Quality detection method of traditional Chinese medicine composition

Abstract

According to the quality detection method of the traditional Chinese medicine composition, rhizoma drynariae, glossy privet fruit, caulis spatholobi and radix cyathulae are identified through a thin-layer chromatography method, and it is determined that the content of naringin (C27H32O14) in the traditional Chinese medicine composition is larger than or equal to 1.79 mg per gram through naringin quantitative determination; according to the method disclosed by the invention, the quality detection of the traditional Chinese medicine composition has integrity, characteristics and stability, the detection sensitivity is high, and the quality control of the traditional Chinese medicine composition can be realized.

Description

Technical Field

[0001] The invention belongs to a technology for detecting the content of active ingredients in traditional Chinese medicines, and particularly relates to a method for detecting the quality of a traditional Chinese medicine composition. Background Art

[0002] Traditional Chinese medicine quality testing relies on sensory observation of the medicinal material's appearance and characteristics, microscopic observation of tissue structure and cell morphology, or detection of specific components through color development and precipitation reactions. These traditional testing techniques are only capable of qualitative testing and cannot achieve quantitative detection. With technological advancements, the quality testing of Chinese medicine has gradually evolved from single-ingredient control to overall quality assessment. With the development of modern instrumental analysis techniques, spectroscopy and chromatography are widely used. However, the problem of low-concentration detection is still limited.

[0003] For example, CN117054588A discloses a tortoise antler and bone strengthening tablet and its identification method, which discloses a detection method for naringin: according to the Chinese Pharmacopoeia General Chapter 0512 High Performance Liquid Chromatography, the chromatographic conditions and system suitability test are tested, octadecylsilane bonded silica gel is used as the filler, and 0.05% acetic acid solution-methanol 65:35 is used as the mobile phase; the detection wavelength for psoralen and isopsoralen is 246 nm, and the detection wavelength for naringin is 284 nm; the column temperature is 25°C, and the number of theoretical plates 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 should be higher than 0.30 mg / g, and isopsoralen should be higher than 0.28 mg / g. On this basis, the total content is reduced by 20% to formulate the content limit of this product, that is, the content of naringin in this product should be higher than 0.72 mg / g, psoralen should be higher than 0.24 mg / g, and isopsoralen should be higher than 0.22 mg / g.

[0004] Thin-layer chromatography and high-performance liquid chromatography (HPLC) have enabled the identification of traditional Chinese medicines to move from empirical judgment to scientific quantification, facilitating precise quality control of traditional Chinese medicines. This invention provides qualitative and quantitative detection methods for a traditional Chinese medicine composition consisting of Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Millettia reticulata, Herba schizonepetae, and Cyathulae chinensis, providing a theoretical basis for research into the quality standards and material basis of the composition. Summary of the Invention

[0005] The present invention aims to provide an effective method for detecting the various components in a traditional Chinese medicine composition, especially the volatile oil contained in naringin in Rhizoma Drynariae, so as to stabilize the therapeutic effect and ensure the quality of the medicine.

[0006] The quality detection method of the traditional Chinese medicine composition 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 limit, and provide a control method for the industrialization of the medicine.

[0007] The technical means adopted in the present invention are:

[0008] A method for detecting the quality of a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition is composed of rhizoma drynariae, cibotrys, glossy privet fruit, millettia spatholobi, herba schizonepetae and cyathulae; the rhizoma drynariae, glossy privet fruit, millettia spatholobi and cyathulae are identified by thin layer chromatography, and the quality control index of the traditional Chinese medicine composition is determined by measuring the content of naringin, wherein the rhizoma drynariae is naringin (C 27 H 32 O 14 ) is greater than or equal to 1.79 mg per gram.

[0009] Furthermore, the raw material compatibility of the traditional Chinese medicine composition is 15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Millettia reticulata, 10 parts of Herba Lycopodii, and 10 parts of Cyathulae chinensis. Furthermore, the preparation method of the traditional Chinese medicine composition is as follows: taking the medicinal pieces according to the compatible dosage, adding water and decocting twice, the first time adding 11 times the amount of water and decocting for 1.5 hours, the second time adding 9 times the amount of water and decocting for 1.5 hours, combining the filtrates obtained from the two decoctions and performing two vacuum concentration treatments, the concentration including one-effect concentration and two-effect concentration, the temperature being 60-80°C, to obtain a clear paste and an extract, the relative density of the clear paste being 1.15-1.25, and the relative density of the extract being 1.30-1.35, mixing the dried extract with the clear paste to obtain.

[0010] Furthermore, the TLC method of the Chinese medicine composition is used to identify Drynaria rhizome, and the specific identification steps are as follows:

[0011] (1) Grind the Chinese herbal medicine composition, add methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, and dissolve the residue in methanol to prepare a test solution;

[0012] (2) Take the naringin reference substance and add methanol to prepare a 1 mg / mL solution as the reference solution;

[0013] (3) Take 5-10 μL of the test solution and reference solution, respectively, 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, take out, dry, spray with aluminum chloride solution, heat at 105 ° C, and observe under 365 nm ultraviolet light. In the chromatogram of the test sample, a main spot of the same color appears at the corresponding position in the chromatogram of the reference sample.

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

[0015] (1) Grind the Chinese herbal medicine composition, add chloroform, and ultrasonically treat for 30 min. Filter, evaporate the filtrate to dryness, and dissolve the residue in chloroform to prepare the test solution.

[0016] (2) Take the Ligustrum lucidum control medicinal material and prepare the Ligustrum lucidum control medicinal material solution by the same method as the test solution preparation method in step (1);

[0017] (3) 5-10 μL of the test solution and control medicinal material solution were taken and spotted on the same silica gel G thin layer plate respectively. Ethyl acetate-methanol-water volume ratio of 100:17:13 was used as the developing solvent. After saturation for 15 minutes, the plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105 °C, and observed under a 254 nm ultraviolet lamp. In the chromatogram of the test sample, a main spot of the same color was obtained at the corresponding position in the chromatogram of the control medicinal material.

[0018] Furthermore, the TLC method of the Chinese medicine composition is used to identify Millettia repens, and the specific identification steps are as follows:

[0019] (1) Take the Chinese medicine composition, grind it into powder, add ethanol, ultrasonically treat it for 30 minutes, filter it, evaporate the filtrate naturally, dissolve the residue in water, shake it with ethyl acetate, evaporate the ethyl acetate, and dissolve the residue in methanol to prepare the test solution;

[0020] (2) Take a control medicinal material of Millettia spatholobi and prepare a control medicinal material solution of Millettia spatholobi by the same operation as the test solution preparation described in step (1);

[0021] (3) 5-10 μL of the test solution and control medicinal material solution were taken and spotted on the same silica gel G thin layer plate respectively. The plate was developed with dichloromethane-acetone-methanol in a volume ratio of 8:1.2:0.3. The plate was taken out and dried. The plate was observed under 254 nm ultraviolet light. In the chromatogram of the test solution, a main spot of the same color was obtained at the corresponding position in the chromatogram of the control medicinal material.

[0022] Furthermore, the Chinese medicine composition is used to identify Cyathula officinalis by thin layer chromatography, and the specific identification steps are as follows:

[0023] (1) Take the Chinese medicine composition, add methanol, ultrasonically treat for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in water, and extract with ethyl acetate twice with 10 mL each time. Combine the ethyl acetate solutions, recover the solvent to dryness, and dissolve the residue in methanol to prepare the test solution.

[0024] (2) Take a Cyathula control medicinal material and prepare a Cyathula control medicinal material solution according to the test solution preparation operation described in step (1);

[0025] (3) 5-10 μL of the test solution and control medicinal material solution were taken and spotted on the same silica gel G thin layer plate. The plate was developed with n-hexane-ethyl acetate-acetone in a volume ratio of 8:5:1. The plate was developed over an area of ​​8 cm. The plate was taken out, dried, and observed under a 365 nm ultraviolet lamp. In the chromatogram of the test solution, a main spot of the same color was obtained at the corresponding position in the chromatogram of the control medicinal material solution.

[0026] Another object of the present invention is to provide a method for detecting the quality of a traditional Chinese medicine composition, wherein the method for determining the content of naringin is achieved by the following steps:

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

[0028] (2) taking the Chinese medicine composition, adding methanol to dissolve it, ultrasonically treating it, and filtering it to obtain a test solution;

[0029] (3) Taking different volumes of the naringin reference solution described in step (1), respectively injecting them into a liquid chromatograph, and determining 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 a 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;

[0030] (4) With the peak area as the ordinate and the naringin content in the naringin reference solution as the abscissa, a standard curve was obtained through regression analysis:

[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) Taking the test solution prepared in step (2), measuring the peak area according to the chromatographic conditions described in step (3), and then using the standard curve obtained in step (4) to obtain the content of naringin in the Chinese medicine 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 Agilent5 TC-C18, with a specification of 4.5×250 mm and a filler particle size of 5 μm.

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

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

[0037] (1) The present invention uses thin layer chromatography to identify Rhizoma Drynariae, Fructus Ligustri Lucidi, Caulis Spatholobi and Cyathulae Radix, and uses the naringin content as a quality control index to determine the content of Rhizoma Drynariae and naringin (C 27 H 32 O 14 ) is calculated with a control limit of not less than 1.79 mg per gram, making the quality inspection of Chinese medicine compositions more feasible, holistic, characteristic and stable, thereby realizing the quality control and identification of Chinese medicine compositions, being more conducive to the monitoring of product quality by manufacturers and supervisory and management departments, and ensuring higher quality of medicinal materials and better clinical efficacy.

[0038] (2) The detection sensitivity of the present invention is high, and it can ensure that naringin shows a good linear relationship in the range of 0.001074 to 0.006444 mg, thereby achieving quantitative and accurate detection of naringin, and has good repeatability. In addition, the test solution is stable and can remain stable within 24 hours.

[0039] (3) Under the detection system of the present invention, the recovery rate of naringin was between 97.03% and 102.99%, with an average recovery rate of 100.94% and an RSD of 2.24%, indicating a good recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a TLC chart of Drynaria Rhizoma examined at 365 nm; (In the figure: 1 is the naringin reference substance; 2 is the test substance YZ202501; 3 is the test substance YZ202502; 4 is the test substance YZ202503; 5 is the Drynaria Rhizoma negative control)

[0041] Figure 2 This is a TLC chart of Ligustrum lucidum (Glycerides ligustrum) examined at 254 nm. (In the figure: 1 is the Ligustrum lucidum (Glycerides ligustrum) counter-medicinal material; 2 is the test sample YZ202501; 3 is the test sample YZ202502; 4 is the test sample YZ202503; 5 is the negative control lacking Ligustrum lucidum)

[0042] Figure 3 This is the TLC chart of Millettia spatholobi under ultraviolet light (254nm); (In the figure: 1 is the control medicinal material of Millettia spatholobi; 2 is the test sample YZ202501; 3 is the test sample YZ202502; 4 is the test sample YZ202503; 5 is the negative control without Millettia spatholobi)

[0043] Figure 4This is a TLC chart of Cyathulae under ultraviolet light (365nm); (In the figure: 1 is the control herb of Cyathulae; 2 is the test sample YZ202501; 3 is the test sample YZ202502; 4 is the test sample YZ202503; 5 is the negative control without Cyathulae)

[0044] Figure 5 This is the standard curve of naringin reference substance. DETAILED DESCRIPTION

[0045] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0046] The Chinese medicine composition of the present invention comprises the following raw materials: 15g of Drynaria fortunei, 12g of Cibotium barometz, 10g of Ligustrum lucidum, 10g of Millettia reticulata, 10g of Herba Lycopodii and 10g of Cyathulae chinensis.

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

[0048] (1) Prepare the raw materials according to the prescription;

[0049] (2) Mix Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Millettia repens, Herba Lycopodii, and Cyathulae chinensis, then add water and boil twice: add 11 times the amount of water for the first time and boil for 1.5 h; add 9 times the amount of water for the second time and boil for 1.5 h;

[0050] (3) combining the filtrates obtained from the two decoctions and performing two vacuum concentration processes, including one-effect concentration and two-effect concentration at a temperature of 60-80° C., to obtain a clear paste and an extract, wherein the relative density of the clear paste is 1.15-1.25, and the relative density of the extract is 1.30-1.35;

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

[0052] Furthermore, the drynaria rhizome, glossy privet fruit, millettia reticulata and cyperus rotundus in the traditional Chinese medicine composition were identified by thin layer chromatography, and the specific identification steps were as follows:

[0053] (1) Identification of Drynaria fortunei:

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

[0055] S2: Take naringin reference substance and add methanol to make a 1 mg / mL solution as reference solution;

[0056] S3: 5-10 μL of the test solution and reference solution were taken and spotted on the same silica gel G thin layer plate respectively. Dichloromethane-methanol-water volume ratio of 8:2:0.5 was used as the developing agent. After saturation for 15 minutes, the plate was developed, taken out, dried, sprayed with aluminum chloride solution, heated at 105°C, and observed under 365nm ultraviolet light. In the chromatogram of the test sample, spots of the same color were obtained at the corresponding positions in the chromatogram of the reference medicinal material.

[0057] (2) Identification of Ligustrum lucidum:

[0058] S1: Take 1g of the Chinese medicine composition, grind it into powder, add 20mL of chloroform, ultrasonicate it for 30min, filter it, evaporate the filtrate to dryness, and dissolve the residue in 1mL of chloroform to prepare the test solution;

[0059] S2: Take 1 g of Ligustrum lucidum control medicinal material and prepare Ligustrum lucidum control medicinal material solution by the same method as the test solution preparation in step S1;

[0060] S3: 5 μL of the test solution and control medicinal material solution were taken and spotted on the same silica gel G thin layer plate respectively. Ethyl acetate-methanol-water volume ratio of 100:17:13 was used as the developing solvent. After saturation for 15 minutes, the plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C, and observed under a 254nm violet lamp. In the chromatogram of the test sample, a main spot of the same color was obtained at the corresponding position in the chromatogram of the control medicinal material.

[0061] (3) Identification of Millettia repens:

[0062] S1: Take 2g of the Chinese medicine composition, grind it into powder, add 40mL of ethanol, ultrasonicate it for 30min, filter it, evaporate the filtrate naturally, dissolve the residue in 10mL of water, shake it with ethyl acetate, evaporate the ethyl acetate, and dissolve the residue in 1mL of methanol to prepare the test solution;

[0063] S2: Take 2 g of Millettia spatholobi control medicinal material and prepare Millettia spatholobi control medicinal material solution by the same procedure as the test solution preparation in step S1;

[0064] S3: Pipette 5-10 μL of the above-mentioned test solution and control medicinal material solution respectively, 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 agent. Develop, take out, dry, and observe under 254 nm ultraviolet light. In the chromatogram of the test solution, a main spot of the same color is obtained at the corresponding position in the chromatogram of the control medicinal material.

[0065] (4) Identification of Cyathula

[0066] S1: Take 3 g of the traditional Chinese medicine composition, add 30 mL of methanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in 10 mL of water, and extract with ethyl acetate twice with 10 mL each time. Combine the ethyl acetate solutions, recover the solvent to dryness, and dissolve the residue in 1 mL of methanol to prepare the test solution;

[0067] S2: Take 1 g of Cyathulae Radix control medicinal material, add 10 mL of methanol, and prepare a Cyathulae Radix control medicinal material solution according to the preparation method of the test solution in step S1;

[0068] S3: 5 μL of the test solution and the control medicinal material solution were taken and spotted on the same silica gel G thin layer plate. The plate was developed with n-hexane-ethyl acetate-acetone in a volume ratio of 8:5:1. The plate was developed over an area of ​​8 cm. The plate was taken out, dried, and observed under a 365 nm ultraviolet lamp. In the chromatogram of the test solution, a main spot of the same color was obtained at the corresponding position in the chromatogram of the control medicinal material solution.

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

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

[0071] (2) taking the Chinese medicine composition, adding methanol to dissolve it, ultrasonically treating it, and filtering it to obtain a test solution;

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

[0073] (4) With the peak area as the ordinate and the naringin content in the naringin reference solution as the abscissa, a standard curve was obtained through regression analysis:

[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) Taking the test solution of step (2) and measuring the peak area under the chromatographic conditions of step (3), and using the standard curve obtained in step (4) to obtain the content of naringin in the Chinese medicine composition of the test solution.

[0077] The present invention determines that the Chinese medicine composition contains naringin (C 27 H 32 O 14 ) per gram, greater than or equal to 1.79 mg (taking into account the reasons of industrial production, the content per gram is calculated as 80% of the measured amount).

[0078] Furthermore, the quality control indexes of the Chinese medicine composition of the present invention are determined according to the above method, aiming to establish the quality control standards of the Chinese medicine composition, and the specific research is as follows:

[0079] 1. Identification of Drynaria Rhizoma, Ligustrum lucidum, Millettia reticulata and Cyathula obtusifolia by thin layer chromatography

[0080] Test drugs: provided by Xi'an Afang Palace Pharmaceutical Co., Ltd., based on the original daily dosage of Chinese medicinal materials, and prepared according to the above preparation method to prepare 1000g of granules, divided into three batches of samples, respectively recorded as YZ202501, YZ202502, and YZ202503.

[0081] Negative test sample: A negative control sample is a Chinese medicine composition that does not contain the medicinal materials to be tested.

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

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

[0084] Take naringin and prepare the reference solution by following the same procedure as above for preparing the test solution;

[0085] According to the thin layer chromatography method (General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 5 μL of the test solution and naringin reference solution were respectively spotted on the same silica gel G thin layer plate, and dichloromethane-methanol-water 8:2:0.5 was used as the developing solvent. After saturation for 15 minutes, the plate was developed, taken out, dried, sprayed with aluminum chloride solution, heated at 105°C, and observed under 365nm ultraviolet light.

[0086] Three batches of samples were tested according to the above operation. At the same time, a negative preparation without Drynaria was prepared. The negative control was prepared by the same method as the test sample. The results are as follows: Figure 1 shown.

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

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

[0089] Take 1 g of the test drug, grind it into powder, add 20 mL of chloroform, ultrasonically treat it for 30 minutes, filter it, evaporate the filtrate to dryness, and add 1 mL of chloroform to dissolve the residue as the test solution.

[0090] Prepare a Ligustrum lucidum control solution using 1 g of Ligustrum lucidum (Ligustrum lucidum) as described for the test solution. This Ligustrum lucidum control solution was provided by the China Food and Drug Administration (CFDA), batch number 121041-202106.

[0091] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 5 μL of the test sample solution and the Ligustrum lucidum control medicinal material solution were taken and spotted on the same silica gel G thin layer plate, respectively, with ethyl acetate-methanol-water (100:17:13) as the developing agent. After saturation for 15 minutes, the plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C, and observed under a 254nm ultraviolet lamp.

[0092] Three batches of samples were tested according to the above operation, and negative preparations without Ligustrum lucidum were prepared at the same time. The negative control was prepared in the same way. The results are as follows: Figure 2 shown.

[0093] The test results show that after heating at 105°C and observing under a 254nm violet lamp, the test sample chromatogram shows spots of the same color at the corresponding positions in the control medicinal material chromatogram, while the accompanying negative control shows no spots at the corresponding positions.

[0094] (3) TLC Identification of Millettia repens

[0095] Take 2.0 g of the test drug, grind it into powder, add 40 mL of ethanol, ultrasonically treat it for 30 minutes, filter it, evaporate the filtrate naturally, dissolve the residue in 10 mL of water, shake and extract with 10 mL of ethyl acetate, evaporate the ethyl acetate, and dissolve the residue in 1 mL of methanol to obtain the test solution.

[0096] Prepare a Spatholobus control solution using 2.0 g of Spatholobus control material using the same procedures as described above for the test solution. This Spatholobus control material was provided by the China Food and Drug Administration, batch number 121065-202408.

[0097] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 5 μL of the test sample solution and the control medicinal material solution of Millettia reticulata were taken and spotted on the same silica gel G thin layer plate, and dichloromethane-acetone-methanol (8:1.2:0.3) was used as the developing agent. The plate was developed, taken out, dried, and inspected under ultraviolet light (254 nm).

[0098] Three batches of samples were tested according to the above operation, and negative preparations without Millettia repens were prepared at the same time. Negative controls were prepared in the same way. The results were as follows: Figure 3 shown.

[0099] Figure 3 The experimental results show that when examined under ultraviolet light (254nm), fluorescent spots of the same color appear in the chromatogram of the test sample at the corresponding positions in the chromatogram of the control medicinal material.

[0100] (4) Thin layer chromatography identification of Cyathula

[0101] Take about 3 g of the test drug, add 30 mL of methanol, ultrasonically treat for 20 minutes, filter, evaporate the filtrate, dissolve the residue in 10 mL of water, and extract with ethyl acetate twice with 10 mL each time. Combine the ethyl acetate liquid, recover the solvent to dryness, and dissolve the residue in 1 mL of methanol to prepare the test solution.

[0102] Prepare a control solution by adding 1.0 g of Cyathulae Radix to 10 mL of methanol, following the same procedures as for the test solution. This control solution was provided by the China Food and Drug Administration (CFDA), batch number 121065-22408.

[0103] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 5 μL of the test sample solution and the Sichuan Cyathula control medicinal material solution were taken and spotted on the same silica gel G thin layer plate. The plate was developed with n-hexane-ethyl acetate-acetone (8:5:1) as the developing agent, developed to 8 cm, taken out, dried, and inspected under ultraviolet light (365 nm).

[0104] Three batches of samples were tested according to the above operation, and negative preparations without Sichuan Cyathula were prepared at the same time. Negative controls were prepared in the same way. The results were as follows: Figure 4 shown.

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

[0106] Second, the naringin (C 27 H 32O 14 ) content determination

[0107] Based on the determination of the content of naringin, a representative component of Drynaria fortunei, the quality inspection and identification of the traditional Chinese medicine composition were investigated. The method for detecting the content of naringin in the traditional Chinese medicine composition of the present invention was obtained, and the precision, repeatability and stability of the detection method were investigated. 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 substance: Naringin reference substance was dissolved in methanol to prepare a naringin reference substance solution with a concentration of 0.02144 mg / mL, which was set aside. The naringin reference substance was provided by the China Food and Drug Administration and had a content of 99.7%.

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

[0112] Test solution: Take 2 g of the Chinese medicine composition, grind it into powder, add 30 mL of methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of methanol to prepare 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 no less than 3000, and naringin in the sample was well separated. The blank sample had no absorption at this wavelength, which did not affect the content determination of the present invention.

[0115] (3) Investigation of linear range

[0116] 2, 4, 6, 8, 10, and 12 μL of the naringin reference solution prepared above were accurately drawn and injected into a liquid chromatograph to measure the peak area. The peak area (Y) was used as the ordinate and the content (X) was used as the abscissa. The results are shown in Tables 1 and 2 below. Figure 5 :

[0117] Table 1 shows the results of linear investigation of Chinese medicine composition granules.

[0118]

[0119] The regression equation of the standard curve is:

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

[0121] The above results show that the peak area value of naringin shows a good linear relationship with the 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 repeat the injection 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] The peak area changes were observed by repeating the injections six times as described in Table 2. The results showed that the relative standard deviation (RSD) of the peak area values ​​of the six injections was 1.43%, indicating that the detection method of the present invention has good precision.

[0127] (5) Repeatability test

[0128] Take 6 samples of the same batch number (batch number: YZ202501), prepare them according to the above test solution preparation method, and determine the content according to the above detection method. The results are shown in Table 3 below:

[0129] Table 3 shows the repeatability test results.

[0130]

[0131]

[0132] As shown in Table 3, the same sample was tested 6 times. The results showed that the average naringin content of the 6 tests was 10.79 mg, and the standard deviation RSD was 1.77%. This shows 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) and prepare a test solution according to the above test sample preparation method. Measure the peak area at 0, 2, 4, 6, 8, 10, 12, and 24 hours using the above chromatographic conditions and method. The results are shown in Table 4 below:

[0135] Table 4 shows the stability test results.

[0136]

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

[0138] (7) Sample recovery test

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

[0140]

[0141] Table 5 is the recovery test results of Chinese medicine composition (n=6)

[0142]

[0143] The test results in Table 5 show that the recovery rate of naringin is between 97.03% and 102.99%, the average recovery rate is 100.94%, and the RSD is 2.24%, indicating a good recovery rate.

[0144] (8) Sample measurement

[0145] Three batches of pilot products of the Chinese medicine composition of the present invention were taken and the content was determined according to the above-mentioned content determination method. 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, considering the source, origin and other factors of the medicinal materials, it was determined that the Chinese medicine composition of the present invention contains rhizoma drynariae and naringin (C 27 H 32 O 14 ) is greater than or equal to 1.79 mg per gram.

[0149] Finally, it should be noted that the above 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 aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent replacements for some of the technical features therein, which should all be included in the scope of protection of the present invention.

Claims

1. A method for detecting the quality of a Chinese medicine composition, characterized in that: The Chinese medicine composition comprises rhizoma drynariae, cibotrys, glossy privet fruit, millettia spatholobi, herba schizonepetae and cyathulae; the rhizoma drynariae, glossy privet fruit, millettia spatholobi and cyathulae are identified by thin layer chromatography, and the quality control index of the Chinese medicine composition is determined by naringin content determination, wherein the rhizoma drynariae contains naringin (C 27 H 32 O 14 ) is greater than or equal to 1.79 mg per gram.

2. The method for quality inspection of a Chinese medicine composition according to claim 1, wherein: The raw material compatibility of the traditional Chinese medicine composition is 15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Millettia reticulata, 10 parts of Herba Lycopodii and 10 parts of Cyathulae chinensis.

3. The quality detection method of the Chinese medicine composition according to claim 2, characterized in that: The preparation method of the traditional Chinese medicine composition comprises the following steps: taking medicinal pieces according to the compatible dosage, adding water and decocting twice, adding 11 times the amount of water for the first time and decocting for 1.5 hours, adding 9 times the amount of water for the second time and decocting for 1.5 hours, combining the filtrates obtained from the two decoctions and performing two vacuum concentration treatments, the concentration including single-effect concentration and double-effect concentration at a temperature of 60-80°C, to obtain a clear paste and an extract, wherein the relative density of the clear paste is 1.15-1.25, and the relative density of the extract is 1.30-1.35, and the dried extract is mixed with the clear paste to obtain the composition.

4. The method for quality inspection of the Chinese medicine composition according to claim 3, wherein: The thin layer chromatography method is used to identify Rhizoma Drynariae, and the specific identification steps are as follows: (1) Grind the Chinese herbal medicine composition, add methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, and dissolve the residue in methanol to prepare a test solution; (2) Take the naringin reference substance and add methanol to prepare a 1 mg / mL solution as the reference solution; (3) Take 5-10 μL of the test solution and reference solution, respectively, 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, take out, dry, spray with aluminum chloride solution, heat at 105 ° C, and observe under 365 nm ultraviolet light. In the chromatogram of the test sample, a main spot of the same color appears at the corresponding position in the chromatogram of the reference sample.

5. The method for quality inspection of the Chinese medicine composition according to claim 3, wherein: The thin layer chromatography method is used to identify the fruits of Ligustrum lucidum, and the specific identification steps are as follows: (1) Grind the Chinese herbal medicine composition, add chloroform, and ultrasonically treat for 30 min. Filter, evaporate the filtrate to dryness, and dissolve the residue in chloroform to prepare the test solution. (2) Take the Ligustrum lucidum control medicinal material and prepare the Ligustrum lucidum control medicinal material solution by the same method as the test solution preparation method in step (1); (3) 5-10 μL of the test solution and control medicinal material solution were taken and spotted on the same silica gel G thin layer plate respectively. Ethyl acetate-methanol-water volume ratio of 100:17:13 was used as the developing solvent. After saturation for 15 minutes, the plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105 °C, and observed under a 254 nm ultraviolet lamp. In the chromatogram of the test sample, a main spot of the same color was obtained at the corresponding position in the chromatogram of the control medicinal material.

6. The method for quality inspection of the Chinese medicine composition according to claim 3, wherein: The thin layer chromatography method is used to identify Millettia repens, and the specific identification steps are as follows: (1) Take the Chinese medicine composition, grind it into powder, add ethanol, ultrasonically treat it for 30 minutes, filter it, evaporate the filtrate naturally, dissolve the residue in water, shake it with ethyl acetate, evaporate the ethyl acetate, and dissolve the residue in methanol to prepare the test solution; (2) Take a control medicinal material of Millettia spatholobi and prepare a control medicinal material solution of Millettia spatholobi by the same operation as the test solution preparation described in step (1); (3) 5-10 μL of the test solution and control medicinal material solution were taken and spotted on the same silica gel G thin layer plate, respectively. The plate was developed with dichloromethane-acetone-methanol in a volume ratio of 8:1.2:0.

3. The plate was taken out and dried. The plate was observed under 254 nm ultraviolet light. In the chromatogram of the test solution, a main spot of the same color was obtained at the corresponding position in the chromatogram of the control medicinal material.

7. The method for quality inspection of a Chinese medicine composition according to claim 3, wherein: The thin layer chromatography method is used to identify Cyathula, and the specific identification steps are as follows: (1) Take the Chinese medicine composition, add methanol, ultrasonically treat for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in water, and extract with ethyl acetate twice with 10 mL each time. Combine the ethyl acetate solutions, recover the solvent to dryness, and dissolve the residue in methanol to prepare the test solution. (2) Take a Cyathula control medicinal material and prepare a Cyathula control medicinal material solution according to the test solution preparation operation described in step (1); (3) 5-10 μL of the test solution and control medicinal material solution were taken and spotted on the same silica gel G thin layer plate. The plate was developed with n-hexane-ethyl acetate-acetone in a volume ratio of 8:5:

1. The plate was developed over an area of ​​8 cm. The plate was taken out, dried, and observed under a 365 nm ultraviolet lamp. In the chromatogram of the test solution, a main spot of the same color was obtained at the corresponding position in the chromatogram of the control medicinal material solution.

8. The method for quality inspection of a traditional Chinese medicine composition according to any one of claims 1 to 7, characterized in that: The method for determining the content of naringin is achieved by the following steps: (1) Take naringin reference substance and dissolve it in methanol to prepare a naringin reference substance solution with a concentration of 0.02144 mg / mL; (2) taking the Chinese medicine composition, adding methanol to dissolve it, ultrasonically treating it, and filtering it to obtain a test solution; (3) Taking different volumes of the naringin reference solution described in step (1), respectively injecting them into a liquid chromatograph, and determining 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 a 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) With the peak area as the ordinate and the naringin content in the naringin reference solution as the abscissa, a standard curve was obtained through regression analysis: 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) Taking the test solution prepared in step (2), measuring the peak area according to the chromatographic conditions described in step (3), and then using the standard curve obtained in step (4) to obtain the content of naringin in the Chinese medicine composition of the test solution.

9. The method for quality inspection of a Chinese medicine composition according to claim 8, wherein: In the step (1), the ultrasonic time is 30 minutes, and filtration is performed using a 0.22 μm microporous membrane. In the step (2), the chromatographic column is an Agilent 5 TC-C18 column with a specification of 4.5×250 mm and a filler particle size of 5 μm.

10. The method for quality inspection of a Chinese medicine composition according to claim 8, wherein: In the step (3), when the naringin content is in the range of 0.001074 to 0.006444 mg, the standard curve is: Y=11960X+0.2146.

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