, the contents of the four extraction times were similar, with 45 minutes and 60 minutes being the best. Considering that 60 minutes is easier to operate, we chose 60 minutes for this experiment.

[0164] 2.7 Water bath oscillation speed investigation

[0165] Under the experimental conditions of water bath oscillation for 60 minutes at 60°C, the water bath oscillation speeds were set at 100 r / min, 150 r / min and 200 r / min, respectively. The experimental results are shown in Table 14.

[0166] Table 14 Investigation of water bath oscillation speed

[0167]

[0168] The results show that the results measured at 100-200 r / min are close, and the effect at 100 r / min is better. Under the condition of temperature 60°C, the speed has limited effect on the experimental results, but higher speed, such as 200 r / min, may cause the test sample to splash out in the experiment, which may easily cause unstable results. Because the water bath oscillation speed has little effect, the speed is not specified in the preparation method of the test sample solution.

[0169] 2.8 Investigation of water bath oscillation solvent amount

[0170] The investigation conditions of water bath oscillation solvent amount were as follows: the test sample was finely ground, about 1.0 g was accurately weighed into a conical flask with a stopper, 40 / 50 / 60 ml of methanol was accurately added, the weight was determined, it was placed in a 60°C water bath environment and oscillated at 100 r / min for 60 minutes, it was cooled, the weight was determined again, the lost weight was made up with methanol, it was shaken uniformly, filtered, and the filtrate was obtained. The analysis results are shown in Table 15.

[0171] Table 15 Investigation of water bath oscillation solvent amount

[0172]

[0173]

[0174] The results show that when the sample amount is 1.0 g, the methanol amounts of 40 ml, 50 ml and 60 ml have little difference. Therefore, the methanol amount is determined to be 50 ml.

[0175] Based on the above research, the preparation method of the test sample solution is determined as follows:

[0176] An appropriate amount of the product was finely ground, about 1.0 g was accurately weighed into a conical flask with a stopper, 50 ml of methanol was accurately added, the weight was determined, it was placed on a 60°C water bath shaker for 1 hour, it was cooled, the weight was determined again, the lost weight was made up with methanol, it was shaken uniformly, filtered, and the filtrate was obtained.

[0177] 3 Negative test

[0178] Considering the low content of tanshinone I in the test sample, the concentration of the reference solution was adjusted to cryptotanshinone and tanshinone II. A The peak areas of the test and reference substances were close at 10 μg / ml and 5 μg / ml of tanshinone I respectively.

[0179] Preparation of reference solution: Take cryptotanshinone, tanshinone I and tanshinone II A An appropriate amount of reference substance was accurately weighed and methanol was added to prepare a solution containing cryptotanshinone and tanshinone II per 1 ml. A Mixed solution of 10 μg each and 5 μg of tanshinone I (the concentrations of the reference solution are: cryptotanshinone 10.44 μg / ml, tanshinone I 5.07 μg / ml and tanshinone II A 10.01 μg / ml).

[0180] Prepare the test solution according to the test solution preparation method.

[0181] Preparation of negative sample solution: Take about 1.0 g of the preparation of Salvia miltiorrhiza, grind it into powder, and prepare the negative sample solution according to the preparation method of the test solution.

[0182] High performance liquid chromatograph: LC-20AD; detector: SPD-M20A diode array detector; column oven: CTO-20AC.

[0183] Chromatographic column: Nacalai tesque COSMOSIL 5C18-MS-Ⅱ 4.6 ID × 250 mm

[0184] Pipette 10 μl each of the reference solution, test solution, and negative sample solution, inject them into the liquid chromatograph, and analyze according to the proposed chromatographic conditions.

[0185] The results showed that the negative samples had no interference at the corresponding locations of the three reference substances.

[0186] Comparative Example: The Jin Gu Tong Xiao Wan of the present invention has the current standard number: WS3-423 (Z-067) -2000Z-2009. The specific contents involved under this standard are as follows:

[0187] 1. TLC identification.

[0188] (1) Take 3g of this product, grind it into powder, add 30ml of ether, shake it, let it stand for 1 hour, filter it, evaporate the filtrate, add 1ml of ethyl acetate to dissolve the residue, and use it as the test solution. Take 0.5g of Danshen as a control medicinal material and prepare a control medicinal material solution in the same way. Then take Tanshinone II AFor the reference substance, add ethyl acetate to prepare a solution containing 2 mg per 1 ml. This serves as the reference solution. According to the thin layer chromatography method (Appendix VI B of the Part I of the Chinese Pharmacopoeia 2005 edition), 5 μl of each of the three solutions was spotted on the same silica gel G thin layer plate. Develop the plate using toluene-ethyl acetate (19:1), remove the plate, and air dry. The test sample chromatogram should show spots of the same color at the corresponding positions in the control herb chromatogram and the reference substance chromatogram.

[0189] (2) Take 3 g of this product, grind it into powder, add 30 ml of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol, add an appropriate amount of neutral alumina and mix well, load it onto a pre-filled neutral alumina column (inner diameter about 1 cm, alumina about 4 g), elute with 40 ml of methanol, discard the first 2 ml of eluate, collect the subsequent eluate, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use it as the test solution. Separately, take the paeoniflorin reference substance, add methanol to make a solution containing 2 mg per 1 ml, and use it as the reference solution. According to the thin layer chromatography method (Appendix VI B of Part I of the Chinese Pharmacopoeia 2005 Edition), take 4 μl of each of the above two solutions and spot them on the same silica gel G thin layer plate, use chloroform-n-butanol-glacial acetic acid (3:3:0.2) as the developing solvent, develop, remove, dry, spray with 5% vanillin sulfuric acid solution, and heat until the spots are clearly colored. In the chromatogram of the test sample, the same blue-purple spot appears at the corresponding position in the chromatogram of the reference sample.

[0190] (3) Take 5g of this product, grind it into powder, add 20ml of ether and soak it for 1 hour, shake it from time to time, filter it, evaporate the filtrate to dryness, add 2ml of ethyl acetate to dissolve the residue, and use it as the test solution. Take another 1g of Cyperus rotundus as the reference medicinal material, and prepare the reference medicinal material solution in the same way. Take α-Cyperus rotundus ketone as the reference material, add ethyl acetate to prepare a solution containing 1μl per 1ml, and use it as the reference solution. According to the thin layer chromatography method (Appendix VI B of the first part of the Chinese Pharmacopoeia 2005 edition), take 2μl of each of the three solutions mentioned above and spot them on the same piece of silica gel GF 254 Develop the plate with petroleum ether (60-90°C)-ethyl acetate (15:1) as the developing solvent, remove the plate, air dry, and examine it under ultraviolet light (254 nm). In the chromatogram of the test sample, spots of the same color will appear at the corresponding positions in the chromatograms of the reference medicinal material and the reference sample.

[0191] (4) Take 2g of this product, grind it into powder, add 20ml of ethanol-10% ammonia solution (50:7) and soak it for 3 hours, shaking occasionally. Filter, recover the ethanol from the filtrate and evaporate to dryness, add 5ml of methanol to dissolve the residue, filter, and use the filtrate as the test solution. Take 1g of the reference medicinal material Gentiana macrophylla and prepare the reference medicinal material solution in the same way. According to the thin layer chromatography method (Appendix VI B of Part I of the 2005 edition of the Chinese Pharmacopoeia), take 2μl of each of the above two solutions and spot them on the same silica gel G thin layer plate. Use toluene-chloroform-acetone-isopropanol (8:4:1:1) as the developing solvent, develop, remove, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test product, the same green fluorescent spot will appear at the corresponding position in the chromatogram of the reference medicinal material.

[0192] (5) Take 2g of this product, grind it into powder, add 40ml of 75% ethanol, reflux extract for 1 hour, filter, evaporate the filtrate to dryness, add 10ml of water to dissolve the residue, shake and extract with 20ml of ether, evaporate the extract to dryness, and add 1ml of anhydrous ethanol to dissolve the residue to prepare the test solution. Take 1g of Sichuan Cyathula as a control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (Appendix VI B of Part I of the Chinese Pharmacopoeia 2005 Edition), take 4μl of each of the above two solutions and spot them on the same silica gel G thin layer plate. Use toluene-chloroform-acetone (8:4:1) as the developing solvent, develop, remove, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test product, the same blue fluorescent spot appears at the corresponding position in the chromatogram of the control medicinal material.

[0193] (6) Take 5g of this product, grind it into powder, transfer it into a 500ml round-bottom flask, add 200ml of water and a few glass beads, mix well, connect it to a volatile oil analyzer (d<1), add water from the top of the analyzer to the scale, and until it overflows into the flask, then add 5ml of petroleum ether (60-90℃), connect it to a reflux condenser, heat to boiling, and keep it boiling for 1 hour, let it cool, and take the petroleum ether layer as the test solution. Take 2g of cinnamon twig control medicinal material and prepare the control medicinal material solution in the same way. Take cinnamaldehyde reference material and add petroleum ether (60-90℃) to make a solution containing 1μl per 1ml, which is used as the reference solution. According to the thin layer chromatography method (Appendix VI B of Part I of the 2005 edition of the Chinese Pharmacopoeia), 2 μl of each of the three solutions was spotted on the same silica gel G thin layer plate. The plate was developed with petroleum ether (60-90°C)-ethyl acetate (17:3). The plate was removed, air-dried, and sprayed with dinitrophenylhydrazine ethanol test solution. In the chromatogram of the test sample, an orange-red spot of the same color appeared at the corresponding position in the chromatogram of the reference sample.

[0194] 2. Content determination.

[0195] Determined by high performance liquid chromatography (Appendix ⅥD of Part I of the Chinese Pharmacopoeia 2005 edition).

[0196] (1) Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel was used as filler; methanol-water (75:25) was used as mobile phase; the detection wavelength was 268 nm. The theoretical plate number was calculated based on the tanshinone II A Peak count should be no less than 2000.

[0197] (2) Preparation of reference solution Tanshinone II A Accurately weigh an appropriate amount of reference substance and add methanol to make a solution containing 10 μg per 1 ml.

[0198] (3) Preparation of test solution: Take the product under the item of difference in filling amount, grind it into powder, take about 0.4 g, weigh it accurately, place it in a stoppered conical flask, add 30 ml of methanol accurately, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 45 minutes. Let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate.

[0199] (4) Determination method: Accurately aspirate 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0200] (5) Calculated on a dry basis, each 1g of this product contains tanshinone II A (C 19 H 18 O3), it shall not be less than 0.34 mg.

[0201] It should be noted that the above-described embodiments are to be understood as illustrative and not limiting of the scope of protection of the present invention, which is subject to the claims. It will be apparent to those skilled in the art that non-essential improvements and adjustments to the present invention, without departing from the spirit and scope of the present invention, still fall within the scope of protection of the present invention.

Claims

1. A quality control method for Jingutongxiao pills, comprising identification and content determination; characterized in that: The identification items include thin layer chromatography identification of Cyperus rotundus, Cinnamomum cassia twig, White Peony Root, Cyathula root, Gentiana macrophylla and Licorice root, as follows: (1) Thin layer chromatography identification method of Cyperus rotundus: Take 10 g of this product, grind it into powder, put it into a 500 ml round-bottom flask, add 200 ml of water and a few glass beads, mix well, connect the volatile oil analyzer, add water from the upper end of the analyzer to the scale, and until it overflows into the flask, then add 2 ml of petroleum ether with a boiling range of 60-90 ° C, connect a reflux condenser, heat to boiling, and keep it boiling for 1 hour, let it cool, and take the petroleum ether layer as the test solution; take another 1 g of Cyperus rotundus reference medicinal material, and prepare the reference medicinal material solution in the same way; then take α-cyperone reference material, add ethyl acetate to prepare a solution containing 1 mg per 1 ml, as the reference material solution; according to the thin layer chromatography test, take 8 μl of the test solution, the reference medicinal material solution and the reference material solution 1 μl, and spot them on the same silica gel GF 254 On a thin layer plate, develop the plate using a mixture of dichloromethane-ethyl acetate-glacial acetic acid in a volume ratio of 80:1:

1. Remove the plate, air dry, and examine it under a 254 nm ultraviolet lamp. In the chromatogram of the test sample, spots of the same color appear at corresponding positions in the chromatograms of the reference medicinal material and the reference sample. Spray with dinitrophenylhydrazine test solution and let it sit for a while, the spots will gradually turn orange-red; (2) Thin layer chromatography identification method of cinnamon twig: take the test solution of the identification item of Cyperus rotundus in (1) as the test solution; take another cinnamaldehyde reference substance, add petroleum ether with a boiling range of 60-90 °C to make a solution containing 1 μl per 1 ml, as the reference solution; according to the thin layer chromatography test, take 4 μl of the test solution and the reference solution, respectively, and spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate with a boiling range of 60-90 °C in a volume ratio of 17:3 as the developing solvent, develop, take out, dry, and spray with dinitrophenylhydrazine ethanol test solution; in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference substance, a spot of the same color appears; (3) Thin layer chromatography identification method of white peony root: take 3 g of this product, grind it into powder, add 30 ml of ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, let it cool, add 30 ml of water to the residue, stir, filter, extract the filtrate twice with 20 ml of n-butanol saturated with water, combine the n-butanol solution, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution; take another paeoniflorin reference substance, add methanol to make a solution containing 2 mg per 1 ml, and use it as the reference solution; according to the thin layer chromatography test, take 5 μl of the test solution and 2 μl of the reference solution, and spot them on the same silica gel G thin layer plate respectively, use ethyl acetate-acetone-formic acid-water with a volume ratio of 15:6:2:0.5 as the developing solvent, develop, take out, dry, spray with 5% vanillin in 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly colored; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference substance; (4) Thin layer chromatography identification method of Cyathula obtusifolia: Take 3 g of this product, grind it into powder, add 40 ml of 75% ethanol, reflux extract for 1 hour, filter, evaporate the filtrate to dryness, add 10 ml of water to dissolve the residue, shake and extract with 20 ml of ether, evaporate the ether solution to dryness, add 1 ml of anhydrous ethanol to dissolve the residue, and use it as the test solution; take another 0.5 g of Cyathula obtusifolia as a control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 4 μl of the test solution and the control medicinal material solution, respectively, and spot them on the same silica gel G thin layer plate, use toluene-chloroform-acetone with a volume ratio of 8:4:1 as the developing solvent, develop, take out, dry, and examine under a 365 nm ultraviolet lamp; in the chromatogram of the test product, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material; (5) Thin layer chromatography identification method of Gentiana macrophylla: Take 1 g of this product, grind it into powder, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution; take another gentiopicroside reference substance, add methanol to make a solution containing 1 mg per 1 ml, and use it as the reference solution; according to the thin layer chromatography test, take 2 μl of the test solution and the reference solution, and spot them on the same silica gel GF 254 On a thin layer plate, develop with ethyl acetate-methanol-water in a volume ratio of 10:2:1, remove, dry, and examine under a 254nm ultraviolet lamp; in the chromatogram of the test sample, a spot of the same color will appear at the corresponding position in the chromatogram of the reference sample; (6) Thin layer chromatography identification method of licorice: take 4 g of this product, grind it into powder, add 30 ml of methanol, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, add 30 ml of 5% sodium carbonate solution to dissolve the residue, wash it twice with ethyl acetate, 30 ml each time, adjust the pH value of the aqueous layer to 2-3 with 10% hydrochloric acid, shake and extract it twice with ethyl acetate, 30 ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution; take another 0.2 g of licorice control medicinal material, and prepare the control medicinal material solution in the same way; take the glycyrrhizin reference substance, add methanol to prepare a solution containing 0.5 mg per 1 ml, and use it as the reference solution; according to the thin layer chromatography test, take 1-2 ml of each of the test solution, control medicinal material solution, and reference solution. 1 μl, spotted separately on the same silica gel G thin layer plate, developed with ethyl acetate-formic acid-glacial acetic acid-water in a volume ratio of 15:1:1:2, removed, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under sunlight and 365nm ultraviolet light respectively; in the chromatogram of the test sample, at the corresponding position in the chromatogram of the control medicinal material, a main spot or fluorescent spot of the same color appeared; in the chromatogram of the control, a spot or fluorescent spot of the same color appeared at the corresponding position; Determination items: Control of cryptotanshinone, tanshinone I and tanshinone II A The total amount of the content limit is: each 1 g of Danshen contains cryptotanshinone, tanshinone I and tanshinone II A The total amount shall not be less than 0.42 mg.

2. The quality control method of a Jin Gu Tong Xiao Wan according to claim 1, characterized in that: The content determination items are determined by liquid chromatography; The chromatographic conditions were as follows: octadecylsilane bonded silica gel as filler, acetonitrile-0.02% phosphoric acid solution as mobile phase, gradient elution, detection wavelength at 270 nm, injection volume of 10 μl; the theoretical plate number was calculated based on the tanshinone II A Peak count should be no less than 20,000; Preparation of reference solution: Take cryptotanshinone, tanshinone I and tanshinone II A An appropriate amount of reference substance was accurately weighed and placed in a brown volumetric flask, and methanol was added to make a volumetric solution containing cryptotanshinone and tanshinone II per 1 ml. A A mixed solution of 10 μg of tanshinone I and 5 μg of tanshinone I was obtained; Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1.0 g, weigh it accurately, place it in a stoppered conical flask, add 50 ml of methanol accurately, stopper it tightly, weigh it accurately, keep it in a 60℃ water bath shaker for 1 hour, let it cool, weigh it accurately again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the product; Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatography instrument, and determine; Calculated on the basis of dry product, each 1 g of Danshen contains cryptotanshinone, tanshinone I and tanshinone II. A The total amount shall not be less than 0.42 mg.

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