Quality detection method of rhizoma corydalis stomach harmonizing granules

Abstract

The invention relates to the technical field of quality detection of traditional Chinese medicines, in particular to a quality detection method of rhizoma corydalis stomach harmonizing granules. The method comprises the steps of thin-layer identification of vinegar-processed rhizoma corydalis, thin-layer identification of rhizoma bletillae and content determination of tetrahydropalmatine. A methodology investigation experiment proves that the detection method has the advantages of strong specificity, high accuracy and good durability, is simple and convenient to operate, and has important significance for improving the product quality.

Description

Technical Field

[0001] The invention relates to the technical field of quality detection of traditional Chinese medicines, and in particular to a quality detection method for Yuanhu Hewei granules. Background Art

[0002] In Traditional Chinese Medicine (TCM), the diagnostic criteria for spleen and stomach disorders include symptoms such as stomach pain, acid reflux, noisy stomach, abdominal distension, vomiting, choking, regurgitation, hiccups, abdominal pain, and diarrhea. Modern medicine primarily treats spleen and stomach disorders with symptomatic supportive care. While effective, this approach is associated with high recurrence rates and numerous adverse reactions. Traditional Chinese medicine preparations for the treatment of spleen and stomach disorders are widely favored by clinicians and patients due to their significant efficacy and minimal side effects.

[0003] Yuanhu Hewei Granules are a prescription formulated by Dr. Yan Huayong, a nationally renowned grassroots Chinese medicine doctor at Puding County Traditional Chinese Medicine Hospital, based on the traditional Chinese medicine philosophy of treating spleen and stomach diseases. This prescription has been approved by our hospital's Medical Department as a hospital agreement prescription and has a long history of use in our hospital. Long-term clinical observations have confirmed that this prescription has significant therapeutic effects in the treatment of spleen and stomach diseases such as moderate to severe superficial gastritis, erosive gastritis, gastric and duodenal ulcers, and has great clinical promotion value.

[0004] The prescription composition of Yuanhu Hewei Granules is: vinegar Corydalis 6g, bran-fried Citrus aurantium 3g, citron 3g, Panax notoginseng 3g, cuttlebone 6g, Cynanchum chinense 6g, Bletilla striata 6g, Gypsophila paniculata 3g, Pseudostellaria pseudoginseng 3g, Tripterygium wilfordii 6g, and Fritillaria thunbergii 3g. The preparation method of the above prescription is as follows: weigh each medicinal material according to the formula, add water and boil it three times, add 9 times the weight of the medicinal material in water for the first time, soak for 1 hour and then boil for 1 hour, add 8 times the weight of the medicinal material in water for the second and third times, and boil for 1 hour each time; filter the decoction, combine the filtrates, and concentrate under reduced pressure to form a clear paste with a density of 1.05 to 1.20 at 80°C, dry and crush in an oven at 105°C to prepare a dry paste powder; take the dry paste powder prepared in the above steps, add a mixed excipient consisting of dextrin and microcrystalline cellulose in a mass ratio of 7:1, and use 70% ethanol solution as a wetting agent to prepare a soft material, wherein the standard of the soft material is that it can be lumped together by hand and falls apart when touched lightly; wherein the mass ratio of the dry paste powder to the mixed excipient is 5:4; squeeze the prepared soft material through a No. 1 sieve to granulate, dry it with air at 80°C, and granulate it with a No. 1 sieve and a No. 5 sieve, and collect the particles between the No. 1 sieve and the No. 5 sieve to obtain the obtained soft material. At present, the quality inspection methods for Yuanhuhewei granules have not yet established methods for identifying relevant medicinal flavors and determining the content of active ingredients, making it difficult to achieve systematic and comprehensive control of drug quality.

[0005] To solve the above problems, the present invention establishes a quality detection method for Yuanhuhewei granules, which includes thin layer identification of vinegared Corydalis yanhusuo, thin layer identification of Bletilla striata and determination of the content of tetrahydropalmatine, aiming to achieve better control of drug quality. Summary of the Invention

[0006] The purpose of the present invention is to provide a quality detection method for Yuanhu Hewei granules.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The quality detection method of the Yuanhu Hewei granules of the present invention includes thin layer identification of vinegared Corydalis yanhusuo, thin layer identification of Bletilla striata and determination of the content of tetrahydropalmatine, which is specifically as follows:

[0009] The thin layer chromatography identification of vinegar Corydalis is as follows: take an appropriate amount of this product, grind it into powder, accurately weigh 10-20g, place it in a conical flask, add 50-100mL of 70% methanol solution, ultrasonically treat it for 20-40min, filter, evaporate the filtrate to dryness, add 5-20mL of 0.5% sodium hydroxide solution to the residue, shake it to dissolve, shake it with 10-30mL of ether for 2-4 times, combine the ether solution, evaporate it to dryness, add 1-3mL of methanol to dissolve the residue, and use it as the test solution; take the corydalis butylene standard substance, add Prepare a solution containing 0.5 mg per 1 mL of methanol as the reference solution; according to the thin layer chromatography test, aspirate 5-10 μL of each of the above two solutions and spot them on the same silica gel G thin layer plate prepared with 2% sodium hydroxide. Use toluene-dichloromethane-ethyl acetate as the developing solvent. Develop the plate, remove the plate, dry it, place it in an iodine jar for 3-5 minutes, then remove it and examine it under ultraviolet light at 365 nm. In the chromatogram of the test sample, a fluorescent spot of the same color will appear at the corresponding position in the chromatogram of the reference sample.

[0010] The thin-layer chromatography identification of Bletilla striata is as follows: prepare the test solution according to the method under the thin-layer chromatography identification of vinegar-corydalis yanhusuo; take 1g of Bletilla striata as a control medicinal material and prepare the control medicinal material solution in the same manner; according to the thin-layer chromatography test, take 5-10μL of each of the above two solutions and spot them on the same silica gel G thin-layer plate, use petroleum ether-dichloromethane-methanol as the developing solvent, develop, remove, dry, spray with 5%-15% sulfuric acid ethanol solution, heat at 105℃ for 3-8 minutes, and let it stand for 20-50 minutes; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material;

[0011] The content of tetrahydropalmatine was determined as follows:

[0012] Chromatographic conditions and system suitability test: The chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution (adjusted to pH 6.0 with triethylamine) was used as mobile phase B, with gradient elution; the flow rate was 0.8-1.2 mL / min; the column temperature was 25-30°C; the detection wavelength was 280 nm; the theoretical plate number calculated based on the tetrahydropalmatine peak should be no less than 3000; the gradient elution procedure was as follows:

[0013]

[0014]

[0015] Preparation of reference solution: Take an appropriate amount of tetrahydropalmatine reference substance, accurately weigh it, and add methanol to make a solution containing 10 μg per 1 mL.

[0016] Preparation of test solution:

[0017] Take an appropriate amount of this product, grind it into powder, accurately weigh 1-3g, place it in a stoppered conical flask, accurately add 5-15mL of methanol, weigh it, and extract it by ultrasonic for 20-40min. Make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the product.

[0018] Determination method: Accurately aspirate 5-10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0019] Preferably, the thin layer identification of vinegar Corydalis in the quality detection method of Yuanhuhewei granules of the present invention is specifically as follows: take an appropriate amount of this product, grind it into powder, accurately weigh 10-15g, place it in a conical flask, add 50-80mL of 70% methanol solution, ultrasonically treat for 30-40min, filter, evaporate the filtrate to dryness, add 5-15mL of 0.5% sodium hydroxide solution to the residue, shake to dissolve, shake and extract with 10-20mL of ether 3-4 times, combine the ether solution, evaporate to dryness, add 1-2mL of methanol to dissolve the residue, and use it as the test solution. liquid; take tetrahydropalmatine reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, which is used as the reference substance solution; according to the thin layer chromatography test, aspirate 8-10 μL of each of the above two solutions, respectively, and spot them on the same silica gel G thin layer plate prepared with 2% sodium hydroxide, use toluene-dichloromethane-ethyl acetate as the developing solvent, develop, take out, dry, place in an iodine cylinder for 4-5 minutes, take out, and examine under ultraviolet light at 365 nm; in the chromatogram of the test substance, at the corresponding position in the chromatogram of the reference substance, a fluorescent spot of the same color will appear.

[0020] Further preferably, the thin layer identification of vinegar Corydalis in the quality detection method of Yuanhuhewei granules of the present invention is specifically as follows: take an appropriate amount of this product, grind it finely, accurately weigh 10g, place it in a conical flask, add 60mL of 70% methanol solution, ultrasonically treat for 30min, filter, evaporate the filtrate to dryness, add 10mL of 0.5% sodium hydroxide solution to the residue, shake to dissolve, shake and extract 3 times with 15mL of ether, combine the ether solution, evaporate to dryness, and add 1mL of methanol to dissolve the residue as the test solution; take Corydalis butylene glycol for 4 minutes. For the reference sample, methanol is added to prepare a solution containing 0.5 mg per 1 mL, which is used as the reference solution; according to the thin layer chromatography test, 8 μL of each of the above two solutions are respectively spotted on the same silica gel G thin layer plate prepared with 2% sodium hydroxide, and developed with toluene-dichloromethane-ethyl acetate in a ratio of 8:2:1. The plate is taken out, dried, placed in an iodine cylinder for 4 minutes, and then taken out and examined under ultraviolet light at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the reference sample.

[0021] Preferably, the thin layer chromatography identification of Bletilla striata in the quality detection method of Yuanhuhewei granules described in the present invention is specifically as follows: prepare a test solution according to the method under the thin layer chromatography identification of vinegar Corydalis yanhusuo; take 1 g of Bletilla striata control medicinal material and prepare a control medicinal material solution in the same way; according to the thin layer chromatography test, take 7 to 9 μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-dichloromethane-methanol as the developing agent, develop, take out, dry, spray with 5% to 10% sulfuric acid ethanol solution, heat at 105°C for 4 to 6 minutes, and let it stand for 20 to 40 minutes; in the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions of the control medicinal material chromatogram.

[0022] Further preferably, the thin layer identification of Bletilla striata in the quality detection method of Yuanhuhewei granules described in the present invention is specifically as follows: prepare a test solution according to the method under the thin layer identification of vinegar Corydalis yanhusuo; take 1 g of Bletilla striata control medicinal material and prepare a control medicinal material solution in the same way; according to the thin layer chromatography test, aspirate 8 μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-dichloromethane-methanol with a ratio of 9:5:2 as the developing agent, develop, take out, dry, spray with 5% sulfuric acid ethanol solution, heat at 105°C for 5 minutes, and let it stand for 30 minutes; in the test sample chromatogram, at the corresponding position of the control medicinal material chromatogram, a fluorescent spot of the same color appears.

[0023] Preferably, in the quality detection method of Yuanhu Hewei granules of the present invention, the boiling range of the petroleum ether is 60-90°C.

[0024] Preferably, in the quality detection method of Yuanhuhewei granules of the present invention, the flow rate of the chromatographic conditions and system suitability test in the determination of tetrahydropalmatine content is 1.0 mL / min and the column temperature is 30°C.

[0025] Preferably, in the quality detection method of Yuanhuhewei granules described in the present invention, the preparation of the test solution described in the content determination of tetrahydropalmatine is specifically as follows: take an appropriate amount of the product, grind it into powder, accurately weigh 2 to 3 g, place it in a stoppered conical flask, accurately add 10 to 15 mL of methanol, weigh the weight, ultrasonically extract for 30 to 40 minutes, make up the lost weight with methanol, shake well, filter; take the filtrate to obtain.

[0026] Further preferably, in the quality detection method of Yuanhuhewei granules described in the present invention, the preparation of the test solution described in the content determination of tetrahydropalmatine is specifically as follows: take an appropriate amount of the product, grind it into powder, accurately weigh 2 g, place it in a stoppered conical flask, accurately add 10 mL of methanol, weigh the weight, ultrasonically extract for 30 minutes, make up the lost weight with methanol, shake well, filter; take the filtrate to obtain.

[0027] Preferably, in the quality detection method of Yuanhuhewei granules of the present invention, the injection volume in the determination of the content of tetrahydropalmatine is 10 μL.

[0028] Beneficial effects of the present invention:

[0029] 1. The present invention establishes a quality detection method for Yuanhuhewei granules, which includes thin layer identification of vinegared Corydalis yanhusuo, thin layer identification of Bletilla striata and determination of tetrahydropalmatine content, which is of great significance to the large-scale production of Yuanhuhewei granules.

[0030] 2. The research team investigated the effects of different mobile phase compositions on chromatographic separation in the determination of tetrahydropalmatine content, and concluded that the optimal mobile phase composition was: acetonitrile as mobile phase A, 0.1% phosphoric acid solution (adjusted to pH 6.0 with triethylamine) as mobile phase B, and gradient elution. Methodological investigation experiments such as specificity tests, linearity tests, recovery tests, precision tests, and durability tests confirmed that the quality detection method provided by the present invention has the advantages of strong specificity, good durability, high accuracy, and good stability. In addition, the method is simple and quick to operate, and can achieve better control of product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the TLC identification spectrum of vinegar-corydalis (in the figure: 1, 2, 3 are three batches of Yuanhuhewei granule samples; 4 is the tetrahydropalmatine reference substance; 5 is the negative sample lacking vinegar-corydalis);

[0032] Figure 2 The TLC identification patterns of vinegar Corydalis yanhusuo under different temperature conditions (in the figure: A is the TLC pattern under 4°C; B is the TLC pattern under 20°C; C is the TLC pattern under 35°C);

[0033] Figure 3 The TLC identification patterns of vinegar Corydalis yanhusuo under different humidity conditions (in the figure: A is the TLC pattern under 20% humidity; B is the TLC pattern under 55% humidity; C is the TLC pattern under 90% humidity);

[0034] Figure 4 The TLC identification patterns of vinegar Corydalis yanhusuo using TLC plates from different manufacturers (in the figure: A is the TLC pattern of the thin layer plate from the Sijia Biochemical Plastic Factory in Luqiao, Taizhou City, Zhejiang Province; B is the TLC pattern of the thin layer plate from Qingdao Bangkai High-tech Materials Co., Ltd.);

[0035] Figure 5 This is the TLC identification spectrum of Bletilla striata (in the figure: 1, 2, and 3 are three batches of Yuanhu Hewei granule samples; 4 is the control medicinal material of Bletilla striata; 5 is the negative sample lacking Bletilla striata);

[0036] Figure 6 The following are the TLC patterns of Bletilla striata under different temperature conditions (in the figure: A is the TLC pattern under 4°C; B is the TLC pattern under 20°C; C is the TLC pattern under 35°C);

[0037] Figure 7 The following are the TLC patterns of Bletilla striata under different humidity conditions (A is the TLC pattern under 20% humidity; B is the TLC pattern under 55% humidity; C is the TLC pattern under 90% humidity);

[0038] Figure 8 The identification patterns of Bletilla striata TLC plates using TLC plates from different manufacturers are shown in Figure 1 (A is the TLC pattern of a TLC plate from the Sijia Biochemical Plastics Factory in Luqiao, Taizhou City, Zhejiang Province; B is the TLC pattern of a TLC plate from Qingdao Bangkai High-tech Materials Co., Ltd.);

[0039] Figure 9 is the ultraviolet spectrum of tetrahydropalmatine;

[0040] Figure 10 is the chromatogram of solvent methanol;

[0041] Figure 11 This is the chromatogram of tetrahydropalmatine reference substance;

[0042] Figure 12 This is the chromatogram of Yuanhu Hewei Granule sample;

[0043] Figure 13 This is the chromatogram of the negative sample of Corydalis yanhusuo lacking vinegar;

[0044] Figure 14 is the standard curve of tetrahydropalmatine;

[0045] Figure 15 is the detection limit determination spectrum;

[0046] Figure 16 The chromatogram is for the quantification limit determination. DETAILED DESCRIPTION

[0047] The following is a detailed description of the technical solution of the present invention in conjunction with specific embodiments. The following embodiments are only for explanation and illustration, and do not constitute a limitation of the technical solution of the present invention.

[0048] Example 1

[0049] The quality inspection method of Yuanhuhewei granules includes thin layer identification of vinegared Corydalis yanhusuo, thin layer identification of Bletilla striata and determination of tetrahydropalmatine content, as follows;

[0050] (1) Thin layer chromatography identification of vinegar Corydalis yanhusuo

[0051] Take an appropriate amount of this product, grind it into powder, accurately weigh 10 g, place it in a conical flask, add 60 mL of 70% methanol solution, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 10 mL of 0.5% sodium hydroxide solution to the residue, shake to dissolve it, extract it with 15 mL of ether by shaking three times, combine the ether solutions, evaporate to dryness, and add 1 mL of methanol to dissolve the residue to prepare the test solution; take the tetrahydropalmatine reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, and use it as the reference solution; according to the thin layer chromatography test, take 8 μL of each of the above two solutions and spot them on the same silica gel G thin layer plate prepared with 2% sodium hydroxide, use toluene-dichloromethane-ethyl acetate in a ratio of 8:2:1 as the developing solvent, take out, dry, place in an iodine cylinder for 4 minutes, take out, and examine under ultraviolet light at 365 nm; in the chromatogram of the test product, at the corresponding position of the chromatogram of the reference substance, a fluorescent spot of the same color will appear.

[0052] (2) Thin layer identification of Bletilla striata

[0053] Prepare the test solution according to the method under the thin layer chromatography identification of vinegar Corydalis yanhusuo; take 1 g of Bletilla striata control medicinal material and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, aspirate 8 μL of each of the above two solutions and spot them on the same silica gel G thin layer plate, use petroleum ether-dichloromethane-methanol in a ratio of 9:5:2 as the developing solvent, develop, take out, dry, spray with 5% sulfuric acid ethanol solution, heat at 105°C for 5 minutes, and let it stand for 30 minutes; in the test sample chromatogram, at the corresponding position of the control medicinal material chromatogram, a fluorescent spot of the same color will appear.

[0054] (3) Determination of Tetrahydropalmatine Content

[0055] Chromatographic conditions and system suitability test: The chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution (adjusted to pH 6.0 with triethylamine) was used as mobile phase B, with gradient elution; the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 280 nm; the theoretical plate number calculated based on the tetrahydropalmatine peak should be no less than 3000; the gradient elution procedure was as follows:

[0056]

[0057] Preparation of reference solution: Take an appropriate amount of tetrahydropalmatine reference substance, accurately weigh it, and add methanol to make a solution containing 10 μg per 1 mL.

[0058] Preparation of test solution:

[0059] Take an appropriate amount of this product, grind it into powder, accurately weigh 2 g, place it in a stoppered conical flask, accurately add 10 mL of methanol, weigh the weight, extract by ultrasonic for 30 minutes, make up the lost weight with methanol, shake well, filter; take the filtrate to obtain the product.

[0060] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0061] Example 2

[0062] The quality inspection method of Yuanhuhewei granules includes thin layer identification of vinegared Corydalis yanhusuo, thin layer identification of Bletilla striata and determination of tetrahydropalmatine content, as follows;

[0063] (1) Thin layer chromatography identification of vinegar Corydalis yanhusuo

[0064] Take an appropriate amount of this product, grind it into powder, accurately weigh 10 g, place it in a conical flask, add 50 mL of 70% methanol solution, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, add 5 mL of 0.5% sodium hydroxide solution to the residue, shake to dissolve it, extract it with 10 mL of ether and shake it three times, combine the ether solution, evaporate to dryness, and add 1 mL of methanol to dissolve the residue to prepare the test solution; take the tetrahydropalmatine reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, and use it as the reference solution; according to the thin layer chromatography test, take 5 μL of each of the above two solutions and spot them on the same silica gel G thin layer plate prepared with 2% sodium hydroxide, use toluene-dichloromethane-ethyl acetate in a ratio of 8:2:1 as the developing solvent, take out, dry, place in an iodine cylinder for 3 minutes, then take out and examine under ultraviolet light at 365 nm; in the chromatogram of the test product, at the corresponding position of the chromatogram of the reference substance, a fluorescent spot of the same color will appear.

[0065] (2) Thin layer identification of Bletilla striata

[0066] Prepare the test solution according to the method under the thin layer identification of vinegar Corydalis yanhusuo; take 1 g of Bletilla striata control medicinal material and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 5 μL of each of the above two solutions and spot them on the same silica gel G thin layer plate, use petroleum ether (60-90°C)-dichloromethane-methanol in a ratio of 9:5:2 as the developing solvent, develop, take out, dry, spray with 5% sulfuric acid ethanol solution, heat at 105°C for 3 minutes, and let it stand for 20 minutes; in the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions in the control medicinal material chromatogram.

[0067] (3) Determination of Tetrahydropalmatine Content

[0068] Chromatographic conditions and system suitability test: The chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution (adjusted to pH 6.0 with triethylamine) was used as mobile phase B, with gradient elution; the flow rate was 0.8 mL / min; the column temperature was 30°C; the detection wavelength was 280 nm; the theoretical plate number calculated based on the tetrahydropalmatine peak should be no less than 3000; the gradient elution procedure was as follows:

[0069]

[0070] Preparation of reference solution: Take an appropriate amount of tetrahydropalmatine reference substance, accurately weigh it, and add methanol to make a solution containing 10 μg per 1 mL.

[0071] Preparation of test solution:

[0072] Take an appropriate amount of this product, grind it into powder, accurately weigh 1g, place it in a stoppered conical flask, accurately add 5mL of methanol, weigh the weight, extract by ultrasonic for 20min, make up the lost weight with methanol, shake well, filter; take the filtrate to obtain the product.

[0073] Determination method: Accurately aspirate 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0074] Example 3

[0075] The quality inspection method of Yuanhuhewei granules includes thin layer identification of vinegared Corydalis yanhusuo, thin layer identification of Bletilla striata and determination of tetrahydropalmatine content, as follows;

[0076] (1) Thin layer chromatography identification of vinegar Corydalis yanhusuo

[0077] Take an appropriate amount of this product, grind it into powder, accurately weigh 20 g, place it in a conical flask, add 100 mL of 70% methanol solution, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, add 20 mL of 0.5% sodium hydroxide solution to the residue, shake to dissolve it, extract it with 30 mL of ether by shaking three times, combine the ether solutions, evaporate to dryness, and add 1 mL of methanol to dissolve the residue to prepare the test solution; take the tetrahydropalmatine reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, and use it as the reference solution; according to the thin layer chromatography test, take 10 μL of each of the above two solutions and spot them on the same silica gel G thin layer plate prepared with 2% sodium hydroxide, use toluene-dichloromethane-ethyl acetate in a ratio of 8:2:1 as the developing solvent, develop, remove, dry, place in an iodine cylinder for 5 minutes, remove, and examine under ultraviolet light at 365 nm; in the chromatogram of the test product, at the corresponding position of the chromatogram of the reference substance, a fluorescent spot of the same color will appear.

[0078] (2) Thin layer identification of Bletilla striata

[0079] Prepare the test solution according to the method under the thin layer identification of vinegar Corydalis yanhusuo; take 1 g of Bletilla striata control medicinal material and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 10 μL of each of the above two solutions and spot them on the same silica gel G thin layer plate, use petroleum ether (60-90°C)-dichloromethane-methanol in a ratio of 9:5:2 as the developing solvent, develop, take out, dry, spray with 15% sulfuric acid ethanol solution, heat at 105°C for 8 minutes, and let it stand for 50 minutes; in the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions in the control medicinal material chromatogram.

[0080] (3) Determination of Tetrahydropalmatine Content

[0081] Chromatographic conditions and system suitability test: The chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution (adjusted to pH 6.0 with triethylamine) was used as mobile phase B, with gradient elution; the flow rate was 1.2 mL / min; the column temperature was 25°C; the detection wavelength was 280 nm; the theoretical plate number calculated based on the tetrahydropalmatine peak should be no less than 3000; the gradient elution procedure was as follows:

[0082]

[0083] Preparation of reference solution: Take an appropriate amount of tetrahydropalmatine reference substance, accurately weigh it, and add methanol to make a solution containing 10 μg per 1 mL.

[0084] Preparation of test solution:

[0085] Take an appropriate amount of this product, grind it into powder, accurately weigh 3 g, place it in a stoppered conical flask, accurately add 15 mL of methanol, weigh the weight, extract by ultrasonic for 40 minutes, make up the lost weight with methanol, shake well, filter; take the filtrate to obtain the product.

[0086] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0087] In order to further verify the reliability of the present invention and screen out the best solution, the inventors conducted a series of experiments, as follows:

[0088] 1. Composition and preparation method of Yuanhu Hewei Granules

[0089] 1.1 Prescription composition

[0090] The prescription composition of Yuanhu Hewei Granules is: vinegar Corydalis 6g, bran-fried Citrus aurantium 3g, citron 3g, Panax notoginseng 3g, cuttlebone 6g, Cynanchum chinense 6g, Bletilla striata 6g, Gypsophila paniculata 3g, Pseudostellaria pseudoginseng 3g, Tripterygium wilfordii 6g, and Fritillaria thunbergii 3g.

[0091] 1.2 Preparation method

[0092] Weigh each medicinal material according to the formula, add water and boil it three times. For the first boiling, add 9 times the weight of the medicinal material in water, soak for 1 hour, and then boil for 1 hour. For the second and third boilings, add 8 times the weight of the medicinal material in water, and boil for 1 hour each time. Filter the decoction, combine the filtrates, let them stand, and concentrate the supernatant under normal pressure to form a thick extract with a density of 1.0 to 1.1. Dry and pulverize it in an oven at 105°C to prepare a dry paste powder. Take the dry paste powder prepared in the above steps, add a mixed excipient consisting of dextrin and microcrystalline cellulose in a mass ratio of 7:1, and use 70% ethanol solution as a wetting agent to prepare a soft material. The standard of the soft material is that it can be lumped together by hand and falls apart when touched lightly. The mass ratio of the dry paste powder to the mixed excipient is 5:4. Extrude the prepared soft material through a No. 1 sieve to produce granules, dry it with air at 80°C, and sieve it with a No. 1 sieve and a No. 5 sieve to resize the granules. Collect the granules between the No. 1 and No. 5 sieves to obtain the obtained soft material.

[0093] 2. Quality standard research

[0094] 2.1 Thin layer chromatography identification of vinegar-cured Corydalis yanhusuo and Bletilla striata

[0095] The research team used the method in the Chinese Pharmacopoeia to perform thin-layer chromatographic identification on vinegar-corydalis and Bletilla striata in this product. The results showed that in the thin-layer chromatographic identification of vinegar-corydalis, spots of the same color appeared at the corresponding positions of the reference substance and reference medicinal materials, but there was interference in the negative sample of vinegar-corydalis. Therefore, based on the method in the Chinese Pharmacopoeia, this study optimized the thin-layer chromatographic identification of vinegar-corydalis and Bletilla striata, as follows:

[0096] 2.1.1 Thin layer chromatography identification of vinegar-cured Corydalis

[0097] Take an appropriate amount of this product, grind it into powder, accurately weigh 10g, place it in a conical flask, add 60mL of 70% methanol solution, ultrasonicate for 30min, filter, evaporate the filtrate to dryness, add 10mL of 0.5% sodium hydroxide solution to the residue, shake to dissolve, extract with 15mL of ether and shake three times, combine the ether solution, evaporate to dryness, and add 1mL of methanol to dissolve the residue as the test solution; take the corydalis butylene standard, add methanol to make a solution containing 0.5mg per 1mL, as the reference solution; according to the thin layer chromatography test, take 8μL of each of the above two solutions, respectively, spot them on the same silica gel G thin layer plate prepared with 2% sodium hydroxide, use toluene-dichloromethane-ethyl acetate in a ratio of 8:2:1 as the developing solvent, develop, take out, dry, place in an iodine cylinder for 4min, take out, and examine under ultraviolet light at 365nm; in the chromatogram of the test product, at the corresponding position of the chromatogram of the reference product, a fluorescent spot of the same color appears. The thin layer identification spectrum of vinegar corydalis butylene is as follows Figure 1 As shown (in the figure: 1, 2, 3 are three batches of Yuanhuhewei granule samples; 4 is the corydalin reference substance; 5 is the vinegar-deficient corydalin negative sample).

[0098] The invention team conducted a TLC test on the durability of the above-mentioned vinegar-corydalis yanhusuo identification method, as follows:

[0099] (1) Temperature investigation

[0100] The effects of developing at 4℃, 20℃ and 35℃ on TLC were investigated respectively. The results are as follows: Figure 2 As shown in the figure (A is the TLC spectrum at 4°C; B is the TLC spectrum at 20°C; C is the TLC spectrum at 35°C). The results show that temperature has no significant effect on the TLC spots.

[0101] (2) Humidity inspection

[0102] The effects of developing TLC at humidity of 20%, 55%, and 90% were investigated. Figure 3 As shown in the figure (A is the TLC spectrum under 20% humidity; B is the TLC spectrum under 55% humidity; C is the TLC spectrum under 90% humidity). The results show that humidity has no obvious effect on TLC spots.

[0103] (3) Inspection of thin-layer boards from different manufacturers

[0104] The effects of thin layer plates from Luqiao Sijia Biochemical Plastics Factory in Taizhou City, Zhejiang Province and Qingdao Bangkai High-tech Materials Co., Ltd. on TLC were investigated. The results are as follows: Figure 4As shown in the figure (A is the TLC spectrum of the thin layer plate from the Sijia Biochemical Plastic Factory in Luqiao, Taizhou City, Zhejiang Province; B is the TLC spectrum of the thin layer plate from Qingdao Bangkai High-tech Materials Co., Ltd.) The results show that the thin layer plates from different manufacturers have no significant effect on the thin layer chromatography spots.

[0105] 2.1.2 Thin layer identification of Bletilla striata

[0106] Prepare the test solution according to the method under the thin layer chromatography identification of vinegar Corydalis yanhusuo; take 1g of Bletilla striata as a control medicinal material and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 8μL of each of the above two solutions and spot them on the same silica gel G thin layer plate, use petroleum ether (60-90℃)-dichloromethane-methanol in a ratio of 9:5:2 as the developing solvent, develop, take out, dry, spray with 5% sulfuric acid ethanol solution, heat at 105℃ for 5min, and let it stand for 30min; in the test sample chromatogram, at the corresponding position of the control medicinal material chromatogram, a fluorescent spot of the same color appears, and the thin layer identification spectrum of Bletilla striata is as follows Figure 5 As shown (in the figure: 1, 2, 3 are three batches of Yuanhu Hewei granule samples; 4 is the Bletilla striata control medicinal material; 5 is the Bletilla striata-deficient and negative sample).

[0107] At the same time, the invention team conducted a method durability investigation on the thin layer identification of Bletilla striata, and the investigation items and conditions were the same as those of the thin layer identification of vinegar Corydalis yanhusuo. The results were as follows: Figures 6 to 8 The results show that different temperature, humidity conditions and thin layer plates from different manufacturers have no significant effect on TLC spots.

[0108] In summary, the thin-layer identification method provided by the present invention has a separation degree that meets the requirements, has no negative interference, and is durable and feasible.

[0109] 2.2 Determination of Tetrahydropalmatine Content

[0110] Yuanhu Hewei Granules are prepared from 11 Chinese medicinal herbs, including vinegar-cured Corydalis yanhusuo, bran-fried immature bitter orange, and citron. The research team used the method in the Chinese Pharmacopoeia to determine the content of tetrahydropalmatine. However, they found that the target peak overlapped with other components, the resolution did not meet the requirements, and the preparation process of the test solution in the Chinese Pharmacopoeia was relatively complex. Therefore, the research team optimized the tetrahydropalmatine determination method and validated it through methodological investigation experiments.

[0111] 2.2.1 Sample processing investigation

[0112] This preparation is a granule. The following methods were investigated for the preparation of the test solution:

[0113] (1) Take an appropriate amount of this product, grind it into powder, accurately weigh 2.5 g, place it in a stoppered conical flask, accurately add 50 mL of a mixed solution of concentrated ammonia test solution and methanol (1:20), weigh the weight, soak it in cold water for 1 hour, heat it under reflux for 1 hour, let it cool, weigh it again, make up the lost weight with a mixed solution of concentrated ammonia test solution and methanol (1:20), shake it well, and filter it; take 20 mL of the filtrate, evaporate it to near dryness, dissolve it with a mixed solution of methanol and 0.1% phosphoric acid solution (adjust the pH to 6.0 with triethylamine) in a volume ratio of 1:1 and transfer it to a 5 mL volumetric flask, make up to the volume, shake it well, filter it, and take the filtrate to obtain the product.

[0114] (2) Take an appropriate amount of this product, grind it into powder, accurately weigh 2 g, place it in a stoppered conical flask, accurately add 10 mL of methanol, weigh the weight, and extract it ultrasonically for 30 minutes. Make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the product.

[0115] By comparing the separation degree, peak area, retention time and other parameters of tetrahydropalmatine, there was no significant difference between the two treatment methods. The second sample treatment method was selected for this preparation.

[0116] 2.2.2 Column selection

[0117] This study selected a commonly used octadecylsilane-bonded silica gel column. Comparison of three sample batches showed that the resolution of the tetrahydropalmatine peak and adjacent peaks was greater than 1.5, and the peak shape was good.

[0118] 2.2.3 Detection wavelength selection

[0119] The ultraviolet absorption spectrum of tetrahydropalmatine was detected by ultraviolet-visible spectrophotometry. The ultraviolet spectrum of tetrahydropalmatine is shown in FIG. Figure 9 As shown, 280 nm was selected as the detection wavelength in this study.

[0120] 2.2.4 Mobile phase and others

[0121] This study used methanol or acetonitrile as mobile phase A and 0.1% phosphoric acid solution (adjusted to pH 6.0 with triethylamine) as mobile phase B. Gradient elution was performed according to the elution schedule in Table 1, with resolution and symmetry as indicators. The results showed that when acetonitrile was used as mobile phase A, the chromatographic peak shape was symmetrical and the resolution was good. Therefore, a gradient elution was performed using acetonitrile as mobile phase A and 0.1% phosphoric acid solution (adjusted to pH 6.0 with triethylamine) as mobile phase B.

[0122] Table 1 Gradient elution program

[0123]

[0124] Chromatographic conditions and system suitability test: The chromatographic column is filled with octadecylsilane bonded silica gel; the detection wavelength is 280 nm; the flow rate is 1.0 mL / min, and the theoretical plate number calculated based on the tetrahydropalmatine peak should be no less than 3000.

[0125] Preparation of reference solution: Take an appropriate amount of corydalin reference substance, accurately weigh it, and add methanol to make a solution containing 10 μg per 1 mL.

[0126] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, accurately weigh 2 g, place it in a stoppered conical flask, accurately add 10 mL of methanol, weigh the weight, extract by ultrasonic for 30 minutes, make up the lost weight with methanol, shake well, filter; take the filtrate to obtain the product.

[0127] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0128] 2.2.5 Methodological validation

[0129] 2.2.5.1 Specificity test

[0130] Accurately draw 10 μL each of the solvent methanol, reference solution, test solution and negative sample solution, and inject them into the chromatography instrument for determination. Figure 10-13 The results show that this method has good specificity and there is no interference from the solvent methanol and the negative test solution.

[0131] 2.2.5.2 Linear relationship test

[0132] An appropriate amount of tetrahydropalmatine reference substance (China Food and Drug Inspection Institute, batch number 110726-202421, purity 99.6%) was accurately weighed and prepared into a 10 μg / mL solution by adding methanol. 5, 10, 15, 20, and 25 μL were injected into the liquid chromatograph, and the peak area was measured. The standard curve regression equation was calculated with the reference substance injection volume as the abscissa and the peak area as the ordinate. The results are shown in Table 2. The tetrahydropalmatine standard curve is shown in Table 2. Figure 14 .

[0133] Table 2 Linear regression results of tetrahydropalmatine

[0134]

[0135]

[0136] 2.2.5.3 Recovery test

[0137] Accurately weigh 1.0001 g of Yuanhu Hewei Granule sample (batch number: 241001), add 1 mL of the reference substance with a concentration of 52.5 μg / mL, mix well, prepare the test solution, inject it into the high performance liquid chromatography for determination, record the peak area, and calculate the content of tetrahydropalmatine. Repeat 6 times, and the results are shown in Table 3. The results show that the recovery rate of the proposed method is good.

[0138] Table 3 Recovery rate experimental results (n=6)

[0139]

[0140] 2.2.5.4 Precision test

[0141] (1) Repeatability test

[0142] The same batch number (batch number: 241001) of Yuanhu Hewei Granules was used to prepare the test solution, which was injected into the HPLC for determination. The peak area was recorded and the content of tetrahydropalmatine was calculated. The results were repeated 6 times. The results are shown in Table 4. The results show that the proposed method has good reproducibility.

[0143] Table 4 Repeatability test results (n=6)

[0144]

[0145]

[0146] (2) Reproducibility test

[0147] Because this preparation is manufactured, sold, and used within our organization, we conducted a reproducibility study through collaborative testing between our R&D and production quality laboratories. This study aimed to examine the reproducibility of the established method across different laboratories. On-site testing was conducted at different locations, times, with different personnel, and using different instruments. The results are shown in Table 5.

[0148] Table 5 Reproducibility test results

[0149]

[0150] 2.2.5.5 Detection limit test

[0151] We used the signal-to-noise ratio method to study the minimum detectable amount of the analyte in the sample. The specific method is: dilute the sample solution by an appropriate multiple and then inject it. When the dilution multiple is 33 times, the signal-to-noise ratio is about 3:1, and the concentration of tetrahydropalmatine is 0.9μg / mL. The detection limit of tetrahydropalmatine is shown in the following figure: Figure 15 shown.

[0152] 2.2.5.6 Limit of Quantitation Test

[0153] We used the signal-to-noise ratio method to study the lowest amount of the analyte in the sample that can be quantitatively determined. The specific method is: dilute the sample solution by an appropriate amount before injection. When the dilution factor is 3.5 times, the signal-to-noise ratio is approximately 10:1, and the concentration of tetrahydropalmatine is 3μg / mL. The quantitative limit of tetrahydropalmatine determination spectrum is as follows: Figure 16 shown.

[0154] 2.2.5.7 Durability test

[0155] By testing at different times of the day, different times of the day, different flow rates, different chromatographic columns, and different column temperatures, the results of tetrahydropalmatine determination under different conditions were compared to evaluate the durability of the method.

[0156] (1) Intraday stability

[0157] Take an appropriate amount of Yuanhuhewei granules (batch number: 241001) to prepare the test solution, measure it at 0, 4h, and 8h, record the peak area, and calculate the content of tetrahydropalmatine. The results are shown in Table 6.

[0158] Table 6 Intraday stability test results

[0159]

[0160] (2) Daytime stability

[0161] Take Yuanhu Hewei Granules (batch number: 241001) to prepare the test solution, measure it at 0, 24, and 48 hours, record the peak area, and calculate the content of tetrahydropalmatine. The results are shown in Table 7. The results show that the sample is stable within 48 hours.

[0162] Table 7 Daytime stability test results

[0163]

[0164] (3) Investigation of different flow rates

[0165] Yuanhu Hewei Granules (Batch No. 241001) were used to prepare test solutions. High-performance liquid chromatography (HPLC) was used to measure the concentration of tetrahydropalmatine at different flow rates. The peak areas were recorded and the tetrahydropalmatine content was calculated. The results are shown in Table 8. The results showed that changes in flow rate did not affect the test results.

[0166] Table 8 Test results at different flow rates

[0167]

[0168] (4) Investigation of different chromatographic columns

[0169] Yuanhu Hewei Granules (Batch No. 241001) were used to prepare the test solution. The test was performed using chromatographic columns filled with octadecylsilane bonded silica gel from different manufacturers. The peak area was recorded and the content of tetrahydropalmatine was calculated. The results are shown in Table 9. The results showed that changing different chromatographic columns did not affect the test results.

[0170] Table 9 Test results of different chromatographic columns

[0171]

[0172] (5) Investigation of different column temperatures

[0173] Yuanhu Hewei Granules (Batch No. 241001) were used to prepare the test solution, and the test was performed using a high performance liquid chromatograph under different column temperature conditions. The peak area was recorded and the content of tetrahydropalmatine was calculated. The results are shown in Table 10. The results show that a small change in column temperature does not affect the test results.

[0174] Table 10 Test results at different column temperatures

[0175]

[0176] Although the present invention has been described in detail above using general descriptions, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A quality detection method for Yuanhu Hewei granules, characterized in that: The quality detection method includes thin layer identification of vinegar Corydalis yanhusuo, thin layer identification of Bletilla striata and determination of tetrahydropalmatine content, which are specifically as follows: The thin layer chromatography identification of vinegar Corydalis is as follows: take an appropriate amount of this product, grind it into powder, accurately weigh 10-20g, place it in a conical flask, add 50-100mL of 70% methanol solution, ultrasonically treat it for 20-40min, filter, evaporate the filtrate to dryness, add 5-20mL of 0.5% sodium hydroxide solution to the residue, shake it to dissolve, shake it with 10-30mL of ether for 2-4 times, combine the ether solution, evaporate it to dryness, add 1-3mL of methanol to dissolve the residue, and use it as the test solution; take the corydalis butylene standard substance, add Prepare a solution containing 0.5 mg per 1 mL of methanol as the reference solution; according to the thin layer chromatography test, aspirate 5-10 μL of each of the above two solutions and spot them on the same silica gel G thin layer plate prepared with 2% sodium hydroxide. Use toluene-dichloromethane-ethyl acetate as the developing solvent. Develop the plate, remove the plate, dry it, place it in an iodine jar for 3-5 minutes, then remove it and examine it under ultraviolet light at 365 nm. In the chromatogram of the test sample, a fluorescent spot of the same color will appear at the corresponding position in the chromatogram of the reference sample. The thin-layer chromatography identification of Bletilla striata is as follows: prepare the test solution according to the method under the thin-layer chromatography identification of vinegar-corydalis yanhusuo; take 1g of Bletilla striata as a control medicinal material and prepare the control medicinal material solution in the same manner; according to the thin-layer chromatography test, take 5-10μL of each of the above two solutions and spot them on the same silica gel G thin-layer plate, use petroleum ether-dichloromethane-methanol as the developing solvent, develop, remove, dry, spray with 5%-15% sulfuric acid ethanol solution, heat at 105℃ for 3-8 minutes, and let it stand for 20-50 minutes; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material; The content of tetrahydropalmatine was determined as follows: Chromatographic conditions and system suitability test: The chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution (adjusted to pH 6.0 with triethylamine) was used as mobile phase B, with gradient elution; the flow rate was 0.8-1.2 mL / min; the column temperature was 25-30°C; the detection wavelength was 280 nm; the theoretical plate number calculated based on the tetrahydropalmatine peak should be no less than 3000; the gradient elution procedure was as follows: Preparation of reference solution: Take an appropriate amount of tetrahydropalmatine reference substance, accurately weigh it, and add methanol to make a solution containing 10 μg per 1 mL. Preparation of test solution: Take an appropriate amount of this product, grind it into powder, accurately weigh 1-3g, place it in a stoppered conical flask, accurately add 5-15mL of methanol, weigh it, and extract it by ultrasonic for 20-40min. Make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the product. Determination method: Accurately aspirate 5-10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

2. The quality inspection method according to claim 1, characterized in that: The thin layer chromatography identification of vinegar Corydalis yanhusuo is specifically as follows: take an appropriate amount of the product, grind it into powder, accurately weigh 10-15g, place it in a conical flask, add 50-80mL of 70% methanol solution, ultrasonically treat it for 30-40min, filter, evaporate the filtrate to dryness, add 5-15mL of 0.5% sodium hydroxide solution to the residue, shake it to dissolve it, shake and extract it with 10-20mL of ether for 3-4 times, combine the ether solution, evaporate it to dryness, add 1-2mL of methanol to dissolve the residue, and use it as the test solution; take Corydalis bianhusuo reference substance , add methanol to make a solution containing 0.5 mg per 1 mL, which is used as the reference solution; according to the thin layer chromatography test, aspirate 8-10 μL of each of the above two solutions and spot them on the same silica gel G thin layer plate prepared with 2% sodium hydroxide, use toluene-dichloromethane-ethyl acetate as the developing solvent, develop, take out, dry, place in an iodine cylinder for 4-5 minutes, then take out and examine under ultraviolet light at 365 nm; in the test chromatogram, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the reference.

3. The quality inspection method according to claim 2, characterized in that: The thin layer chromatography identification of the vinegar Corydalis yanhusuo is specifically as follows: take an appropriate amount of the product, grind it into powder, accurately weigh 10g, place it in a conical flask, add 60mL of 70% methanol solution, ultrasonically treat for 30min, filter, evaporate the filtrate to dryness, add 10mL of 0.5% sodium hydroxide solution to the residue, shake to dissolve, shake and extract with 15mL of ether three times, combine the ether solution, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution; take the corydalis butyl amine reference substance, add methanol to make a solution containing 0.5mg per 1mL, and use it as the reference solution; according to the thin layer chromatography test, take 8μL of each of the above two solutions, respectively, and spot them on the same silica gel G thin layer plate prepared with 2% sodium hydroxide, use toluene-dichloromethane-ethyl acetate in a ratio of 8:2:1 as the developing solvent, develop, take out, dry, place in an iodine cylinder for 4min, take out, and place under ultraviolet light for inspection at 365nm; in the chromatogram of the test product, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the reference substance.

4. The quality inspection method according to claim 1, characterized in that: The thin layer identification of Bletilla striata is specifically as follows: prepare the test solution according to the method under the thin layer identification of vinegar Corydalis yanhusuo; take 1g of Bletilla striata control medicinal material and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 7 to 9 μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-dichloromethane-methanol as the developing agent, develop, take out, dry, spray with 5% to 10% sulfuric acid ethanol solution, heat at 105°C for 4 to 6 minutes, and let it stand for 20 to 40 minutes; in the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions in the control medicinal material chromatogram.

5. The quality inspection method according to claim 4, characterized in that: The thin layer identification of Bletilla striata is specifically as follows: prepare the test solution according to the method under the thin layer identification of vinegar Corydalis yanhusuo; take 1g of Bletilla striata control medicinal material and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 8μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-dichloromethane-methanol with a ratio of 9:5:2 as the developing agent, develop, take out, dry, spray with 5% sulfuric acid ethanol solution, heat at 105℃ for 5min, and let it stand for 30min; in the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions in the control medicinal material chromatogram.

6. The quality inspection method according to any one of claims 1, 4 or 5, characterized in that: The boiling range of the petroleum ether is 60-90°C.

7. The quality inspection method according to claim 1, characterized in that: The chromatographic conditions and system suitability test for the determination of tetrahydropalmatine were as follows: a flow rate of 1.0 mL / min and a column temperature of 30°C.

8. The quality inspection method according to claim 1, characterized in that: The preparation of the test solution described in the determination of the content of tetrahydropalmatine is specifically as follows: take an appropriate amount of the product, grind it into powder, accurately weigh 2 to 3 g, place it in a stoppered conical flask, accurately add 10 to 15 mL of methanol, weigh the weight, ultrasonically extract for 30 to 40 minutes, make up the lost weight with methanol, shake well, filter; and take the filtrate to obtain the product.

9. The quality inspection method according to claim 8, characterized in that: The preparation of the test solution described in the determination of the content of tetrahydropalmatine is specifically as follows: take an appropriate amount of the product, grind it into powder, accurately weigh 2 g, place it in a stoppered conical flask, accurately add 10 mL of methanol, weigh the weight, ultrasonically extract for 30 minutes, make up the lost weight with methanol, shake well, filter; and take the filtrate to obtain.

10. The quality inspection method according to claim 1, characterized in that: The injection volume in the assay method for the determination of tetrahydropalmatine content was 10 μL.

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