A quality detection method of a traditional Chinese medicine composition

Abstract

The present invention relates to a method for quality detection of a traditional Chinese medicine composition, which identifies Drynaria fortunei, Ligustrum lucidum, Spatholobus suberectus, and Achyranthes bidentata by thin-layer chromatography, and determines the presence of Drynaria fortunei and naringin (C) in the traditional Chinese medicine composition by quantitative determination. 27 H 32 O 14 The method of the present invention provides the quality detection of traditional Chinese medicine compositions with integrity, characteristics and stability, and high detection sensitivity, and can realize the quality control of traditional Chinese medicine compositions. The content is greater than or equal to 1.79 mg per gram.

Description

Technical Field

[0001] This invention pertains to the detection technology of active ingredients in traditional Chinese medicine, specifically relating to a method for quality testing of a traditional Chinese medicine composition. Background Technology

[0002] Traditional Chinese medicine (TCM) quality testing relies on sensory observation of the medicinal materials' appearance and microscopic observation of tissue structure and cell morphology, or on colorimetric and precipitation reactions to detect specific components. These traditional techniques can only perform qualitative analysis and cannot achieve quantitative detection. With technological advancements, TCM quality testing techniques have gradually shifted from controlling single components to overall quality evaluation. This is accompanied by the development of modern instrumental analysis techniques, such as spectroscopy and chromatography, which are widely used. However, limitations in low-concentration detection still exist.

[0003] For example, CN117054588A discloses a tortoise antler bone-strengthening tablet and its identification method. The method for detecting naringin is as follows: high performance liquid chromatography is used according to the Chinese Pharmacopoeia General Chapter 0512. The chromatographic conditions and system suitability test are as follows: octadecylsilane-bonded silica gel is used as the packing material, and 0.05% acetic acid solution-methanol 65:35 is used as the mobile phase; the detection wavelength of psoralen and isopsoralen is 246 nm, and the detection wavelength of naringin is 284 nm; the column temperature is 25℃, and the theoretical plate number calculated based on the psoralen peak is greater than 3000. The patent specification clearly states that the content of naringin in this product should be higher than 0.90 mg / g, psoralen higher than 0.30 mg / g, and isopsoralen higher than 0.28 mg / g. Based on these, the total content is reduced by 20% to set the content limit of this product, that is, the content of naringin in this product should be higher than 0.72 mg / g, psoralen higher than 0.24 mg / g, and isopsoralen higher than 0.22 mg / g.

[0004] Thin-layer chromatography and high-performance liquid chromatography have enabled the identification of traditional Chinese medicine (TCM) to move from "experience-based judgment" to "scientific quantification," facilitating precise quality control. This invention provides a qualitative and quantitative detection method for a TCM composition consisting of Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Spatholobus suberectus, Pyrola rotundifolia, and Achyranthes bidentata, providing a theoretical basis for the quality standards and material basis research of the TCM composition described in this invention. Summary of the Invention

[0005] The purpose of this invention is to address the issue that traditional Chinese medicine compositions contain numerous components, particularly the volatile oil contained in naringin in Drynaria fortunei, which requires an effective method for detection in order to stabilize their efficacy and ensure the quality of medication.

[0006] The quality testing method for traditional Chinese medicine compositions of the present invention enables better control of product quality, and has high detection sensitivity and good repeatability. It can not only complete the qualitative detection of medicinal materials, but also realize the quantitative detection of effective active ingredients, determine the quality control limits, and provide a control method for the industrialization of the drug.

[0007] The technical means adopted in this invention are:

[0008] A method for quality testing of a traditional Chinese medicine composition, wherein the composition comprises Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Spatholobus suberectus, Pyrola rotundifolia, and Achyranthes bidentata; the composition is identified by thin-layer chromatography, and the quality control indicators of the composition are determined by naringin content determination, wherein Drynaria fortunei contains naringin (C... 27 H 32 O 14 The concentration is greater than or equal to 1.79 mg per gram.

[0009] Further, the raw materials of the traditional Chinese medicine composition are: 15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Spatholobus suberectus, 10 parts of Pyrola rotundifolia, and 10 parts of Achyranthes bidentata. Further, the preparation method of the traditional Chinese medicine composition is as follows: take the medicinal materials according to the prescribed dosage, add water and decoct twice. The first time, add 11 times the amount of water and decoct for 1.5 hours; the second time, add 9 times the amount of water and decoct for 1.5 hours. Combine the filtrates obtained from the two decoctions and perform two vacuum concentration treatments, including first-effect concentration and second-effect concentration, at a temperature of 60-80℃, to obtain a clear extract and an extract. The relative density of the clear extract is 1.15-1.25, and the relative density of the extract is 1.30-1.35. Mix the dried extract with the clear extract to obtain the final product.

[0010] Furthermore, the thin-layer chromatography method of the traditional Chinese medicine composition is used to identify Drynaria fortunei. The specific identification steps are as follows:

[0011] (1) Take the Chinese herbal medicine composition, grind it into a fine powder, add methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, add methanol to dissolve the residue, and prepare the test solution.

[0012] (2) Prepare a 1 mg / mL solution of naringin reference standard with methanol and use it as the reference solution;

[0013] (3) Take 5-10 μL of the above test solution and reference solution and spot them on the same silica gel G thin layer plate. Use dichloromethane-methanol-water volume ratio of 8:2:0.5 as the developing solvent. After saturation for 15 minutes, develop the plate, remove it, air dry it, spray it with aluminum trichloride solution, heat it at 105℃ and observe it under 365nm ultraviolet light. In the chromatogram of the test sample, the main spot of the same color appears at the corresponding position as in the chromatogram of the reference sample.

[0014] Furthermore, the thin-layer chromatography method of the traditional Chinese medicine composition is used to identify Ligustrum lucidum. The specific identification steps are as follows:

[0015] (1) Take the Chinese herbal medicine composition, grind it into a fine powder, add chloroform, sonicate for 30 min, filter, evaporate the filtrate to dryness, add chloroform to dissolve the residue, and use it as the test solution.

[0016] (2) Take the privet fruit reference material and prepare the privet fruit reference material solution in the same way as the test solution preparation operation described in step (1);

[0017] (3) Take 5-10 μL of the above test solution and reference medicinal material solution and spot them on the same silica gel G thin layer plate. Use ethyl acetate-methanol-water volume ratio of 100:17:13 as the developing solvent. After saturation for 15 minutes, develop the plate, remove it, air dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ and observe it under a 254nm ultraviolet lamp. In the chromatogram of the test sample, the main spot of the same color as the reference medicinal material chromatogram is obtained at the corresponding position.

[0018] Furthermore, the thin-layer chromatography method is used to identify *Spatholobus suberectus* using the aforementioned traditional Chinese medicine composition. The specific identification steps are as follows:

[0019] (1) Take the Chinese herbal medicine composition, grind it into a fine powder, add ethanol, sonicate for 30 min, filter, let the filtrate evaporate naturally, dissolve the residue in water, extract with ethyl acetate by shaking, evaporate the ethyl acetate, dissolve the residue in methanol to make it into the test solution.

[0020] (2) Take the chicken blood vine reference material and prepare the chicken blood vine reference material solution in the same way as the test sample solution preparation operation described in step (1);

[0021] (3) Take 5-10 μL of the above test solution and reference medicinal material solution and spot them on the same silica gel G thin layer plate. Use dichloromethane-acetone-methanol volume ratio of 8:1.2:0.3 as the developing solvent, develop, remove, air dry, and observe under 254 nm ultraviolet light. In the chromatogram of the test solution, the main spot of the same color as the reference medicinal material is obtained at the corresponding position.

[0022] Furthermore, the traditional Chinese medicine composition is used to identify Achyranthes bidentata by thin-layer chromatography. The specific identification steps are as follows:

[0023] (1) Take the Chinese herbal composition, add methanol, sonicate for 20 min, filter, evaporate the filtrate to dryness, add water to dissolve the residue, extract twice with ethyl acetate, 10 mL each time, combine the ethyl acetate solutions, recover the solvent to dryness, add methanol to dissolve the residue, and use it as the test solution.

[0024] (2) Take the reference herb Achyranthes bidentata and prepare the reference herb solution Achyranthes bidentata according to the test solution preparation operation described in step (1);

[0025] (3) Take 5-10 μL of the above test solution and reference medicinal material solution and spot them on the same silica gel G thin layer plate. Use hexane-ethyl acetate-acetone volume ratio of 8:5:1 as the developing solvent, develop to 8 cm, take it out, air dry, and observe under a 365 nm ultraviolet lamp. In the chromatogram of the test solution, the main spot of the same color as the reference medicinal material solution is obtained at the corresponding position.

[0026] Another objective of this invention is to provide a method for quality testing of a traditional Chinese medicine composition, wherein the method for determining the naringin content is implemented by the following steps:

[0027] (1) Take naringin reference standard and dissolve it in methanol to prepare a naringin reference standard solution with a concentration of 0.02144 mg / mL;

[0028] (2) Take the Chinese herbal medicine composition, dissolve it in methanol, sonicate it, filter it, and obtain the test solution;

[0029] (3) Take different volumes of naringin reference solution from step (1) and inject them into the liquid chromatograph. Determine the peak area of ​​naringin reference solution under the following chromatographic conditions: Agilent 5 TC-C18 column, mobile phase is 0.1% phosphoric acid solution-acetonitrile with a volume ratio of 75:25, detection wavelength is 280 nm, and flow rate is 1.0 mL / min.

[0030] (4) A standard curve was obtained by regression analysis with peak area as the ordinate and the naringin content in the naringin reference solution as the abscissa:

[0031] Y = kX + b;

[0032] Where: Y is the peak area, X is the content of naringin in the naringin reference solution; k is the regression coefficient; b is the intercept;

[0033] (5) Take the test solution prepared in step (2), determine the peak area according to the chromatographic conditions described in step (3), and then use the standard curve obtained in step (4) to obtain the content of naringin in the herbal composition of the test solution.

[0034] Preferably, in step (1), the ultrasonic time is 30 min, and filtration is performed using a 0.22 μm microporous membrane; in step (2), the chromatographic column is an Agilent 5 TC-C18 column with a specification of 4.5 × 250 mm and a packing particle size of 5 μm.

[0035] Preferably, in step (3), the standard curve is Y = 11960X + 0.2146 when the naringin content is in the range of 0.001074 to 0.006444 mg.

[0036] Compared with the prior art, the beneficial effects of the present invention mainly include the following aspects:

[0037] (1) This invention identifies Drynaria fortunei, Ligustrum lucidum, Spatholobus suberectus, and Achyranthes bidentata by thin-layer chromatography, and determines the presence of Drynaria fortunei in the herbal composition by using naringin content as a quality control indicator. 27 H 32 O 14 The control limit of not less than 1.79 mg per gram makes the quality testing of traditional Chinese medicine compositions more feasible, holistic, characteristic and stable. This allows for the quality control and identification of traditional Chinese medicine compositions, which is more conducive to the monitoring of product quality by manufacturers and supervisory departments, resulting in higher quality medicinal materials and better clinical efficacy.

[0038] (2) The present invention has high detection sensitivity, which can ensure that naringin exhibits a good linear relationship in the range of 0.001074 to 0.006444 mg, thus achieving accurate quantitative detection of naringin. Moreover, it has good repeatability and the test solution has good stability, remaining stable within 24 hours.

[0039] (3) Under the detection system of the present invention, the recovery rate of naringin is between 97.03% and 102.99%, with an average recovery rate of 100.94% and an RSD of 2.24%, indicating good recovery rate. Attached Figure Description

[0040] Figure 1 The image shows a TLC plot of *Drynaria fortunei* at 365 nm. (In the image: 1 is naringin reference standard; 2 is test sample YZ202501; 3 is test sample YZ202502; 4 is test sample YZ202503; 5 is negative control of *Drynaria fortunei*.)

[0041] Figure 2 The image shows a TLC image of Ligustrum lucidum viewed at 254 nm. (In the image: 1 represents Ligustrum lucidum as a medicinal material; 2 represents test sample YZ202501; 3 represents test sample YZ202502; 4 represents test sample YZ202503; 5 represents a negative control without Ligustrum lucidum.)

[0042] Figure 3 The image shows the TLC pattern of *Spatholobus suberectus* under ultraviolet light (254 nm); (In the image: 1 is *Spatholobus suberectus* control material; 2 is test sample YZ202501; 3 is test sample YZ202502; 4 is test sample YZ202503; 5 is negative control without *Spatholobus suberectus*)

[0043] Figure 4The image shows the TLC pattern of Achyranthes bidentata under ultraviolet light (365nm); (In the image: 1 is the reference material of Achyranthes bidentata; 2 is the test sample YZ202501; 3 is the test sample YZ202502; 4 is the test sample YZ202503; 5 is the negative control without Achyranthes bidentata)

[0044] Figure 5 This is a standard curve of naringin reference standard. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] The raw material composition of the traditional Chinese medicine composition of the present invention is as follows: 15g of Drynaria fortunei, 12g of Cibotium barometz, 10g of Ligustrum lucidum, 10g of Spatholobus suberectus, 10g of Pyrola rotundifolia, and 10g of Achyranthes bidentata.

[0047] The traditional Chinese medicine composition is prepared according to the following steps:

[0048] (1) Prepare each ingredient according to the prescription;

[0049] (2) Mix Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Spatholobus suberectus, Pyrola rotundifolia and Achyranthes bidentata, then add water and decoct twice. The first time, add 11 times the amount of water and decoct for 1.5 hours. The second time, add 9 times the amount of water and decoct for 1.5 hours.

[0050] (3) The filtrates obtained from the two decoctions are combined and subjected to two vacuum concentration treatments, including first-effect concentration and second-effect concentration, at a temperature of 60-80℃, to obtain a clear extract and an extract. The clear extract has a relative density of 1.15-1.25 and the extract has a relative density of 1.30-1.35.

[0051] (4) Mix the dried extract with the clear extract to obtain the final product.

[0052] Furthermore, thin-layer chromatography was used to identify Drynaria fortunei, Ligustrum lucidum, Spatholobus suberectus, and Achyranthes bidentata in the traditional Chinese medicine composition. The specific identification steps are as follows:

[0053] (1) Identification of Drynaria fortunei:

[0054] S1: Take 2g of the Chinese herbal medicine composition, grind it into a fine powder, add 30mL of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution.

[0055] S2: Prepare a 1 mg / mL solution of naringin reference standard with methanol and use it as the reference solution;

[0056] S3: Take 5-10 μL of the above test solution and reference solution and spot them separately on the same silica gel G thin-layer plate. Use dichloromethane-methanol-water in a volume ratio of 8:2:0.5 as the developing solvent. After saturation for 15 minutes, develop the plate, remove it, air dry it, spray it with aluminum trichloride solution, heat it at 105℃, and then observe it under a 365nm ultraviolet light. In the chromatogram of the test sample, spots of the same color as those in the chromatogram of the reference medicinal material should be obtained at the corresponding positions.

[0057] (2) Identification of privet fruit:

[0058] S1: Take 1g of the Chinese herbal composition, grind it into a fine powder, add 20mL of chloroform, sonicate for 30min, filter, evaporate the filtrate to dryness, add 1mL of chloroform to the residue to dissolve it, and use it as the test solution.

[0059] S2: Take 1g of Ligustrum lucidum reference material and prepare the Ligustrum lucidum reference material solution using the same procedure as described in step S1.

[0060] S3: Take 5 μL each of the above test solution and reference medicinal material solution and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-methanol-water volume ratio of 100:17:13 as the developing solvent. After saturation for 15 minutes, develop the plate, remove it, air dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ and observe it under a 254nm ultraviolet lamp. In the chromatogram of the test sample, a main spot of the same color as the reference medicinal material chromatogram is obtained at the corresponding position.

[0061] (3) Identification of Chicken Blood Vine:

[0062] S1: Take 2g of the Chinese herbal medicine composition, grind it into a fine powder, add 40mL of ethanol, sonicate for 30min, filter, let the filtrate evaporate naturally, add 10mL of water to the residue to dissolve it, extract with ethyl acetate by shaking, let the ethyl acetate evaporate, add 1mL of methanol to the residue to dissolve it, and use it as the test solution.

[0063] S2: Take 2g of chicken blood vine reference material and prepare chicken blood vine reference material solution using the same test solution preparation operation as described in step S1;

[0064] S3: Take 5-10 μL of the above test solution and reference medicinal material solution and spot them separately on the same silica gel G thin layer plate. Use dichloromethane-acetone-methanol volume ratio of 8:1.2:0.3 as the developing solvent, develop, remove, air dry, and observe under 254 nm ultraviolet light. In the chromatogram of the test solution, a main spot of the same color as the reference medicinal material chromatogram is obtained at the corresponding position.

[0065] (4) Identification of Achyranthes bidentata:

[0066] S1: Take 3g of the Chinese herbal medicine composition, add 30mL of methanol, sonicate for 20min, filter, evaporate the filtrate to dryness, add 10mL of water to dissolve the residue, extract twice with ethyl acetate, 10mL each time, combine the ethyl acetate solutions, recover the solvent to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution.

[0067] S2: Take 1g of Achyranthes bidentata reference material, add 10mL of methanol, and prepare the Achyranthes bidentata reference material solution according to the test solution preparation method in step S1;

[0068] S3: Take 5 μL each of the above test solution and reference herb solution and spot them on the same silica gel G thin-layer plate. Use hexane-ethyl acetate-acetone volume ratio of 8:5:1 as the developing solvent, develop to 8 cm, remove, air dry, and observe under a 365 nm ultraviolet lamp. In the chromatogram of the test solution, a main spot of the same color as the reference herb solution is obtained at the corresponding position.

[0069] Furthermore, the quality control of the traditional Chinese medicine composition of the present invention is based on the determination of the content of naringin, a representative component of Drynaria fortunei, to determine the quality control indicators of the traditional Chinese medicine composition. The specific detection steps are as follows:

[0070] (1) Take the stock solution of naringin reference standard, dissolve and dilute it with methanol to prepare a naringin reference standard solution with a concentration of 0.02144 mg / mL;

[0071] (2) Take the Chinese herbal medicine composition, dissolve it in methanol, sonicate it, filter it, and obtain the test solution;

[0072] (3) Take different volumes of naringin reference solution from step (1) and inject them into the liquid chromatograph. Determine the peak area of ​​naringin reference solution under the following chromatographic conditions: the chromatographic column is an Agilent 5 TC-C18 column; the mobile phase is 0.1% phosphoric acid solution-acetonitrile with a volume ratio of 75:25; the detection wavelength is 280 nm; the flow rate is 1.0 mL / min; the theoretical plate number is not less than 3000 for naringin.

[0073] (4) A standard curve was obtained by regression analysis with peak area as the ordinate and the naringin content in the naringin reference solution as the abscissa:

[0074] Y = kX + b;

[0075] Where: Y is the peak area, X is the content of naringin in the naringin reference solution; k is the regression coefficient; b is the intercept;

[0076] (5) Take the test solution from step (2) and determine the peak area under the chromatographic conditions of step (3). Use the standard curve obtained in step (4) to obtain the content of naringin in the herbal composition of the test solution.

[0077] This invention, through quantitative detection of naringin as a representative compound, determines that the traditional Chinese medicine composition contains naringin (C) from Drynaria fortunei. 27 H 32 O 14 The content is calculated to be greater than or equal to 1.79 mg per gram (considering industrial production reasons, the content per gram is calculated as 80% of the measured amount).

[0078] Furthermore, the quality control indicators for the traditional Chinese medicine composition of the present invention were determined according to the above method, with the aim of constructing a quality control standard for the traditional Chinese medicine composition. The specific research is as follows:

[0079] I. Identification of Drynaria fortunei, Ligustrum lucidum, Spatholobus suberectus, and Achyranthes bidentata by thin-layer chromatography

[0080] Test drugs: provided by Xi'an Afanggong Pharmaceutical Co., Ltd. The amount of Chinese medicinal materials required to prepare 1000g of granules was determined based on the original daily dosage of Chinese medicinal materials. The test drugs were divided into three batches of samples, which were designated as YZ202501, YZ202502 and YZ202503 respectively.

[0081] Negative test sample: The formula containing no tested medicinal material in the traditional Chinese medicine composition is used as the negative control sample.

[0082] (1) Thin-layer chromatography identification of Drynaria fortunei

[0083] Take 2g of the test drug, grind it into a fine powder, add 30mL of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol, and use it as the test solution.

[0084] Prepare a reference solution by taking naringin and following the same procedure as above for preparing the test solution;

[0085] According to the thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition), 5 μL each of the above test solution and naringin reference solution were spotted onto the same silica gel G thin-layer plate. The developing solvent was dichloromethane-methanol-water 8:2:0.5. After saturation for 15 min, the plate was developed, removed, dried, sprayed with aluminum trichloride solution, heated at 105℃, and observed under 365nm ultraviolet light.

[0086] The three batches of samples were tested separately according to the above procedure. Simultaneously, a negative control preparation without *Drynaria fortunei* was prepared using the same method as the test sample. The results are as follows: Figure 1 As shown.

[0087] The results showed that after spraying with aluminum trichloride solution, heating at 105℃ and observing under 365nm ultraviolet light, the test sample chromatogram showed yellow-green fluorescent spots of the same color at the corresponding positions as the reference medicinal material chromatogram, while the accompanying negative control did not show fluorescent spots.

[0088] (2) Thin-layer chromatography identification of Ligustrum lucidum

[0089] Take 1g of the test reagent, grind it into a fine powder, add 20mL of chloroform, sonicate for 30min, filter, evaporate the filtrate to dryness, add 1mL of chloroform to dissolve the residue, and use it as the test solution.

[0090] Take 1g of Ligustrum lucidum reference material and prepare the Ligustrum lucidum reference material solution using the same procedure as the preparation of the test solution described above. The Ligustrum lucidum reference material was provided by the China National Institutes for Food and Drug Control, batch number: 121041-202106.

[0091] According to the thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition), 5 μL each of the above-mentioned test solution and the Ligustrum lucidum reference material solution were spotted separately onto the same silica gel G thin-layer plate. Ethyl acetate-methanol-water (100:17:13) was used as the developing solvent. After saturation for 15 min, the plate was developed, removed, and dried. It was then sprayed with 10% sulfuric acid ethanol solution, heated at 105℃, and observed under a 254 nm ultraviolet lamp.

[0092] The above procedures were followed to test three batches of samples. Simultaneously, a negative control formulation without privet fruit was prepared, and a negative control was prepared using the same method. The results are as follows: Figure 2 As shown.

[0093] The experimental results showed that after heating at 105℃ and observing under a 254nm ultraviolet lamp, the chromatogram of the test sample showed spots of the same color at the corresponding positions as the chromatogram of the reference medicinal material, while the accompanying negative control did not show spots at the corresponding positions.

[0094] (3) Identification of Chicken Blood Vine by Thin Layer Chromatography

[0095] Take 2.0g of the test drug, grind it into a fine powder, add 40mL of ethanol, sonicate for 30min, filter, let the filtrate evaporate naturally, dissolve the residue in 10mL of water, extract with 10mL of ethyl acetate by shaking, let the ethyl acetate evaporate, dissolve the residue in 1mL of methanol to obtain the test solution.

[0096] Take 2.0g of *Spatholobus suberectus* reference material and prepare a *Spatholobus suberectus* reference material solution using the same method as the test sample solution preparation described above. The *Spatholobus suberectus* reference material was provided by the China National Institutes for Food and Drug Control, batch number: 121065-202408.

[0097] According to the thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition), 5 μL each of the above-mentioned test solution and the chicken blood vine reference medicinal material solution were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using dichloromethane-acetone-methanol (8:1.2:0.3) as the developing solvent. The plate was then removed, dried, and examined under ultraviolet light (254 nm).

[0098] The above procedures were followed to test three batches of samples separately. Simultaneously, a negative control formulation without *Spatholobus suberectus* was prepared, and a negative control was prepared using the same method. The results are as follows: Figure 3 As shown.

[0099] Figure 3 Experimental results show that, when examined under ultraviolet light (254 nm), the test sample chromatogram shows fluorescent spots of the same color at the corresponding positions as the reference medicinal material chromatogram.

[0100] (4) Thin-layer chromatography identification of Achyranthes bidentata

[0101] Take about 3g of the test drug, add 30mL of methanol, sonicate for 20min, filter, evaporate the filtrate to dryness, add 10mL of water to dissolve the residue, extract twice with ethyl acetate, 10mL each time, combine the ethyl acetate solutions, recover the solvent to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution.

[0102] Take 1.0 g of Achyranthes bidentata reference material, add 10 mL of methanol, and prepare the reference material solution using the same method as the test solution preparation method described above. The Achyranthes bidentata reference material was provided by the China National Institutes for Food and Drug Control, batch number: 121065-22408.

[0103] According to the thin-layer chromatography method (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition), 5 μL each of the test solution and the reference herb solution of Achyranthes bidentata were spotted on the same silica gel G thin-layer plate. Hexane-ethyl acetate-acetone (8:5:1) was used as the developing solvent. The plate was developed to 8 cm, removed, air-dried, and examined under ultraviolet light (365 nm).

[0104] The three batches of samples were tested separately according to the above procedure. A negative control preparation without Achyranthes bidentata was also prepared using the same method. The results are as follows: Figure 4 As shown.

[0105] Figure 4 The experimental results showed that when examined under ultraviolet light (365nm), the test sample chromatogram showed spots of the same color at the corresponding positions as the reference medicinal material chromatogram, while the accompanying negative control did not show spots of the same color at the corresponding positions.

[0106] II. Analysis of naringin (C10) in a traditional Chinese medicine composition using high performance liquid chromatography. 27 H 32O 14 Content determination

[0107] Based on the determination of naringin, a representative component of Drynaria fortunei, this invention investigates the quality detection and identification of traditional Chinese medicine compositions, resulting in a method for detecting naringin content in the traditional Chinese medicine compositions. The precision, repeatability, and stability of the detection method are then examined. The specific verification process is as follows:

[0108] (1) Instruments and reagents

[0109] Instruments: UltiMate 3000 high performance liquid chromatography system; UltiMate 3000 Pump; UltiMate 3000 PDA Detector UV detector; UltiMate 3000 Controller system chromatography workstation.

[0110] Reference standard: Naringin reference standard was dissolved in methanol to prepare a naringin reference standard solution with a concentration of 0.02144 mg / mL for later use; the naringin reference standard was provided by the China National Institutes for Food and Drug Control, with a purity of 99.7%;

[0111] Reagents: Methanol, acetonitrile, and phosphoric acid were of chromatographic grade; water was redistilled water.

[0112] Test solution: Take 2g of the traditional Chinese medicine composition, grind it into a fine powder, add 30mL of methanol, sonicate for 30min, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution.

[0113] (2) Chromatographic conditions

[0114] The chromatographic column was an Agilent 5 TC-C18 column (4.5 × 250 mm, 5 μm); the mobile phase was 0.1% phosphoric acid solution-acetonitrile (75:25); the detection wavelength was 280 nm; and the flow rate was 1.0 mL / min. Under these conditions, the theoretical plate number for naringin was not less than 3000, and naringin in the sample was well separated. The blank sample showed no absorption at this wavelength and did not affect the content determination of this invention.

[0115] (3) Examination of linear range

[0116] Accurately pipette 2, 4, 6, 8, 10, and 12 μL of the prepared naringin reference solution were injected into the liquid chromatograph, and the peak area was measured. The results are shown in Table 1 below, with peak area (Y) as the ordinate and concentration (X) as the abscissa. Figure 5 :

[0117] Table 1 shows the results of the linearity study of the traditional Chinese medicine composition particles.

[0118]

[0119] The regression equation for obtaining the standard curve is:

[0120] Y = 11960X + 0.2146, where R 2 =0.9992, n=6

[0121] The above results indicate that naringin shows a good linear relationship between peak area and injection volume in the range of 0.001074-0.006444 mg.

[0122] (4) Precision test

[0123] Accurately pipette 10 μL of the reference solution and inject it 6 times under the above chromatographic conditions. Record the peak area. The results are shown in Table 2 below:

[0124] Table 2 shows the results of the precision test.

[0125]

[0126] By observing the peak area changes through the six repeated injections described in Table 2, the results show that the relative standard deviation (RSD) of the peak area values ​​for the six injections is 1.43%, indicating that the detection method of the present invention has good precision.

[0127] (5) Repeatability test

[0128] Six samples from the same batch (batch number: YZ202501) were prepared according to the above-described method for preparing the test solution, and the content was determined according to the above-described detection method. The results are shown in Table 3 below:

[0129] Table 3 shows the results of the repeatability test.

[0130]

[0131]

[0132] Table 3 shows that the same sample was tested six times. The results showed that the average content of naringin in the six tests was 10.79 mg, and the standard deviation RSD was 1.77%. This indicates that the detection method of the present invention has good repeatability.

[0133] (6) Stability test

[0134] Take a sample of this product (batch number: YZ202501), prepare a test solution according to the above-described test solution preparation method, and determine the peak area at 0, 2, 4, 6, 8, 10, 12, and 24 h under the same chromatographic conditions and methods described above. The results are shown in Table 4 below:

[0135] Table 4 shows the results of the stability test.

[0136]

[0137] As shown in Table 4, when the sample was injected and measured according to the chromatographic conditions of the embodiment within 0-24h, the RSD of the peak area of ​​naringin in the test solution was 2.45%, indicating that the test solution of the invention has good stability within 24h at room temperature.

[0138] (7) Recovery test

[0139] Six samples (batch number: YZ202501) with known content were taken, each approximately 2.5 g, accurately weighed and placed in stoppered conical flasks. 2.50 mL of naringin reference standard stock solution (concentration 2.16 mg / mL) and 27.50 mL of methanol were accurately added to each sample. The determination was performed according to the above detection method. The results are shown in Table 5. The recovery rate was calculated using the following formula:

[0140]

[0141] Table 5 shows the results of the recovery rate test of the traditional Chinese medicine composition (n=6).

[0142]

[0143] Table 5 shows that the recovery rate of naringin ranged from 97.03% to 102.99%, with an average recovery rate of 100.94% and an RSD of 2.24%, indicating good recovery.

[0144] (8) Sample determination

[0145] Three batches of pilot-scale products of the traditional Chinese medicine composition of the present invention were taken, and the content was determined according to the content determination method determined above. The results are shown in Table 6 below:

[0146] Table 6 shows the results of sample content determination.

[0147]

[0148] After testing three batches of samples, and considering the influence of the source, origin, and other factors of the medicinal materials, it was determined that the traditional Chinese medicine composition of this invention contains *Drynaria fortunei* with naringin (C...). 27 H 32 O 14 The concentration is greater than or equal to 1.79 mg per gram.

[0149] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features, all of which should be included within the protection scope of the present invention.

Claims

1. A method for quality testing of a traditional Chinese medicine composition, characterized in that, The traditional Chinese medicine composition comprises Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Spatholobus suberectus, Pyrola rotundifolia, and Achyranthes bidentata. The composition is identified by thin-layer chromatography of Drynaria fortunei, Ligustrum lucidum, Spatholobus suberectus, and Achyranthes bidentata, and the quality control indicators are determined by naringin content assay. Drynaria fortunei contains naringin (C... 27 H 32 O 14 (Calculated, each gram is greater than or equal to 1.79 mg;) The method for determining the naringin content is implemented by the following steps: (1) Take naringin reference standard and dissolve it in methanol to prepare a naringin reference standard solution with a concentration of 0.02144 mg / mL; (2) Take the Chinese herbal medicine composition, dissolve it in methanol, sonicate it, filter it, and obtain the test solution; (3) Take different volumes of the naringin reference solution described in step (1) and inject them into the liquid chromatograph. Determine the peak area of ​​the naringin reference solution under the following chromatographic conditions: the chromatographic column is an Agilent 5 TC-C18 column, the mobile phase is 0.1% phosphoric acid solution-acetonitrile with a volume ratio of 75:25, the detection wavelength is 280 nm, and the flow rate is 1.0 mL / min; (4) A standard curve was obtained by regression analysis with peak area as the ordinate and the naringin content in the naringin reference solution as the abscissa: Y = kX + b; Where: Y is the peak area, X is the content of naringin in the naringin reference solution; k is the regression coefficient; b is the intercept; (5) Take the test solution prepared in step (2), determine the peak area according to the chromatographic conditions described in step (3), and then use the standard curve obtained in step (4) to obtain the content of naringin in the herbal composition of the test solution.

2. The method for quality testing of the traditional Chinese medicine composition according to claim 1, characterized in that, The raw materials of the traditional Chinese medicine composition are 15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Spatholobus suberectus, 10 parts of Pyrola rotundifolia, and 10 parts of Achyranthes bidentata.

3. The method for quality testing of the traditional Chinese medicine composition according to claim 2, characterized in that, The preparation method of the traditional Chinese medicine composition is as follows: take the medicinal materials according to the compatibility dosage, add water and decoct twice. The first time, add 11 times the amount of water and decoct for 1.5 hours. The second time, add 9 times the amount of water and decoct for 1.5 hours. Combine the filtrates obtained from the two decoctions and perform two vacuum concentration treatments, including first-effect concentration and second-effect concentration, at a temperature of 60-80 ℃, to obtain a clear extract and an extract. The relative density of the clear extract is 1.15-1.25, and the relative density of the extract is 1.30-1.

35. Mix the dried extract and the clear extract to obtain the final product.

4. The method for quality testing of the traditional Chinese medicine composition according to claim 3, characterized in that, The thin-layer chromatography method is used to identify Drynaria fortunei, and the specific identification steps are as follows: (1) Take the Chinese herbal medicine composition, grind it into a fine powder, add methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, and prepare the test solution. (2) Prepare a 1 mg / mL solution of naringin reference standard with methanol and use it as the reference solution; (3) Take 5-10 μL of the above test solution and reference solution and spot them on the same silica gel G thin layer plate. Use dichloromethane-methanol-water volume ratio of 8:2:0.5 as the developing solvent. After saturation for 15 minutes, develop the plate, remove it, air dry it, spray it with aluminum trichloride solution, heat it at 105 °C and observe it under 365 nm ultraviolet light. In the chromatogram of the test sample, the main spot of the same color appears at the corresponding position as in the chromatogram of the reference sample.

5. The method for quality testing of the traditional Chinese medicine composition according to claim 3, characterized in that, The thin-layer chromatography method is used to identify privet fruit. The specific identification steps are as follows: (1) Take the Chinese herbal medicine composition, grind it into a fine powder, add chloroform, sonicate for 30 min, filter, evaporate the filtrate to dryness, add chloroform to the residue to dissolve it, and use it as the test solution. (2) Take the privet fruit reference material and prepare the privet fruit reference material solution in the same way as the test solution preparation operation described in step (1); (3) Take 5-10 μL of the above test solution and reference medicinal material solution and spot them on the same silica gel G thin layer plate. Use ethyl acetate-methanol-water volume ratio of 100:17:13 as the developing solvent. After saturation for 15 minutes, develop the plate, remove it, air dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105 ℃ and observe it under a 254 nm ultraviolet lamp. In the chromatogram of the test sample, the main spot of the same color as the reference medicinal material is obtained at the corresponding position.

6. The method for quality testing of the traditional Chinese medicine composition according to claim 3, characterized in that, The thin-layer chromatography method is used to identify *Spatholobus suberectus*, and the specific identification steps are as follows: (1) Take the Chinese herbal medicine composition, grind it into a fine powder, add ethanol, sonicate for 30 min, filter it, let the filtrate evaporate naturally, dissolve the residue in water, extract it with ethyl acetate by shaking, let the ethyl acetate evaporate, dissolve the residue in methanol, and use it as the test solution. (2) Take the chicken blood vine reference material and prepare the chicken blood vine reference material solution in the same way as the test solution preparation operation described in step (1); (3) Take 5-10 μL of the above test solution and reference medicinal material solution and spot them on the same silica gel G thin layer plate. Use dichloromethane-acetone-methanol volume ratio of 8:1.2:0.3 as the developing solvent, develop, remove, air dry, and observe under 254 nm ultraviolet light. The test solution chromatogram shows the same main spot of the same color at the corresponding position as the reference medicinal material chromatogram.

7. The method for quality testing of the traditional Chinese medicine composition according to claim 3, characterized in that, The thin-layer chromatography method is used to identify Achyranthes bidentata, and the specific identification steps are as follows: (1) Take the Chinese herbal medicine composition, add methanol, sonicate for 20 min, filter, evaporate the filtrate to dryness, add water to dissolve the residue, extract twice with ethyl acetate, 10 mL each time, combine the ethyl acetate solutions, recover the solvent to dryness, add methanol to dissolve the residue, and use it as the test solution. (2) Take the reference herb Achyranthes bidentata and prepare the reference herb solution Achyranthes bidentata according to the test solution preparation operation described in step (1); (3) Take 5-10 μL of the above test solution and reference medicinal material solution and spot them on the same silica gel G thin layer plate. Use hexane-ethyl acetate-acetone volume ratio of 8:5:1 as the developing solvent, develop to 8 cm, take it out, air dry, and observe under 365 nm ultraviolet light. In the chromatogram of the test solution, the main spot of the same color as the reference medicinal material solution is obtained at the corresponding position.

8. The method for quality testing of the traditional Chinese medicine composition according to claim 1, characterized in that, In step (2), the ultrasonic time is 30 min, and filtration is performed using a 0.22 μm microporous membrane; in step (3), the chromatographic column is an Agilent 5 TC-C18 column with a specification of 4.5 × 250 mm and a packing particle size of 5 µm.

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