Quality control method of traditional Chinese medicine composition capable of relieving pain and numbness

Abstract

The invention relates to the technical field of traditional Chinese medicine quality control, in particular to a quality control method of a traditional Chinese medicine composition capable of relieving pain and numbness. The traditional Chinese medicine composition is prepared from the following components in parts by weight: 2-8 parts of caulis spatholobi, 3-10 parts of rhizoma corydalis, 4-10 parts of caulis sinomenii, 2-5 parts of frankincense, 2-8 parts of radix zanthoxyli, 2-7 parts of ligusticum wallichii, 1-6 parts of sappan wood, 0.5-3 parts of borneol, 3-9 parts of turmeric, 1-7 parts of radix achyranthis bidentatae, 0.5-4 parts of pepper and 1-5 parts of cassia twig. A mobile phase A (acetonitrile)-B (0.1% phosphoric acid aqueous solution) is used for gradient elution, and triethylamine is used for adjusting pH. The sample quality control method which is good in specificity, high in separation degree and good in accuracy is obtained by optimizing preparation conditions and chromatographic conditions of a test solution, and the method is suitable for quality control of the traditional Chinese medicine composition capable of relieving pain and numbness.

Description

Technical Field

[0001] The invention relates to the technical field of quality control of traditional Chinese medicine, and in particular to a quality control method for a traditional Chinese medicine composition capable of alleviating pain and numbness. Background Art

[0002] Limb pain and numbness is one of the common clinical symptoms. In addition to traumatic injuries, the affected parts include the face, mouth, limbs, fingertips, skin, shoulders, back, chest and abdomen. The main manifestations are numbness of the affected part, no pain or itching, as if there are worms crawling in the flesh, or even itching without feeling pain, loss of sensation, and thickness like wood. The cause of the disease is relatively complicated. There are many methods for treating pain and numbness on the market, including Western medicine, Chinese medicine, acupuncture and combined treatment. Western medicine is often accompanied by side effects during use, and some drugs such as opioid painkillers are prone to drug dependence and can easily cause adverse reactions to the human body. There are many Chinese medicine treatment methods, but the effects are often uncertain.

[0003] Chinese invention patent CN116832132 B discloses a Chinese medicine composition, preparation, preparation method and application thereof that can relieve pain and numbness. The Chinese medicine composition is made of the following components in parts by weight: 2-8 parts of Millettia reticulata, 3-10 parts of Corydalis yanhusuo, 4-10 parts of Caulis Sinomenii, 2-5 parts of frankincense, 2-8 parts of Zanthoxylum bungeanum, 2-7 parts of Chuanxiong, 1-6 parts of Sappanwood, 0.5-3 parts of Borneolum, 3-9 parts of Curcuma longa, 1-7 parts of Achyranthes bidentata, 0.5-4 parts of Capsicum and 1-5 parts of Cinnamomum cassia. The components are more streamlined, which can better relieve limb pain and numbness, and can be used for limb pain and numbness caused by diabetic peripheral neuropathy and rheumatic immune-related diseases. However, there is currently no quality detection method applicable to the drug, and it is difficult to ensure the stability and efficacy of the preparation. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a quality control method for a Chinese medicine composition that can relieve pain and numbness, thereby ensuring the stability and efficacy of the Chinese medicine composition preparation of Chinese invention patent CN116832132 B.

[0005] The present invention is achieved through the following technical solutions: One of the purposes of the present invention is to provide a quality control method for a traditional Chinese medicine composition capable of relieving pain and numbness. The traditional Chinese medicine composition is prepared from the following components in parts by weight: 2-8 parts of Millettia reticulata, 3-10 parts of Corydalis yanhusuo, 4-10 parts of Caulis Sinomenii, 2-5 parts of frankincense, 2-8 parts of Zanthoxylum bungeanum, 2-7 parts of Chuanxiong, 1-6 parts of Sappanwood, 0.5-3 parts of Borneolum, 3-9 parts of Curcuma longa, 1-7 parts of Achyranthes bidentata, 0.5-4 parts of Capsicum and 1-5 parts of Cinnamomum cassia. The quality control method includes the determination of the contents of tetrahydropalmatine, curcumin and borneol; The chromatographic conditions for determining the content of tetrahydropalmatine are as follows: Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: A is acetonitrile, B is 0.1% phosphoric acid aqueous solution, and triethylamine is used to adjust the pH to 7.0; elution gradient: 0-30 min, A is 40%, B is 60%; 30-35 min, A is 40%→60%, B is 60%→40%; 35-40 min, A is 60%→40%, B is 40%→60%; 40-45 min, A is 40%, B is 60%; detection wavelength is 282 nm; column temperature is 30°C; flow rate is 1 mL / min.

[0006] Preferably, the step of determining the content of tetrahydropalmatine is: (1) Preparation of reference solution: Take an appropriate amount of corydaline reference substance, weigh accurately, and add methanol to make a 20 µg / mL solution. (2) Preparation of test solution: Take the test sample, extract, wash it, and then make up the volume; (3) Determination method: Accurately aspirate the reference solution and the test solution respectively, inject them into the liquid chromatograph, determine, and calculate.

[0007] Further preferably, the test solution for the determination of the tetrahydropalmatine content is prepared by the following steps: (1) Extraction: Take 2 g of the test sample and extract it with 25-50 mL of 0.1-0.3 mol / L hydrochloric acid solution by heating and refluxing for 25-35 minutes, cool it, make up the weight with the solvent, centrifuge it for 4-6 minutes at a centrifugal speed of 3000-5000 rpm, and obtain the supernatant; (2) Extraction: Take the supernatant and extract it 2-4 times with an equal volume of dichloromethane, and combine the dichloromethane solutions; (3) Washing: Wash the dichloromethane solution with ammonia solution for 2-4 times, discard the washing solution, evaporate the dichloromethane solution to dryness, dissolve the residue in methanol, transfer to a 5 mL volumetric flask, and dilute to the mark. Shake well, filter, and take the filtrate to obtain the final product.

[0008] More preferably, the steps for preparing the test solution for the determination of the content of tetrahydropalmatine are specifically as follows: (1) Extraction: Take about 2 g of the Chinese medicine composition, weigh it accurately, put it in a stoppered conical flask, add 50 mL of 0.2 mol / L hydrochloric acid solution accurately, stopper it tightly, weigh it, heat it under reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with 0.2 mol / L hydrochloric acid solution, shake it well, centrifuge it for 5 minutes at a speed of 4000 rpm, and obtain the supernatant; (2) Extraction: Accurately measure 25 mL of the supernatant and extract with dichloromethane three times, 25 mL each time, and combine the dichloromethane solutions; (3) Washing: Wash the dichloromethane solution with ammonia test solution three times, 20 mL each time, discard the washing solution, evaporate the dichloromethane solution to dryness, dissolve the residue in methanol, transfer to a 5 mL volumetric flask, and dilute to the scale. Shake well, filter, and take the filtrate to obtain the product.

[0009] Preferably, the method for determining the content of curcumin is: Liquid chromatography conditions: the chromatographic column was filled with octadecylsilane bonded silica gel; mobile phase A was acetonitrile, and mobile phase B was 0.1% phosphoric acid aqueous solution; elution gradient: 0-20min, A was 42%, B was 58%; 20-35min, A was 42%→55%, B was 58%→45%; 35-40min, A was 55%→42%, B was 45%→58%; 40-45min, A was 42%, B was 58%; detection wavelength was 430nm; column temperature was 30℃; flow rate was 1mL / min; Preparation of reference solution: Take an appropriate amount of curcumin reference substance, accurately weigh it, and add methanol to make a 20µg / mL solution; Preparation of test solution: Take the test sample, extract with solvent, and make up to volume; Determination method: Accurately aspirate the reference solution and the test solution respectively, inject into the liquid chromatograph, determine, calculate, and obtain the result.

[0010] Further preferably, the test solution for the determination of curcumin content is prepared by the following steps: taking 0.5 g of the traditional Chinese medicine composition, dissolving it in petroleum ether, adding 25-50 mL of methanol, ultrasonically treating it for 15-30 minutes, cooling it, weighing it again, making up the lost weight with methanol, shaking it well, filtering it, and taking the filtrate to obtain it.

[0011] Further preferably, the preparation step of the test solution for the determination of curcumin content is specifically as follows: take about 0.5g of the traditional Chinese medicine composition, accurately weigh it, place it in a stoppered conical flask, add 0.5mL of 60-90℃ petroleum ether and heat it slightly to dissolve the sample, then accurately add 50mL of methanol, weigh it, treat it under 300W, 40kHz ultrasound for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0012] Preferably, the method for determining the content of borneol is: Gas chromatography conditions: chromatographic column: Agilent DB-WAX (30 m, 0.32 mm, 0.25 μm); temperature program: initial temperature 130 °C, hold for 15 min, increase to 230 °C at a rate of 20 °C per minute, hold for 5 min; injection port temperature 200 °C; detector temperature 240 °C; Gas flow rate: constant flow mode, 1.0mL / min; Detector: FID; Injection volume: 1 μL; Preparation of reference solution: Take an appropriate amount of borneol reference, weigh accurately, and add ethyl acetate to make a 50µg / mL solution; Preparation of test solution: Take the test sample, extract with solvent, and make up to volume; Determination method: Accurately aspirate the reference solution and the test solution respectively, inject into the gas chromatograph, determine, calculate, and obtain the result.

[0013] Further preferably, the test solution for the borneol content determination is prepared by the following steps: take 0.5 g of the traditional Chinese medicine composition, dissolve it in petroleum ether, add 25-50 mL of ethyl acetate, ultrasonically treat it for 15-30 minutes, cool it, weigh it again, make up the lost weight with ethyl acetate, shake it well, filter it, and take the filtrate to obtain it.

[0014] Further preferably, the preparation steps of the test solution for the determination of borneol content are specifically as follows: take about 0.5 g of the traditional Chinese medicine composition, accurately weigh it, place it in a stoppered conical flask, add 0.5 mL of 60-90 ° C petroleum ether and heat it slightly to dissolve the sample, then accurately add 50 mL of ethyl acetate, weigh the weight, treat it under 300 W, 40 kHz ultrasound for 30 minutes, cool it, weigh it again, make up the lost weight with ethyl acetate, shake it well, filter it, and take the filtrate to obtain it.

[0015] Preferably, a method for preparing a Chinese medicine composition capable of relieving pain and numbness comprises the following steps: S1. Mix the formulated amounts of Millettia spatholobi, Corydalis yanhusuo, Caulis qingfengcao, frankincense, Zanthoxylum bungeanum, Sappan wood, Ligusticum chuanxiong, Curcuma longa, Achyranthes bidentata, Capsicum annuum and cinnamon twig and extract with alcohol to obtain an extract; S2, concentrating the extract obtained in step S1, standing and separating to obtain a fat layer and a water layer; S3, adding auxiliary materials to the oil layer obtained in step S2 to disperse and obtain an oil intermediate; S4, precipitating the aqueous layer obtained in step S2 with alcohol, and concentrating the supernatant to obtain a concentrated paste; S5. Add the concentrated cream obtained in step S4 and the formulated amount of borneol to the oil intermediate obtained in step S3, and mix them to obtain the Chinese medicine composition.

[0016] Further preferably, the alcohol extraction in step S1 is: adding an ethanol solution with an ethanol volume fraction of 30%-90% for extraction, and the amount of the ethanol solution is 15-30 times the total weight of the Millettia reticulata, Corydalis yanhusuo, Caulis Sinomenii, frankincense, Zanthoxylum bungeanum, Sappan wood, Ligusticum chuanxiong, Curcuma longa, Achyranthes bidentata, Capsicum annuum and cinnamon twig.

[0017] Further preferably, the concentration in step S2 is to a relative density of 1.10-1.30.

[0018] Further preferably, the alcohol precipitation in step S4 is: adding ethanol so that the volume fraction of ethanol in the water layer is 30-90%, and letting it stand.

[0019] Further preferably, the concentration in step S4 is performed to a relative density of 1.10-1.30.

[0020] The beneficial effects of the present invention are as follows: by optimizing the extraction conditions, extraction conditions, washing conditions and chromatographic conditions of tetrahydropalmatine, curcumin and borneol in the sample solution, the sample preparation time is short, the determination method has good specificity, high separation degree and good accuracy, and the quality standard of the traditional Chinese medicine composition that can relieve pain and numbness can be formulated through the above method, thereby realizing the quality detection of the traditional Chinese medicine composition that can relieve pain and numbness. Experiments have proved that by using this method as a standard to detect the traditional Chinese medicine composition that can relieve pain and numbness, the stability of the preparation can be well guaranteed, thereby ensuring its efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Experimental Example 1 HPLC chromatogram of blank solution for specificity investigation of the method for determining the content of tetrahydropalmatine; Figure 2 : Experimental Example 1 Tetrahydropalmatine content determination method specificity investigation reference substance solution high performance liquid chromatogram; Figure 3 : Experimental Example 1 HPLC chromatogram of the test solution for the specificity investigation of the method for determining the content of tetrahydropalmatine; Figure 4 : Experimental Example 1 Specificity study of the method for determining the content of corydalis butylene glycol, high performance liquid chromatogram of vinegar corydalis negative test solution; Figure 5 : Experimental Example 1: UV absorption diagram of tetrahydropalmatine reference substance in the method specificity investigation; Figure 6 : Experimental Example 1 Method specificity investigation of the ultraviolet absorption graph of the chromatographic peak of tetrahydropalmatine in the test sample; Figure 7 : HPLC chromatogram of the content determination of tetrahydropalmatine in batch 1 of comparative example 1; Figure 8 : Experimental Example 2 High Performance Liquid Chromatogram of Blank Solution for Specificity Investigation of Curcumin Content Determination Method; Fig. 9 : Experimental Example 2 High Performance Liquid Chromatogram of Reference Solution for Specificity Investigation of Curcumin Content Determination Method; Fig.10: Experimental Example 2 High Performance Liquid Chromatogram of the Test Solution for Specificity Investigation of Curcumin Content Determination Method; Fig.11 : Experimental Example 2 Curcumin content determination method specificity investigation of turmeric negative test solution HPLC chromatogram; Fig.12 : Experimental Example 2: UV absorption graph of curcumin reference substance for method specificity investigation; Fig.13 : Experimental Example 2 Method specificity investigation of the UV absorption graph of the chromatographic peak of curcumin in the test sample; Fig.14 : Experimental Example 3 Specificity investigation of borneol content determination method blank solution HPLC chromatogram; Fig.15 : Experimental Example 3 Specificity study of borneol content determination method, reference solution HPLC chromatogram; Fig.16 : Experimental Example 3 Specificity investigation of borneol content determination method for test sample solution HPLC chromatogram; Fig.17 : Experimental Example 3 Specificity investigation of borneol content determination method, borneol negative test solution HPLC chromatogram.

[0022] It is worth mentioning that: Figure 1-Figure 4 In the investigation of the specificity of the method for determining the content of tetrahydropalmatine, the peak near the retention time of 24.45min is the peak corresponding to tetrahydropalmatine. The peak height of the left peak is too high or too low to provide a complete spectrum, but the corresponding spectrum does not affect the determination of tetrahydropalmatine. Figure 7 In the determination of tetrahydropalmatine content of the test sample of the present invention according to the pharmacopoeia method, the peak with a retention time of 46.857 min is the peak corresponding to tetrahydropalmatine. The peak heights on the left and right sides of the peak are too high to provide a complete spectrum, but the corresponding spectrum does not affect the determination of tetrahydropalmatine. Figure 14-17 In the investigation of the specificity of the borneol content determination method, the peak near the retention time of 4.1min is the borneol peak. The peak height on the left side of this peak is too high to provide a complete spectrum, but the corresponding spectrum does not affect the determination of borneol. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0024] The specific composition and preparation method of the Chinese medicinal composition capable of relieving pain and numbness in the following experimental examples and embodiments are all referenced to Example 4 of Patent CN116832132 B.

[0025] Experimental Example 1: Study on the determination method of vinegar Corydalis content in a traditional Chinese medicine composition that can relieve pain and numbness Vinegar Corydalis is the main drug in the prescription, and its representative component is tetrahydropalmatine, so the content determination method of tetrahydropalmatine in the Chinese medicine composition was studied. After preliminary condition exploration, the chromatographic conditions of tetrahydropalmatine were determined as follows: Chromatographic column: Medium Spectrum Red RD C18 (5μm, 250×4.6mm, SN: 018X22590); Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution (triethylamine adjusted to pH 7.0) as mobile phase B, gradient elution according to Table 1; Flow rate: 1.0mL / min; Column temperature: 30℃; Detection wavelength: 282nm; Injection volume: 10μL.

[0026] Table 1: Gradient elution program for the determination of tetrahydropalmatine content

[0027] Preparation of reference solution: Take an appropriate amount of corydaline reference substance, weigh accurately, and add methanol to make a solution containing 20µg per 1mL.

[0028] 1. Study on the preparation method of test solution Samples of the traditional Chinese medicine composition capable of relieving pain and numbness were taken, and the content of tetrahydropalmatine was used as an indicator. The above-mentioned chromatographic determination method was used to carry out the investigation of the extraction solvent, extraction method, extraction time, extraction solvent volume, extraction solvent, extraction times, washing times, etc., as follows: (1) Investigation of extraction solvent Take about 2g of the Chinese medicine composition, a total of 3 groups, accurately weigh, put in a stoppered conical flask, accurately add 0.1mol / L hydrochloric acid solution, 0.2mol / L hydrochloric acid solution, 0.3mol / L hydrochloric acid solution 50mL, respectively, stopper, weigh, heat reflux for 30 minutes, cool, weigh again, make up the lost weight with 0.1mol / L hydrochloric acid solution, 0.2mol / L hydrochloric acid solution, 0.3mol / L hydrochloric acid solution, shake well, centrifuge (4000 rpm) for 5 minutes, accurately measure 25mL of supernatant, shake and extract 3 times with dichloromethane, 25mL each time, combine the dichloromethane solution, wash 3 times with ammonia test solution, 20mL each time, discard the washing solution, evaporate the dichloromethane solution, dissolve the residue in methanol, transfer to a 5mL volumetric flask, dilute to the scale, shake well, filter, and take the filtrate to obtain. The contents of tetrahydropalmatine under different extraction solvents are shown in Table 2.

[0029] Table 2: Results of the extraction solvent test for the content of tetrahydropalmatine

[0030] Result analysis: There was no significant difference in the content of tetrahydropalmatine in the test solution when the extraction solvent was 0.1mol / L hydrochloric acid solution and 0.2mol / L hydrochloric acid solution, and it was higher than when the extraction solvent was 0.3mol / L hydrochloric acid solution. Considering the batch differences, 0.2mol / L hydrochloric acid solution was selected as the optimal extraction solvent.

[0031] (2) Investigation of extraction methods Take about 2g of the Chinese medicine composition, a total of 2 groups, accurately weigh, put in a stoppered conical bottle, accurately add 50mL of 0.2mol / L hydrochloric acid solution, seal, weigh, heat and reflux for 30 minutes / ultrasonic treatment (300W, 40kHz) for 30 minutes, take out, cool, weigh again, add 0.2mol / L hydrochloric acid solution to make up for the lost weight, shake well, centrifuge (4000 rpm) for 5 minutes, accurately measure 25mL of supernatant, shake and extract 3 times with dichloromethane, 25mL each time, combine the dichloromethane solution, wash 3 times with ammonia test solution, 20mL each time, discard the washing solution, evaporate the dichloromethane solution, dissolve the residue in methanol, transfer to a 5mL volumetric bottle, dilute to the scale, shake well, filter, and take the filtrate. The content of tetrahydropalmatine under different extraction methods is shown in Table 3.

[0032] Table 3: Results of the investigation on the extraction method for the determination of tetrahydropalmatine content

[0033] Result analysis: When the extraction method was ultrasonic treatment, the content of tetrahydropalmatine in the test solution was 0.011 mg / g, and the RD% of the content measured in two parallel samples was 4.45%, which was greater than 3%; when the extraction method was heating reflux, the content of tetrahydropalmatine in the test solution was 0.065 mg / g, which was much greater than ultrasonic extraction, and the parallelism of ultrasonic extraction was poor, so reflux extraction was selected as the extraction method.

[0034] (3) Extraction time investigation Take about 2g of the Chinese medicine composition, a total of 3 groups, accurately weigh, put in a stoppered conical bottle, accurately add 50mL of 0.2mol / L hydrochloric acid solution, weigh, heat and reflux for 15 minutes, 30 minutes, and 45 minutes respectively, take out, cool, weigh again, add 0.2mol / L hydrochloric acid solution to make up the lost weight, shake well, centrifuge (4000 rpm) for 5 minutes, accurately measure 25mL of supernatant, shake and extract 3 times with dichloromethane, 25mL each time, combine the dichloromethane solution, wash 3 times with ammonia test solution, 20mL each time, discard the washing solution, evaporate the dichloromethane solution, dissolve the residue in methanol, transfer to a 5ml volumetric bottle, dilute to the scale, shake well, filter, and take the filtrate. The content of tetrahydropalmatine at different extraction times is shown in Table 4.

[0035] Table 4: Determination of the extraction time of tetrahydropalmatine

[0036] Result analysis: There was no significant difference in the content of tetrahydropalmatine in the test solution when the extraction time was 15 minutes, 30 minutes, and 45 minutes. Considering the difference between sample batches and saving time, 30 minutes was selected as the optimal extraction time.

[0037] (4) Investigation of extraction solvent volume Take about 2g of the Chinese medicine composition, a total of 3 groups, accurately weigh, put in a stoppered conical bottle, accurately add 25mL, 50mL, and 100mL of 0.2mol / L hydrochloric acid solution, weigh, heat and reflux for 30 minutes, take out, cool, weigh again, add 0.2mol / L hydrochloric acid solution to make up the lost weight, shake well, centrifuge (4000 rpm) for 5 minutes, accurately measure 15mL, 25mL, and 50mL of the supernatant, shake and extract 3 times with dichloromethane, 25mL each time, combine the dichloromethane solution, wash 3 times with ammonia test solution, 20mL each time, discard the washing solution, evaporate the dichloromethane solution, dissolve the residue in methanol, transfer to a 5mL volumetric bottle, dilute to the scale, shake well, filter, and take the filtrate. The content of tetrahydropalmatine under different extraction solvent volumes is shown in Table 5.

[0038] Table 5: Investigation of extraction solvent volume for the determination of tetrahydropalmatine content

[0039] Result analysis: There was no significant difference in the content of tetrahydropalmatine in the test solution when the extraction solvent volume was 25mL, 50mL, and 100mL. Considering the differences between sample batches and saving solvent, 50mL was selected as the optimal extraction solvent volume.

[0040] (5) Investigation of extraction solvent Take about 2g of the Chinese medicine composition, a total of 3 groups, accurately weigh, put in a stoppered conical bottle, accurately add 50mL of 0.2mol / L hydrochloric acid solution, weigh, heat and reflux for 30 minutes, take out, cool, weigh again, add 0.2mol / L hydrochloric acid solution to make up the lost weight, shake well, centrifuge (4000 rpm) for 5 minutes, accurately measure 25mL of the supernatant, shake and extract 3 times with ethyl acetate, dichloromethane, and chloroform, 25mL each time, combine the extraction solvents, wash 3 times with ammonia test solution, 20mL each time, discard the washing liquid, evaporate the extraction solvent, dissolve the residue in methanol, transfer to a 5mL volumetric bottle, dilute to the scale, shake well, filter, and take the filtrate. The content of tetrahydropalmatine under different extraction solvents is shown in Table 6.

[0041] Table 6: Investigation of extraction solvents for the determination of tetrahydropalmatine content

[0042] Result analysis: When the extraction solvent was ethyl acetate, the content of tetrahydropalmatine in the test solution was low. When the extraction solvent was dichloromethane or chloroform, there was no significant difference in the content of tetrahydropalmatine in the test solution. Considering the high toxicity of chloroform, dichloromethane was selected as the extraction solvent.

[0043] (6) Investigation of extraction times Take about 2g of the Chinese medicine composition, a total of 3 groups, accurately weigh, put in a stoppered conical bottle, accurately add 50mL of 0.2mol / L hydrochloric acid solution, weigh, heat and reflux for 30 minutes, take out, cool, weigh again, add 0.2mol / L hydrochloric acid solution to make up the lost weight, shake well, centrifuge (4000 rpm) for 5 minutes, accurately measure 25mL of the supernatant, shake and extract with dichloromethane 2, 3, and 4 times, 25mL each time, combine the dichloromethane solution, wash 3 times with ammonia test solution, 20mL each time, discard the washing solution, evaporate the dichloromethane solution, dissolve the residue in methanol, transfer to a 5mL volumetric bottle, dilute to the scale, shake well, filter, and take the filtrate. The content of tetrahydropalmatine under different extraction times is shown in Table 7.

[0044] Table 7: Investigation on extraction times for determination of tetrahydropalmatine content

[0045] Result analysis: There was no significant difference in the content of tetrahydropalmatine in the test solution when the extraction times were 2, 3, and 4. Considering the differences between sample batches and saving solvents, 3 extraction times was the best.

[0046] (7) Washing frequency investigation Take about 2g of the Chinese medicine composition, a total of 3 groups, accurately weigh, put in a stoppered conical bottle, accurately add 50mL of 0.2mol / L hydrochloric acid solution, weigh, heat and reflux for 30 minutes, take out, cool, weigh again, add 0.2mol / L hydrochloric acid solution to make up the lost weight, shake well, centrifuge (4000 rpm) for 5 minutes, accurately measure 25mL of the supernatant, shake and extract 3 times with dichloromethane, 25mL each time, combine the dichloromethane solution, wash with ammonia test solution 2, 3, and 4 times, 20mL each time, discard the washing solution, evaporate the dichloromethane solution, dissolve the residue in methanol, transfer to a 5mL volumetric bottle, dilute to the scale, shake well, filter, and take the filtrate. The content of tetrahydropalmatine under different washing times is shown in Table 8.

[0047] Table 8: Determination of Tetrahydropalmatine Content by Washing Times

[0048] Result analysis: There was no significant difference in the content of tetrahydropalmatine in the test solution when the washing times were 2, 3, and 4 times. Considering the differences between sample batches and saving solvents, 3 washing times was the best.

[0049] 2. Validation of the methodological approach for the determination of corydaline content The specificity, instrument precision, linearity, precision (repeatability, intermediate precision), and accuracy of the method for determining the content of tetrahydropalmatine were investigated as follows: Except for special instructions, the test solution was prepared according to the method in the following corydaline content verification experiment: about 2g of the Chinese medicine composition was taken, accurately weighed, placed in a stoppered conical flask, 50mL of 0.2mol / L hydrochloric acid solution was accurately added, the flask was sealed, the weight was determined, the mixture was heated to reflux for 30 minutes, cooled, and the weight was determined again, the lost weight was supplemented with 0.2mol / L hydrochloric acid solution, shaken, centrifuged (4000 rpm) for 5 minutes, 25mL of the supernatant was accurately measured, and the mixture was extracted 3 times with dichloromethane by shaking, 25mL each time, the dichloromethane solution was combined, and the mixture was washed 3 times with ammonia test solution, 20mL each time, the washing solution was discarded, the dichloromethane solution was evaporated to dryness, the residue was dissolved in methanol, transferred to a 5mL volumetric flask, diluted to the scale, shaken, filtered, and the filtrate was obtained.

[0050] Except for special instructions in the following corydalis butylene content verification experiment, the rest are prepared according to this method: take an appropriate amount of corydalis butylene butylene reference substance, accurately weigh it, add methanol to make a 20µg / mL solution.

[0051] (1) Exclusivity Tested by high performance liquid chromatography (DAD detector).

[0052] Blank solvent: methanol Preparation of negative sample solution of vinegar Corydalis yanhusuo: take about 2g of negative sample of vinegar Corydalis yanhusuo, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of 0.2mol / L hydrochloric acid solution, stopper it, weigh it, heat it under reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with 0.2mol / L hydrochloric acid solution, shake it well, centrifuge it (4000 rpm) for 5 minutes, accurately measure 25ml of supernatant, shake it with dichloromethane to extract it 3 times, 25ml each time, combine the dichloromethane solution, wash it 3 times with ammonia test solution, 20ml each time, discard the washing solution, evaporate the dichloromethane solution, dissolve the residue with methanol, transfer it to a 5ml volumetric flask, and dilute it to the scale, shake it well, filter it, and take the filtrate to obtain it.

[0053] Determination method: Accurately pipette 10 μL of blank solvent, reference solution, test solution and vinegar Corydalis negative sample solution respectively, inject into chromatograph, and determine. The corresponding chromatogram results are shown in Figure 1-Figure 6 .

[0054] Result analysis: Figure 3 In the chromatogram of the test sample, a chromatographic peak was detected at the corresponding position of the corydaline reference substance, and the retention time of the main peak of the test sample was 24.393 minutes. Figure 2 The retention time of the main peak of the reference substance is 24.450 minutes. The main peak is clearly separated from the adjacent chromatographic peaks, with a resolution greater than 1.5. The peak purity is 999, greater than 980 (the peak purity of the THERMO instrument is based on 980, and a value greater than 980 indicates that the purity of the peak meets the requirements, the same below). The spectral absorption of the main peak is consistent with that of the reference substance. Figure 1 The blank solvent and Figure 4 In the chromatogram of the negative sample of acetocopherol, no chromatographic peak was detected at the corresponding retention time position of the main peaks of the test sample and the reference sample, indicating that the specificity of this method is good.

[0055] (2) Instrument precision Accurately pipette 10 μL of the reference solution, inject it into the chromatograph, inject 6 times in a row, and measure. The results are shown in Table 9.

[0056] Table 9: Instrument precision results of the method for determining the content of tetrahydropalmatine

[0057] Result analysis: The RSD% of the peak area of ​​tetrahydropalmatine after 6 consecutive injections was 0.18%, less than 2.0%; the RSD% of the retention time was 0.04%, less than 1.0%, which met the requirements and the instrument precision was good.

[0058] (3) Linear relationship Take an appropriate amount of corydaline reference substance, prepare it into a mother solution with methanol, and then dilute it into a series of concentrations of 200 μg / mL, 100 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL.

[0059] Accurately pipette 10 L of each of the above concentration reference solution and inject it into the liquid chromatograph, and draw a linear relationship graph with the logarithm of the concentration (μg / mL) as the abscissa (X) and the logarithm of each peak area as the ordinate (Y). The results are shown in Table 10.

[0060] Table 10: Results of linear relationship investigation for the verification of the method for determining the content of tetrahydropalmatine

[0061] Result analysis: With the concentration of the reference substance (μg / ml) as the horizontal coordinate (X) and the peak area as the vertical coordinate (Y), a straight line was plotted and a regression equation was drawn. The regression equation was Y=8588.3079X+1871.0501, and the correlation coefficient was 1.0000, which was greater than 0.9990, meeting the requirements. The linearity was good in the range of 4.9829μg / mL-199.3150μg / mL.

[0062] (4) Precision Repeatability experiment: Take 6 portions of the test sample and prepare the test sample solution according to the preparation method of the test sample solution. Accurately pipette 10 μL of the reference solution and the test sample solution respectively, inject into the chromatograph, and measure. The results are shown in Table 11.

[0063] Table 11: Results of repeatability study on the precision verification of the method for determining the content of tetrahydropalmatine

[0064] Result analysis: The average content of 6 test solutions was 0.063 mg / g, and the RSD% was 1.56%, which was less than 6%, meeting the requirements and having good repeatability.

[0065] Intermediate precision: Different personnel used different brands of instruments at different times and conducted the test according to the repeatability method. The results are shown in Tables 12 and 13.

[0066] Table 12: Intermediate precision inspection results of the precision verification of the method for determining the content of tetrahydropalmatine 1

[0067] Table 13: Results of precision verification, repeatability and intermediate precision of the method for determining the content of tetrahydropalmatine 2

[0068] Result analysis: The average content of 6 test solutions with intermediate precision was 0.064 mg / g, and the RSD% was 3.33%, which was less than 6%. The RSD% of 12 test solutions with repeatability and intermediate precision was 2.57%, which was less than 11%, which met the requirements and the intermediate precision was good.

[0069] (5) Accuracy Preparation of recovery reference solution: Take an appropriate amount of corydaline reference substance, accurately weigh it, and add 0.2 mol / L hydrochloric acid solution to make a solution containing 1.3 µg per 1 mL.

[0070] Preparation of test solution: Take 6 portions of Chinese medicine composition, about 1 g each, accurately weigh, place in a stoppered conical flask, accurately add 50 mL of recovery reference solution, seal, weigh, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with 0.2 mol / L hydrochloric acid solution, shake well, centrifuge (4000 rpm) for 5 minutes, accurately measure 25 mL of supernatant, shake and extract 3 times with dichloromethane, 25 mL each time, combine the dichloromethane solution, wash 3 times with ammonia test solution, 20 mL each time, discard the washing solution, evaporate the dichloromethane solution, dissolve the residue in methanol, transfer to a 5 mL volumetric flask, and dilute to the scale, shake well, filter, and take the filtrate to obtain.

[0071] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into chromatograph, and determine. The results are shown in Table 14.

[0072] Table 14: Results of the accuracy test of the method for determining the content of tetrahydropalmatine

[0073] Result analysis: The recoveries of the six test solutions were in the range of 89.69%-95.01%, all in the range of 80%-115%, the average recovery was 93.2%, and the RSD% was 1.95%, which was less than 6%, meeting the requirements and having good accuracy.

[0074] Example 1: Method for determining the content of vinegar Corydalis in a Chinese medicinal composition capable of alleviating pain and numbness The best method for determination is as follows: Vinegar Corydalis: Determination by high performance liquid chromatography.

[0075] Chromatographic conditions and system suitability: Octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution (triethylamine adjusted pH to 7.0) was used as mobile phase B, gradient elution was performed according to the provisions in Table 1, and the detection wavelength was 282 nm. The theoretical plate number calculated based on the tetrahydropalmatine peak should be no less than 5000.

[0076] Preparation of reference solution: Take an appropriate amount of corydaline reference substance, weigh accurately, and add methanol to make a solution containing 20µg per 1mL.

[0077] Preparation of test solution: Take about 2 g of each sample of three batches of pilot Chinese herbal composition for relieving pain and numbness, accurately weigh, put in a stoppered conical flask, accurately add 50 mL of 0.2 mol / L hydrochloric acid solution, seal, weigh, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with 0.2 mol / L hydrochloric acid solution, shake well, centrifuge (4000 rpm) for 5 minutes, accurately measure 25 mL of supernatant, shake and extract 3 times with dichloromethane, 25 mL each time, combine the dichloromethane solution, wash 3 times with ammonia test solution, 20 mL each time, discard the washing solution, evaporate the dichloromethane solution, dissolve the residue in methanol, transfer to a 5 mL volumetric flask, and dilute to the scale, shake well, filter, and take the filtrate to obtain three batches of test solution.

[0078] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into chromatograph, record chromatogram, and calculate by peak area according to external standard method. The results are shown in Table 15.

[0079] Table 15: Results of content determination of three batches of pilot Chinese medicine compositions

[0080] It can be seen from the results that according to the measurement results of the above batches of pilot samples, the average content of vinegar Corydalis in the traditional Chinese medicine composition is about 0.062 mg / g in terms of Corydalis butyl.

[0081] It is proposed that each 1g of the Chinese medicinal composition for relieving pain and numbness contains 1g of vinegar corydalis and 1g of corydalis ethyl (C 21 H 25 NO4), shall not be less than 0.050 mg.

[0082] Comparative Example 1: The content determination test of tetrahydropalmatine in the traditional Chinese medicine composition that can relieve pain and numbness was carried out with reference to the content determination method under the item of corydalis in the 2020 edition of the Chinese Pharmacopoeia, as follows: Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler, acetonitrile-0.1% phosphoric acid solution (triethylamine adjusted pH to 7.0) (55:45) was used as mobile phase, and the detection wavelength was 282nm. The theoretical plate number calculated based on the tetrahydropalmatine peak should be no less than 3000.

[0083] Preparation of reference solution: Take an appropriate amount of corydaline reference substance, weigh accurately, and add methanol to make a solution containing 46µg per 1mL.

[0084] Preparation of test solution: Take appropriate amount of three batches of Chinese medicine composition (approximately equivalent to 0.5g of vinegar Corydalis crude drug), accurately weigh, place in a stoppered conical flask, accurately add 50mL of concentrated ammonia test solution-methanol mixed solution (1:20), stopper, weigh, heat under reflux for 1 hour, cool, weigh again, make up the lost weight with concentrated ammonia test solution-methanol mixed solution (1:20), shake well, filter, accurately measure 25mL of supernatant, evaporate to dryness, dissolve the residue in methanol, transfer to a 5mL volumetric flask, and dilute to the scale, shake well, filter, and take the filtrate.

[0085] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0086] A map of one of the batches is shown in Figure 7 The integration results of the three batches of spectra are shown in the table below.

[0087] Table 16: Integration results of HPLC spectra obtained by the method of Comparative Example 1 for three batches of Chinese medicine composition samples

[0088] Results: According to the above spectrum and integration results, the baseline at the chromatographic peak of tetrahydropalmatine in the chromatogram of the test sample is uneven and the matching value of the peak is less than 980, indicating that the chromatographic peak is impure.

[0089] Experimental Example 2: Study on the determination method of turmeric content in a traditional Chinese medicine composition that can relieve pain and numbness Turmeric is the main ingredient in the prescription, and contains high levels of volatile oil and fat-soluble components. The method for determining the content of curcumin was studied. After preliminary exploration of the conditions, the chromatographic conditions for curcumin were determined as follows: Chromatographic column: Phenol Titank C18 (5μm, 250×4.6mm, SN: 113AB20422); mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the program in the following table; flow rate: 1.0ml / min; column temperature: 30℃; detection wavelength: 430nm; injection volume: 5μL.

[0090] Table 17: Gradient elution program for determination of curcumin content

[0091] Preparation of reference solution: Take an appropriate amount of curcumin reference substance, weigh accurately, and add methanol to make a solution containing 20µg per mL.

[0092] 1. Study on the preparation method of test solution Samples of the Chinese medicinal composition capable of relieving pain and numbness were taken, and the content of curcumin was used as an indicator to conduct an investigation on the extraction solvent, extraction method, extraction time, extraction solvent volume, etc. using the above-mentioned chromatographic determination method, as follows: (1) Investigation of extraction solvent Take about 0.5g of the Chinese medicine composition, a total of 4 groups, accurately weigh, put in a stoppered conical bottle, add 0.5mL of petroleum ether (60-90℃), slightly heat to dissolve the sample, then accurately add 25% methanol, 50% methanol, 75% methanol, and 50mL of methanol, weigh, ultrasonically treat (300W, 40kHz) for 30 minutes, take out, cool, weigh again, add the corresponding extraction solvent to make up for the lost weight, shake well, filter, and obtain. The results are shown in the table below.

[0093] Table 18: Investigation of extraction solvents for determination of curcumin content

[0094] Result analysis: When the extraction solvent was methanol, the curcumin content in the test solution was higher than that when the extraction solvent was 25% methanol, 50% methanol, and 75% methanol, and the test solution was easier to filter, so methanol was selected as the extraction solvent.

[0095] (2) Investigation of extraction methods Take about 0.5g of the Chinese medicine composition, a total of 2 groups, accurately weigh, put in a stoppered conical flask, add 0.5mL of petroleum ether (60-90℃), slightly heat to dissolve the sample, then accurately add 50mL of methanol, weigh, heat and reflux for 30 minutes / ultrasonic treatment (300W, 40kHz) for 30 minutes, take out, cool, weigh again, add methanol to make up for the lost weight, shake well, filter, and obtain. The results are shown in the table below.

[0096] Table 19: Investigation of extraction methods for determination of curcumin content

[0097] Result analysis: The extraction method was heating reflux. There was no significant difference in the curcumin content in the test solution during ultrasonic treatment. Considering that ultrasonic treatment is relatively simple, ultrasonic treatment was selected as the extraction method.

[0098] (3) Extraction time investigation Take about 0.5g of the Chinese medicine composition, a total of 3 groups, accurately weigh, put in a stoppered conical bottle, add 0.5mL of petroleum ether (60-90℃), slightly heat to dissolve the sample, then accurately add 50mL of methanol, weigh, ultrasonically treat (300W, 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively, take out, cool, weigh again, add methanol to make up for the lost weight, shake well, filter, and obtain. The results are shown in the table below.

[0099] Table 20: Curcumin content determination and extraction time investigation

[0100] Result analysis: There was no significant difference in the curcumin content in the test solution when the extraction time was 15 minutes and 30 minutes, and the content was higher than when the extraction time was 45 minutes; considering the differences between sample batches, 30 minutes was selected as the optimal extraction time.

[0101] (4) Investigation of extraction solvent volume Take about 0.5g of the Chinese medicine composition, a total of 3 groups, accurately weigh, put in a stoppered conical bottle, add 0.5mL of petroleum ether (60-90℃), slightly heat to dissolve the sample, then accurately add 25mL, 50mL, and 100mL of methanol respectively, weigh, ultrasonically treat (300W, 40kHz) for 30 minutes, take out, cool, weigh again, add methanol to make up for the lost weight, shake well, filter, and obtain. The results are shown in the table below.

[0102] Table 21: Investigation of extraction solvent volume for determination of curcumin content

[0103] Result analysis: There was no significant difference in the curcumin content in the test solution when the extraction solvent volume was 25mL, 50mL, and 100mL. Considering the differences between sample batches and saving solvent, 50mL was selected as the optimal extraction solvent volume.

[0104] 2. Validation of the methodological approach for determination of curcumin content The specificity, instrument precision, linearity, precision (repeatability, intermediate precision), and accuracy of the curcumin content determination method were investigated as follows: Except for special instructions, the test solution in the following curcumin content verification experiment is prepared according to this method: take about 0.5g of the Chinese medicine composition, accurately weigh it, put it in a stoppered conical flask, add 0.5mL of petroleum ether (60-90℃) and slightly heat to dissolve the sample, then accurately add 50mL of methanol, weigh it, ultrasonically treat it (300W, 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and obtain it.

[0105] In the following curcumin content verification experiments, unless otherwise specified, the reference solution is prepared according to this method: take an appropriate amount of curcumin reference substance, accurately weigh it, and add methanol to make a solution containing 20μg per mL.

[0106] (1) Exclusivity Tested by high performance liquid chromatography (DAD detector).

[0107] Preparation of turmeric negative sample solution: Take about 0.5 g of turmeric negative sample, accurately weigh it, put it in a stoppered conical flask, add 0.5 mL of petroleum ether (60-90°C) and slightly heat to dissolve the sample, then accurately add 50 mL of methanol, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and obtain it.

[0108] Blank solvent: methanol Determination method: Accurately pipette 5 μL of blank solvent, reference solution, test solution and turmeric negative sample solution respectively, inject into chromatograph, and determine. The chromatogram is as follows: Figure 8-Figure 13 shown.

[0109] Result analysis: Fig.10 In the chromatogram of the test sample, a chromatographic peak was detected at the corresponding retention time position of the curcumin reference substance, and the retention time of the main peak of the test sample was 31.783 minutes. Fig. 9 The retention time of the main peak of the reference substance is 31.787 minutes, the separation degree between the main peak and the adjacent chromatographic peak is 3.45, which is greater than 1.5, the peak purity is 1000, which is greater than 980, and the spectral absorption of the main peak is consistent with that of the reference substance. Figure 8 Blank solvent and Fig.11 In the chromatogram of the turmeric negative sample, no chromatographic peaks were detected at the corresponding retention time positions of the main peaks of the test sample and the reference sample, indicating that the method has good specificity.

[0110] (2) Instrument precision Determination method: Accurately pipette 5 μL of the reference solution, inject into the chromatograph, inject 6 times in a row, and determine. The results are shown in the table below.

[0111] Table 22: Instrument precision results for the verification of the method for determining the content of curcumin

[0112] Result analysis: The RSD% of the curcumin peak area after 6 consecutive injections was 0.34%, less than 2.0%; the RSD% of the retention time was 0.03%, less than 1.0%, which met the requirements and the instrument precision was good.

[0113] (3) Linear relationship Take the curcumin reference substance, weigh it accurately, dissolve it in methanol and dilute it to prepare a series of curcumin reference substance concentrations of 200μg / mL, 100μg / mL, 40μg / mL, 20μg / mL, 10μg / mL, and 5μg / mL. Accurately take 5μL of each concentration of the reference substance solution, inject it into the liquid chromatograph, and measure it. The results of the linear relationship investigation are shown in the table below.

[0114] Table 23: Results of linear relationship investigation for verification of curcumin content determination methodology

[0115] Result analysis: With the concentration of the reference substance (μg / mL) as the abscissa (X) and the peak area as the ordinate (Y), a straight line was plotted and a regression equation was drawn. The regression equation was Y=44829.7495X+21231.7593, and the correlation coefficient was 1.0000, which was greater than 0.9990 and met the requirements, indicating that curcumin had a good linear relationship in the range of 5.0325μg / mL-201.3012μg / mL.

[0116] (4) Precision Repeatability: Take 6 samples, each about 0.5g, weigh accurately, put in a stoppered conical bottle, add 0.5mL of petroleum ether (60-90℃), heat slightly to dissolve the sample, then accurately add 50mL of methanol, weigh the weight, ultrasonically treat (300W, 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and get 6 test solutions. Accurately take 5μL of reference solution and test solution respectively, inject into the chromatograph, and measure. The results are shown in the table below.

[0117] Table 24: Results of repeatability study on the methodological validation of curcumin content determination

[0118] Result analysis: The average content of 6 test solutions was 1.455 mg / g, and the RSD% was 1.4%, which was less than 3%, meeting the requirements and having good repeatability.

[0119] Intermediate precision: Different personnel use instruments of different brands at different times and test according to the method under repeatability.

[0120] Table 25: Intermediate precision study results of curcumin content determination method validation 1

[0121] Table 26: Intermediate precision study results of curcumin content determination method validation 2

[0122] Result analysis: The average content of 6 test solutions with intermediate precision was 1.319 mg / g, and the RSD% was 2.72%, which was less than 3%. The RSD% of 12 test solutions with repeatability and intermediate precision was 5.47%, which was less than 6%, which met the requirements and the intermediate precision was good.

[0123] (5) Accuracy Preparation of recovery reference solution: Take an appropriate amount of curcumin reference, accurately weigh it, and add methanol to make a solution containing 7.5µg per mL.

[0124] Preparation of test solution: Take 6 portions of the test sample, about 0.25g each, accurately weigh, place in a stoppered conical flask, add 0.5mL of petroleum ether (60~90℃), slightly heat to dissolve the sample, then accurately add 50mL of recovery reference solution, ultrasonically treat (300W, 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and obtain.

[0125] Determination method: Accurately pipette 5 μL of reference solution and test solution respectively, inject into chromatograph, and determine. The results are shown in the table below.

[0126] Table 27: Results of accuracy study on the methodological validation of curcumin content determination

[0127] Result analysis: The recoveries of the six test solutions were in the range of 105.12%-106.68%, all in the range of 90%-108%, the average recovery was 106.2%, and the RSD% was 0.58%, less than 3%, which met the requirements and had good accuracy.

[0128] Example 2: Method for determining the content of turmeric in a Chinese medicinal composition capable of alleviating pain and numbness Turmeric: Determination by HPLC.

[0129] Chromatographic conditions and system suitability: Octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was performed as specified in Table 17, and the detection wavelength was 430 nm; the theoretical plate number calculated based on the curcumin peak should be no less than 5000.

[0130] Preparation of reference solution: Take an appropriate amount of curcumin reference substance, weigh accurately, and add methanol to make a solution containing 20µg per mL.

[0131] Preparation of test solution: Take about 0.5 g of each sample of three batches of pilot Chinese herbal composition for relieving pain and numbness, accurately weigh, put in a stoppered conical flask, add 0.5 mL of petroleum ether (60-90°C), slightly heat to dissolve the sample, accurately add 50 mL of methanol, weigh, ultrasonically treat (300 W, 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and obtain three batches of test solution.

[0132] Determination method: Accurately pipette 5 μL of reference solution and test solution respectively, inject into chromatograph, record chromatogram, and calculate by peak area according to external standard method. The results are shown in the table below.

[0133] Table 28: Results of determination of curcumin content in three batches of pilot Chinese medicine compositions

[0134] It can be seen from the results that the average content of multiple batches of pilot samples of the Chinese medicine composition is about 1.57 mg / g.

[0135] Proposed: Each 1g of the Chinese medicinal composition for relieving pain and numbness contains turmeric and curcumin (C 21 H 20 O6), not less than 1 mg.

[0136] Experimental Example 3: Study on the determination method of borneol content in a traditional Chinese medicine composition that can relieve pain and numbness Borneol is volatile, and its content may change during storage. A method for determining the content of borneol in a traditional Chinese medicine composition that can relieve pain and numbness was studied. After preliminary exploration of conditions, the gas chromatography conditions for borneol were determined as follows: Chromatographic column: Agilent DB-WAX (30m, 0.32mm, 0.25μm), column number: US1121622H; temperature program: initial temperature 130°C, hold for 15 minutes, increase to 230°C at a rate of 20°C per minute, hold for 5 minutes; injection port temperature 200°C; detector temperature 240°C; gas flow: constant flow mode, 1.0mL / min; detector: FID; injection volume: 1μL. The theoretical plate number calculated based on the borneol peak is not less than 10,000.

[0137] Preparation of reference solution: Take an appropriate amount of borneol reference, weigh accurately, and add ethyl acetate to make a solution containing 50µg per mL.

[0138] 1. Study on the preparation method of test solution A sample of a Chinese medicine composition capable of relieving pain and numbness was taken, and the extraction solvent, extraction method, extraction time, extraction solvent volume, etc. were investigated using the above-mentioned gas chromatography determination method, with the borneol content as an indicator, as follows: (1) Investigation of extraction solvent Take about 0.5g of the Chinese medicine composition, a total of 3 groups, accurately weigh, place in a stoppered conical bottle, add 0.5mL of petroleum ether (60-90℃), slightly heat to dissolve the sample, then accurately add 50mL of methanol, ethanol, and ethyl acetate, weigh the weight, ultrasonically treat (300W, 40kHz) for 30 minutes, take out, cool, weigh again, add the corresponding extraction solvent to make up for the lost weight, shake well, filter, and get it. Accurately take 1μL of the reference solution and the test solution, inject into the chromatograph, and measure, and get it. The results are shown in the table below.

[0139] Table 29: Investigation of extraction solvents for borneol content determination

[0140] Result analysis: When the extraction solvent is ethyl acetate, the borneol content in the test solution is higher than that when the extraction solvent is methanol or ethanol, so ethyl acetate is selected as the extraction solvent.

[0141] (2) Investigation of extraction methods Take about 0.5g of the Chinese medicine composition, a total of 2 groups, accurately weigh, place in a stoppered conical bottle, add 0.5mL of petroleum ether (60-90℃), slightly heat to dissolve the sample, then accurately add 50mL of ethyl acetate, weigh, heat and reflux for 30 minutes / ultrasonic treatment (300W, 40kHz) for 30 minutes, take out, cool, weigh again, add ethyl acetate to make up for the lost weight, shake well, filter, and get it. Accurately take 1μL of the reference solution and the test solution, inject into the chromatograph, and measure, and get it. The results are shown in the table below.

[0142] Table 30: Investigation of extraction methods for determination of borneol content

[0143] Result analysis: There was no significant difference in the borneol content in the test solution when the extraction method was heating reflux or ultrasonic treatment. Considering that ultrasonic treatment is relatively simple, ultrasonic treatment was selected as the extraction method.

[0144] (3) Extraction time investigation Take about 0.5g of the Chinese medicine composition, a total of 3 groups, accurately weigh, place in a stoppered conical flask, add 0.5mL of petroleum ether (60-90℃), slightly heat to dissolve the sample, then accurately add 50mL of ethyl acetate, weigh the weight, ultrasonically treat (300W, 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively, take out, cool, weigh again, add ethyl acetate to make up for the lost weight, shake well, filter, and get it. Accurately aspirate 1μL of the reference solution and the test solution respectively, inject into the chromatograph, and measure to get it. The results are shown in the table below.

[0145] Table 31: Determination of borneol content and extraction time

[0146] Result analysis: There was no significant difference in the borneol content in the test solution when the extraction time was 15 minutes, 30 minutes, and 45 minutes. Considering the differences between sample batches and saving time, 30 minutes was selected as the optimal extraction time.

[0147] (4) Investigation of extraction solvent volume Take about 0.5g of the Chinese medicine composition, a total of 3 groups, accurately weigh, place in a stoppered conical flask, add 0.5mL of petroleum ether (60~90℃), heat slightly to dissolve the sample, then accurately add 25mL, 50mL, and 100mL of ethyl acetate, weigh, ultrasonically treat (300W, 40kHz) for 30 minutes, take out, cool, weigh again, add ethyl acetate to make up for the lost weight, shake well, filter, and get it. Accurately aspirate 1μL of the reference solution and the test solution, inject into the chromatograph, and measure to get it. The results are shown in the table below.

[0148] Table 32: Determination of borneol content extraction solvent volume investigation

[0149] Result analysis: There was no significant difference in the borneol content in the test solution when the extraction solvent volume was 25mL, 50mL, and 100mL. Considering the differences between sample batches and saving solvent, 50mL was selected as the optimal extraction solvent volume.

[0150] 2. Validation of borneol content determination methodology The specificity, instrument precision, linearity, precision (repeatability, intermediate precision), and accuracy of the borneol content determination method of the traditional Chinese medicine composition (batch number: DYTB-0101-230824) that can relieve pain and numbness were investigated as follows: In the following curcumin content verification experiment, unless otherwise specified, the reference solution is prepared according to this method: take an appropriate amount of borneol reference substance, accurately weigh it, and add ethyl acetate to make a solution containing 50 μg per mL.

[0151] In the following borneol content verification experiment, unless otherwise specified, the test solution is prepared according to this method: take about 0.5g of the traditional Chinese medicine composition, place it in a stoppered conical flask, add 0.5mL of petroleum ether (60-90°C) and heat it slightly to dissolve, then accurately add 50mL of ethyl acetate, weigh the weight, ultrasonically treat (300W, 40kHz) for 30 minutes, make up the lost weight with ethyl acetate, shake well, filter, and take the filtrate to obtain.

[0152] (1) Exclusivity Preparation of borneol negative sample solution: Take about 0.5 g of borneol negative sample, weigh it accurately, put it in a stoppered conical flask, add 0.5 mL of petroleum ether (60-90°C) and heat it slightly to dissolve the sample, then accurately add 50 mL of ethyl acetate, weigh it, ultrasonically treat it (300 W, 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and you are done.

[0153] Blank solvent: ethyl acetate Determination method: Accurately pipette 1 μl of blank solvent, reference solution, test solution and borneol negative sample solution respectively, inject into gas chromatograph, and determine. Figure 14-17 .

[0154] Result analysis: Fig.16 In the chromatogram of the test sample, a chromatographic peak was detected at the corresponding retention time position of the borneol reference substance, and the retention time of the main peak of the test sample was 4.107 minutes. Fig.15 The retention time of the main peak of the reference substance was 4.112 minutes, and the separation degree between the main peak and the adjacent chromatographic peaks was 2.740, which was greater than 1.5. Fig.14 Blank solvent and Fig.17 In the chromatogram of the borneol-negative sample, no chromatographic peak was detected at the corresponding retention time position of the main peaks of the test sample and the reference sample, indicating that this method has good specificity.

[0155] (2) Instrument precision Preparation of reference solution: Take an appropriate amount of borneol reference, weigh accurately, and add ethyl acetate to make a solution containing 50 μg per 1 ml.

[0156] Determination method: Accurately pipette 1 μL of the reference solution, inject it into the gas chromatograph, inject 6 times continuously, and determine. The results are shown in the table below.

[0157] Table 33: Instrument precision results for borneol content determination

[0158] Result analysis: The RSD% of borneol peak area after 6 consecutive injections was 1.00%, less than 2.0%; the RSD% of retention time was 0.06%, less than 1.0%, indicating that the instrument had good precision.

[0159] (3) Linear relationship Take borneol reference substance, accurately weigh it, add ethyl acetate to dissolve it, and prepare a series of reference substance solutions with concentrations of 200 μg / mL, 120 μg / mL, 100 μg / mL, 60 μg / mL, 40 μg / mL, and 20 μg / mL.

[0160] Determination method: Accurately pipette 1 μL of each of the above series of concentration reference solutions, inject into gas chromatograph, and determine. The results are shown in the table below.

[0161] Table 34: Results of linear relationship investigation of borneol content determination

[0162] Result analysis: With the concentration of the reference substance (μg / ml) as the horizontal coordinate (X) and the peak area as the vertical coordinate (Y), a straight line was plotted and a regression equation was drawn. The regression equation was Y=1893.9081X-564.4268, and the correlation coefficient was 1.0000, which was greater than 0.9990, indicating that borneol had a good linear relationship in the range of 19.8403μg / mL-198.4032μg / mL.

[0163] (4) Precision Repeatability Preparation of reference solution: Take an appropriate amount of borneol reference, weigh accurately, and add ethyl acetate to make a solution containing 50 μg per 1 ml.

[0164] Preparation of test solution: Take 6 samples, each about 0.5 g, put them in a stoppered conical flask, add 0.5 mL of petroleum ether (60-90°C), heat slightly to dissolve, then accurately add 50 mL of ethyl acetate, weigh the weight, ultrasonically treat (300 W, 40 kHz) for 30 minutes, make up the lost weight with ethyl acetate, shake well, filter, and take the filtrate to obtain the solution.

[0165] Determination method: Accurately measure 1 μL of reference solution and test solution respectively, inject into gas chromatograph, and determine. The results are shown in the table below.

[0166] Table 35: Results of repeatability test of borneol content determination

[0167] Result analysis: The average content of 6 test solutions was 12.028 mg / g, and the RSD% was 0.83%, which was less than 2%, meeting the requirements and having good repeatability.

[0168] Intermediate precision: Different people used different brands of instruments at different times and conducted tests according to the repeatability method. The experimental results are shown in the table below.

[0169] Table 36: Results of intermediate precision study on borneol content determination 1

[0170] Table 37: Results of intermediate precision study of borneol content determination 2

[0171] Result analysis: The average content of 6 test solutions with intermediate precision was 11.508 mg / g, and the RSD% was 1.81%, which was less than 2%. The RSD% of 12 test solutions with repeatability and intermediate precision was 2.66%, which was less than 4%, which met the requirements and the intermediate precision was good.

[0172] (5) Accuracy Preparation of reference solution: Take an appropriate amount of borneol reference, accurately weigh it, and add ethyl acetate to make a solution containing 50 μg per 1 mL.

[0173] Preparation of recovery reference solution: Take an appropriate amount of borneol reference, accurately weigh it, and add ethyl acetate to make a solution containing 53µg per 1mL.

[0174] Preparation of test solution: Take 6 portions of Chinese medicine composition, about 0.25g each, accurately weigh, place in a stoppered conical flask, add 0.5mL of petroleum ether (60~90℃), heat slightly to dissolve the sample, then accurately add 50mL of recovery reference solution, ultrasonically treat (300W, 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with ethyl acetate, shake well, filter, and obtain.

[0175] Determination method: Accurately pipette 1 μL of reference solution and test solution respectively, inject into gas chromatograph, and determine.

[0176] Table 38: Results of accuracy test of borneol content

[0177] Result analysis: The recoveries of the six test solutions were in the range of 100.37% to 104.26%, all in the range of 92% to 105%, with an average recovery of 102.2%, and an RSD% of 1.54%, less than 2.0%, which met the requirements and had good accuracy.

[0178] Example 3: Method for determining the content of borneol in a Chinese medicinal composition capable of alleviating pain and numbness The method for determining the content of borneol in traditional Chinese medicine compositions is proposed as follows: Borneol: Determined by gas chromatography.

[0179] Chromatographic conditions and system suitability test: polyethylene glycol 20M (or similar polarity) is used as the stationary phase; the temperature program is: the initial temperature is 130°C, maintained for 15 minutes, and the temperature is increased to 230°C at a rate of 20°C per minute, and maintained for 5 minutes; the injection port temperature is 200°C; the detector temperature is 240°C. The theoretical plate number is not less than 10,000 calculated based on the borneol peak.

[0180] Preparation of reference solution: Take an appropriate amount of borneol reference, accurately weigh it, and add ethyl acetate to make a solution containing 50 μg per 1 mL.

[0181] Preparation of test solution: Take about 0.5 g of each sample of three batches of pilot Chinese medicine compositions of the Chinese medicine composition capable of relieving pain and numbness, place in a stoppered conical flask, add 0.5 mL of petroleum ether (60-90°C), heat slightly to dissolve, then accurately add 50 mL of ethyl acetate, weigh the weight, ultrasonically treat (300 W, 40 kHz) for 30 minutes, make up the lost weight with ethyl acetate, shake well, filter, and take the filtrate to obtain the sample test solutions of the three batches of pilot Chinese medicine compositions.

[0182] Determination method: Accurately pipette 1 μL of reference solution and test solution respectively, inject into gas chromatograph, record chromatogram, and calculate by peak area according to external standard method. The results are shown in the table below.

[0183] Table 39: Content determination results of three batches of Chinese medicine compositions in the pilot test

[0184] It can be seen from the results that the average content of multiple batches of pilot samples of the Chinese medicine composition is 12.55 mg / g.

[0185] Proposed: Each 1g of the Chinese medicine composition that can relieve pain and numbness contains borneol and borneol (C 10 H 18 O) shall not be less than 10 mg.

[0186] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for controlling the quality of a traditional Chinese medicine composition that can relieve pain and numbness, wherein the traditional Chinese medicine composition is made of the following components in parts by weight: 2-8 parts of Millettia reticulata, 3-10 parts of Corydalis yanhusuo, 4-10 parts of Caulis Sinomenii, 2-5 parts of frankincense, 2-8 parts of Zanthoxylum bungeanum, 2-7 parts of Chuanxiong, 1-6 parts of Sappanwood, 0.5-3 parts of Borneolum, 3-9 parts of Curcuma longa, 1-7 parts of Achyranthes bidentata, 0.5-4 parts of Capsicum and 1-5 parts of Cinnamomum cassia, characterized in that: The quality control method includes the determination of the contents of tetrahydropalmatine, curcumin and borneol; The chromatographic conditions for determining the content of tetrahydropalmatine are as follows: Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: A is acetonitrile, B is 0.1% phosphoric acid aqueous solution, and triethylamine is used to adjust the pH to 7.0; elution gradient: 0-30 min, A is 40%, B is 60%; 30-35 min, A is 40%→60%, B is 60%→40%; 35-40 min, A is 60%→40%, B is 40%→60%; 40-45 min, A is 40%, B is 60%; detection wavelength is 282 nm; column temperature is 30°C; flow rate is 1 mL / min.

2. The quality control method according to claim 1, characterized in that: The content determination steps of tetrahydropalmatine are as follows: Preparation of reference solution: Take an appropriate amount of tetrahydropalmatine reference substance, accurately weigh it, and add methanol to make a 20µg / mL solution; Preparation of test solution: Take the test sample of the Chinese medicine composition, extract, extract, wash in sequence, and then make up to volume; Determination method: Accurately aspirate the reference solution and the test solution respectively, inject into the liquid chromatograph, determine, calculate, and obtain the result.

3. The quality control method according to claim 2, characterized in that: The preparation of the test solution is carried out through the following steps: (1) Extraction: Take 2 g of the test sample and heat it under reflux for 15-45 minutes in 25-50 mL of 0.1-0.3 mol / L hydrochloric acid solution, cool it, make up the weight with the solvent, centrifuge it for 4-6 minutes at a centrifugal speed of 3000-5000 rpm to obtain the supernatant; (2) Extraction: Take the supernatant and extract it 2-4 times with an equal volume of dichloromethane, and combine the dichloromethane solutions; (3) Washing: Wash the dichloromethane solution with ammonia solution for 2-4 times, discard the washing solution, evaporate the dichloromethane solution to dryness, dissolve the residue in methanol, transfer to a 5 mL volumetric flask, and dilute to the mark. Shake well, filter, and take the filtrate to obtain the final product.

4. The quality control method according to claim 3, characterized in that: The preparation of the test solution is specifically as follows: (1) Extraction: Take about 2 g of the Chinese medicine composition, weigh it accurately, put it in a stoppered conical flask, add 50 mL of 0.2 mol / L hydrochloric acid solution accurately, stopper it tightly, weigh it, heat it under reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with 0.2 mol / L hydrochloric acid solution, shake it well, centrifuge it for 5 minutes at a speed of 4000 rpm, and obtain the supernatant; (2) Extraction: Accurately measure 25 mL of the supernatant and extract with dichloromethane three times, 25 mL each time, and combine the dichloromethane solutions; (3) Washing: Wash the dichloromethane solution with ammonia test solution three times, 20 mL each time, discard the washing solution, evaporate the dichloromethane solution to dryness, dissolve the residue in methanol, transfer to a 5 mL volumetric flask, and dilute to the scale. Shake well, filter, and take the filtrate to obtain the product.

5. The quality control method according to claim 1, characterized in that: The content determination method of the curcumin is: Liquid chromatography conditions: the chromatographic column was filled with octadecylsilane bonded silica gel; mobile phase A was acetonitrile, and mobile phase B was 0.1% phosphoric acid aqueous solution; elution gradient: 0-20min, A was 42%, B was 58%; 20-35min, A was 42%→55%, B was 58%→45%; 35-40min, A was 55%→42%, B was 45%→58%; 40-45min, A was 42%, B was 58%; detection wavelength was 430nm; column temperature was 30℃; flow rate was 1mL / min; Preparation of reference solution: Take an appropriate amount of curcumin reference substance, accurately weigh it, and add methanol to make a 20µg / mL solution; Preparation of test solution: Take the test sample, extract with solvent, and make up to volume; Determination method: Accurately aspirate the reference solution and the test solution respectively, inject into the liquid chromatograph, determine, calculate, and obtain the result.

6. The quality control method according to claim 5, characterized in that: When determining the curcumin, the test solution is prepared by the following steps: taking 0.5 g of the traditional Chinese medicine composition, dissolving it in petroleum ether, adding 25-50 mL of methanol, ultrasonically treating it for 15-30 minutes, cooling it, weighing it again, making up the lost weight with methanol, shaking it well, filtering it, and taking the filtrate to obtain it.

7. The quality control method according to claim 6, characterized in that: The preparation steps of the test solution for the determination of curcumin are specifically as follows: taking about 0.5 g of the Chinese medicine composition, accurately weighing it, placing it in a stoppered conical flask, adding 0.5 mL of 60-90° C. petroleum ether and slightly heating it to dissolve the sample, then accurately adding 50 mL of methanol, weighing it, treating it under ultrasound at 300 W and 40 kHz for 30 minutes, cooling it, weighing it again, making up the lost weight with methanol, shaking it well, filtering it, and taking the filtrate to obtain it.

8. The quality control method according to claim 1, characterized in that: The content determination method of borneol is: Gas chromatography conditions: chromatographic column: Agilent DB-WAX; temperature program: initial temperature: 130°C, hold for 15 min, increase to 230°C at a rate of 20°C per minute, hold for 5 min; injection port temperature: 200°C; detector temperature: 240°C; Gas flow rate: constant flow mode, 1.0mL / min; Detector: FID; Injection volume: 1 μL; Preparation of reference solution: Take an appropriate amount of borneol reference, weigh accurately, and add ethyl acetate to make a 50µg / mL solution; Preparation of test solution: Take the test sample, extract with solvent, and make up to volume; Determination method: Accurately aspirate the reference solution and the test solution respectively, inject into the gas chromatograph, determine, calculate, and obtain the result.

9. The quality control method according to claim 8, characterized in that: The test solution for the borneol determination is prepared by the following steps: taking 0.5 g of the traditional Chinese medicine composition, dissolving it in petroleum ether, adding 25-50 mL of ethyl acetate, ultrasonically treating it for 15-30 minutes, cooling it, weighing it again, making up the lost weight with ethyl acetate, shaking it well, filtering it, and taking the filtrate to obtain it.

10. The quality control method according to claim 9, characterized in that: The specific steps for preparing the test solution for the borneol determination are as follows: take about 0.5 g of the traditional Chinese medicine composition, accurately weigh it, put it in a stoppered conical flask, add 0.5 mL of 60-90°C petroleum ether and heat it slightly to dissolve the sample, then accurately add 50 mL of ethyl acetate, weigh it, treat it under ultrasound at 300 W and 40 kHz for 30 minutes, cool it, weigh it again, make up the lost weight with ethyl acetate, shake it well, filter it, and take the filtrate to obtain it.

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