Quality detection method of pharmaceutical composition for osteoarthritis

Abstract

The invention relates to the technical field of medicine detection, in particular to a quality detection method of a medicine composition for osteoarthritis. The quality detection method of the pharmaceutical composition for osteoarthritis comprises the following steps: carrying out thin-layer identification on roots of Japanese bananas, carrying out thin-layer identification on pseudo-ginseng, and determining the contents of lupenone, psoralen and isopsoralen. A thin-layer identification part of pseudo-ginseng is optimized on the basis of an existing quality standard, meanwhile, a method capable of simultaneously determining the content of lupenone, psoralen and isopsoralen is established, methodological experiments prove that the method is good in repeatability, high in accuracy, high in stability, simple and rapid to operate and high in accuracy, and the method is suitable for large-scale popularization and application. The quality detection standard of the composition is further improved, so that the product quality is better controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a quality detection method for a drug composition for osteoarthritis. Background Art

[0002] The pharmaceutical composition for osteoarthritis, which is made from five medicinal materials including banana root, oxalis, psoralea corylifolia, dipsacus root, and Panax notoginseng as raw materials, has the effects of nourishing the liver and kidneys, strengthening tendons and bones, and dredging meridians and relieving pain. It is used for patients with fractures, osteoarthritis, and osteoporosis caused by liver and kidney deficiency and meridian stasis.

[0003] The current quality standard of the composition (standard number WS-10464 (ZD-0464) -2005-2012Z) includes the thin layer identification of banana root, the thin layer identification of Panax notoginseng, and the determination of psoralen and isopsoralen content. The thin layer identification of Panax notoginseng is specifically as follows: take 5g of the content of this product, add 40mL of methanol, heat and reflux for 1h, filter, evaporate the filtrate to dryness, add 15mL of water to the residue to dissolve, shake and extract with 20mL of chloroform, discard the chloroform solution, shake and extract the aqueous solution with water-saturated n-butanol twice (20mL, 15mL), combine the n-butanol solution, wash with ammonia test solution twice (30mL, 15mL), discard the ammonia solution, evaporate the n-butanol solution, add 1mL of methanol to the residue to dissolve, and use it as the test solution. Take another 1g of Panax notoginseng control medicinal material, add 20mL of methanol, and prepare the control medicinal material solution in the same way. Then take the reference substances of Panax notoginseng saponin R1 and ginsenoside Rg1, add methanol to make a mixed solution containing 2 mg per 1 mL, as the reference substance solution. According to the thin layer chromatography method (Appendix VI B of the 2010 edition of the Chinese Pharmacopoeia), 2-5 μL of each of the above three solutions are taken and spotted on the same silica gel G thin layer plate, and the lower layer solution placed overnight at below 10°C in chloroform-ethyl acetate-methanol-water (15:40:22:10) is used as the developing agent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots are clearly colored. In the chromatogram of the test product, spots of the same color are shown at the corresponding positions of the chromatograms of the reference medicinal materials and the reference substance. The thin layer identification of Panax notoginseng in Gukang Capsules using the above method is complicated and time-consuming, which reduces the detection efficiency of the experimenter.

[0004] Based on the above problems, in order to improve the quality standard of the pharmaceutical composition for osteoarthritis, the present invention combines the current edition of the "Chinese Pharmacopoeia", through in-depth research and investigation, establishes a quality detection method for the composition, optimizes the thin layer identification method of Panax notoginseng in the composition, and establishes a content determination method that can simultaneously determine lupeinone, psoralen and isopsoralen, aiming to further improve the quality standard of the pharmaceutical composition for osteoarthritis and better control product quality. Summary of the invention

[0005] The object of the present invention is to provide a method for detecting the quality of a pharmaceutical composition for osteoarthritis.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The prescription of the pharmaceutical composition for osteoarthritis of the present invention is: 600g of banana root, 400g of oxalis, 600g of psoralea corylifolia, 300g of dipsacus root and 150g of notoginseng; the preparation method of the pharmaceutical composition for osteoarthritis is: among the above five flavors, 30g of banana root and notoginseng are respectively crushed into fine powder; the remaining banana root and the other three flavors such as psoralea corylifolia are decocted with water for three times, each time for 1 hour, the decoction is combined, filtered, and the filtrate is concentrated to a thick paste with a relative density of 1.31-1.35 (60°C), the above fine powder is added, mixed, dried, and an appropriate amount of starch is added to adjust the total amount to 400g, crushed into fine powder, mixed, and filled into capsules to make 1000 capsules.

[0008] The quality detection method of the pharmaceutical composition for osteoarthritis of the present invention comprises thin layer identification of banana root, thin layer identification of Panax notoginseng, and determination of the contents of lupeone, psoralen and isopsoralen, as follows:

[0009] 1) Thin layer chromatography identification of banana root: Take 4.5-5.5g of the content of this product, add 25-35mL of methanol, ultrasonically treat for 20-40min, filter, and concentrate the filtrate to 1mL as the test solution; take another 0.8-1.2g of banana root as a control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 4-6μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate as the developing agent, develop, take out, dry, spray with 5%-15% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine them under daylight and ultraviolet light at 365nm respectively. In the chromatogram of the test product, spots of the same color or fluorescent spots appear at the corresponding positions of the chromatogram of the control medicinal material.

[0010] 2) Thin layer identification of Panax notoginseng: Take 4-6g of the content of this product, add 15-25mL of petroleum ether, ultrasonically treat for 20-40min, filter, add 15-25mL of n-butanol to the residue, ultrasonically treat for 20-40min, filter, wash the filtrate with a saturated aqueous solution of n-butanol for 1-3 times, take the n-butanol layer, evaporate to dryness, and make up the volume of the residue with methanol as the test solution; take another 0.5-1.5g of Panax notoginseng reference medicinal material, add 10-20mL of petroleum ether, and prepare the reference medicinal material solution in the same way; then take notoginseng saponin R1 and ginseng Saponin Rg1 reference substance, methanol is added to prepare a mixed solution containing 2 mg of each saponin Rg1 per 1 mL, which is used as the reference substance solution; according to the thin layer chromatography test, 4-10 μL of each of the three solutions mentioned above are respectively spotted on the same silica gel G thin layer plate, and the lower layer solution of dichloromethane-n-butanol-water-ammonia water is used as the developing agent. The plate is developed, taken out, dried, sprayed with 5%-15% ethanolic sulfuric acid solution, and heated at 105°C until the spots are clearly colored; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the reference medicinal material and the reference substance.

[0011] 3) Determination of the content of lupine ketone, psoralen and isopsoralen:

[0012] Preparation of test solution: Accurately weigh 5 g of the product, place it in a stoppered conical flask, add 50 mL of methanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of methanol and transfer to a 10 mL volumetric flask, make up to volume, shake well, filter, and obtain.

[0013] Preparation of reference solution: Accurately weigh 0.02 g of lupine ketone reference substance, place it in a 100 mL volumetric flask, dissolve it in ethanol and make up to the mark to obtain a lupine ketone reference substance mother solution with a concentration of 0.2 mg / mL; Accurately weigh 0.03 g of psoralen and 0.02 g of isopsoralea corylifolia, place them in a 10 mL volumetric flask, accurately transfer 1 mL of lupine ketone reference substance mother solution, add methanol to make up to the mark, shake well, and obtain a mixed reference solution.

[0014] Chromatographic conditions: Chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 35-45℃; flow rate: 0.8-1.2mL / min; detection wavelength: 225nm; using a mixed solution of methanol and acetonitrile in a volume ratio of 45-55:45-55 as mobile phase A and a 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution was performed, and the gradient elution procedure was as follows:

[0015] 0-14min, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0016] At 14-16 min, the volume ratio of mobile phase A to mobile phase B was changed from 30:70 to 98:2;

[0017] 16-25min, the volume ratio of mobile phase A to mobile phase B was 98:2;

[0018] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 98:2 to 30:70;

[0019] Determination method: Accurately pipette 5 μL of the mixed reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0020] Preferably, in the quality detection method for the pharmaceutical composition for osteoarthritis described in the present invention, the thin layer identification of the banana root is specifically as follows: take 5g of the content of the product, add 30mL of methanol, ultrasonically treat for 30min, filter, and concentrate the filtrate to 1mL as the test solution; take 1g of banana root control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 5μL of the above two solutions respectively, spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and place them under daylight and ultraviolet light at 365nm for inspection. In the test sample chromatogram, spots of the same color or fluorescent spots appear at the corresponding positions of the control medicinal material chromatogram.

[0021] Preferably, in the quality detection method of the pharmaceutical composition for osteoarthritis of the present invention, the boiling range of the petroleum ether in the thin layer identification of banana roots is 60-90°C; and the ratio of petroleum ether to acetate is 20:1.

[0022] Preferably, in the quality detection method of the pharmaceutical composition for osteoarthritis described in the present invention, the thin layer identification of Panax notoginseng is specifically as follows: take 4.5-5.5g of the content of the product, add 20-25mL of petroleum ether, ultrasonically treat for 25-35min, filter, add 20-25mL of n-butanol to the residue, ultrasonically treat for 25-35min, filter, wash the filtrate with a saturated aqueous solution of n-butanol for 2-3 times, take the n-butanol layer, evaporate to dryness, and dilute the residue with methanol as the test solution; take another 0.8-1.2g of Panax notoginseng control medicinal material, add 10-15mL of petroleum ether, and carry out the same method. A control medicinal material solution is prepared; then notoginsenoside R1 and ginsenoside Rg1 reference substances are taken, methanol is added to prepare a mixed solution containing 2 mg of each substance per 1 mL, as a reference substance solution; according to the thin layer chromatography test, 6-8 μL of each of the three solutions is taken, and they are respectively spotted on the same silica gel G thin layer plate, and a lower layer solution of dichloromethane-n-butanol-water-ammonia water is used as a developing agent, and the plate is developed, taken out, dried, sprayed with 8%-12% ethanolic sulfuric acid solution, and heated at 105°C until the spots are clearly colored; in the chromatogram of the test substance, spots of the same color appear at the corresponding positions of the chromatograms of the control medicinal material and the reference substance.

[0023] Further preferably, in the quality detection method of the pharmaceutical composition for osteoarthritis described in the present invention, the thin layer identification of Panax notoginseng is specifically as follows: take 5g of the content of the product, add 20mL of petroleum ether, ultrasonically treat for 30min, filter, add 20mL of n-butanol to the residue, ultrasonically treat for 30min, filter, wash the filtrate twice with a saturated aqueous solution of n-butanol, take the n-butanol layer, evaporate to dryness, and make up the volume of the residue with methanol as the test solution; take another 1g of Panax notoginseng control medicinal material, add 15mL of petroleum ether, and prepare a control medicinal material solution in the same way; then take For reference substances of Panax notoginsenoside R1 and ginsenoside Rg1, methanol was added to prepare mixed solutions containing 2 mg of each substance per 1 mL, which were used as reference substance solutions. According to the thin layer chromatography test, 7 μL of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and the lower layer solution of dichloromethane-n-butanol-water-ammonia water was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test substance, spots of the same color appeared at the corresponding positions of the chromatograms of the reference medicinal material and the reference substance.

[0024] Preferably, in the quality detection method of the pharmaceutical composition for osteoarthritis described in the present invention, the ratio of dichloromethane-n-butanol-water-ammonia water in the thin layer identification of Panax notoginseng is 15:6:2:1.

[0025] Preferably, in the quality detection method of the pharmaceutical composition for osteoarthritis described in the present invention, the chromatographic conditions for determining the contents of lupetone, psoralen and isopsoralen are as follows: the column temperature is 40-45° C. and the flow rate is 1.0-1.2 mL / min.

[0026] Further preferably, in the quality detection method of the pharmaceutical composition for osteoarthritis described in the present invention, the chromatographic conditions in the determination of the contents of lupetone, psoralen and isopsoralen are as follows: the column temperature is 40° C. and the flow rate is 1.0 mL / min.

[0027] Preferably, in the quality detection method of the pharmaceutical composition for osteoarthritis described in the present invention, the volume ratio of methanol to acetonitrile in the mobile phase A in the determination of the contents of lupetone, psoralen and isopsoralen is 45-50:50-55.

[0028] Further preferably, in the quality detection method of the pharmaceutical composition for osteoarthritis described in the present invention, the volume ratio of methanol and acetonitrile in the mobile phase A in the determination of the contents of lupetone, psoralen and isopsoralen is 45:55.

[0029] Beneficial effects of the present invention:

[0030] 1. The present invention provides a quality inspection method for a pharmaceutical composition for osteoarthritis, which optimizes the thin layer identification part of Panax notoginseng. Compared with the method in the existing standard, the thin layer identification method provided by the present invention is simple to operate, can quickly complete drug detection, and greatly improves the detection efficiency of experimenters.

[0031] 2. The inventors have conducted extensive research on the thin-layer identification method of Panax notoginseng, as follows: (1) By investigating the effects of extraction solvents such as n-butanol, ethyl acetate, and dichloromethane on the chromatographic separation effect, it was found that when the extraction solvent was n-butanol, the spots were clearest, and the separation degree and Rf value met the requirements; (2) By investigating the effects of extraction methods such as reflux extraction and ultrasonic extraction on the chromatographic separation effect, it was found that when the extraction method was ultrasonic extraction, the thin-layer spots were clearer than those of the reflux extraction method; (3) By investigating the effects of extraction times of 20 min, 30 min, and 40 min on the chromatographic separation effect, The results show that the optimal extraction time is 30 minutes; (4) By investigating the influence of dichloromethane-n-butanol-water-ammonia water, chloroform-n-butanol-water-ammonia water, and dichloromethane-ethyl acetate-water-ammonia water on the chromatographic separation effect, it is found that the chromatographic separation effect is best when the developing agent is dichloromethane-n-butanol-water-ammonia water; (5) By investigating the influence of the developing agent dichloromethane-n-butanol-water-ammonia water ratio of 10:6:2:1, 15:6:2:1, and 20:6:2:1, it is found that the optimal ratio of the developing agent is 15:6:2:1. Under the above optimal experimental conditions, the thin layer identification of Panax notoginseng was carried out on the pharmaceutical composition for osteoarthritis. At the corresponding positions of the chromatograms of the control medicinal material and the reference substance, spots of the same color appeared, and the spots were clear, the negative had no interference, the separation degree met the requirements, and the Rf value was moderate.

[0032] 3. The present invention establishes a method for simultaneously determining the content of lupine ketone, psoralen and isopsoralen, and determines the optimal HPLC parameters for the determination of the above-mentioned effective ingredients through condition screening experiments: column temperature of 40°C, mobile phase flow rate of 1.0 mL / min, and volume ratio of methanol to acetonitrile in mobile phase A of 45:55. Methodological experiments have confirmed that the method has high precision, strong stability, good repeatability, and simple and quick operation, which further improves the quality inspection standard of the pharmaceutical composition for osteoarthritis and is conducive to better control of product quality. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail below in conjunction with specific embodiments. The following embodiments are only used for explanation and illustration, and do not constitute a limitation on the technical solution of the present invention.

[0034] Example 1

[0035] The quality detection method of the pharmaceutical composition for osteoarthritis includes thin layer identification of banana root, thin layer identification of Panax notoginseng, and determination of the contents of lupine ketone, psoralen and isopsoralen, as follows:

[0036] 1) Thin layer chromatography identification of banana root: Take 5g of the content of this product, add 30mL of methanol, ultrasonically treat for 30min, filter, and concentrate the filtrate to 1mL as the test solution; take 1g of banana root as the control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 5μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate (20:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine them under daylight and ultraviolet light at 365nm respectively. In the chromatogram of the test product, spots of the same color or fluorescent spots appear at the corresponding positions of the chromatogram of the control medicinal material.

[0037] 2) Thin layer identification of Panax notoginseng: Take 5g of the content of this product, add 20mL of petroleum ether, ultrasonically treat for 30min, filter, add 20mL of n-butanol to the residue, ultrasonically treat for 30min, filter, wash the filtrate twice with a saturated aqueous solution of n-butanol, take the n-butanol layer, evaporate to dryness, and dilute the residue with methanol as the test solution; take another 1g of Panax notoginseng reference medicinal material, add 15mL of petroleum ether, and prepare the reference medicinal material solution in the same way; then take Panax notoginseng saponin R1 and ginsenoside Rg1 reference substances, add methanol to prepare A mixed solution containing 2 mg of each substance per 1 mL was used as the reference substance solution. According to the thin layer chromatography test, 7 μL of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and a lower layer solution of dichloromethane-n-butanol-water-ammonia water (15:6:2:1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test substance, spots of the same color appeared at the corresponding positions of the chromatograms of the reference medicinal material and the reference substance.

[0038] 3) Determination of the content of lupine ketone, psoralen and isopsoralen:

[0039] Preparation of test solution: accurately weigh 5 g of the product, place in a stoppered conical flask, add 50 mL of methanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of methanol and transfer to a 10 mL volumetric flask, make up to volume, shake well, filter, and obtain;

[0040] Preparation of reference solution: accurately weigh 0.02 g of lupin ketone reference substance, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to obtain a lupin ketone reference substance mother solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of psoralen and 0.02 g of isopsoralea corylifolia, place them in a 10 mL volumetric flask, accurately transfer 1 mL of lupin ketone reference substance mother solution, add methanol to dilute to the mark, shake well, and obtain a mixed reference substance solution;

[0041] Chromatographic conditions: Chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 40℃; flow rate: 1.0mL / min; detection wavelength: 225nm; using a mixed solution of methanol and acetonitrile in a volume ratio of 45:55 as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution was performed, and the gradient elution program was as follows:

[0042] 0-14min, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0043] At 14-16 min, the volume ratio of mobile phase A to mobile phase B was changed from 30:70 to 98:2;

[0044] 16-25min, the volume ratio of mobile phase A to mobile phase B was 98:2;

[0045] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 98:2 to 30:70;

[0046] Determination method: Accurately pipette 5 μL of the mixed reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0047] Example 2

[0048] The quality detection method of the pharmaceutical composition for osteoarthritis includes thin layer identification of banana root, thin layer identification of Panax notoginseng, and determination of the contents of lupine ketone, psoralen and isopsoralen, as follows:

[0049] 1) Thin layer chromatography identification of banana root: Take 4.5g of the content of this product, add 25mL of methanol, ultrasonically treat for 20min, filter, and concentrate the filtrate to 1mL as the test solution; take another 0.8g of banana root as the control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 4μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate (20:1) as the developing agent, develop, take out, dry, spray with 5% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine them under daylight and ultraviolet light at 365nm respectively. In the chromatogram of the test product, spots of the same color or fluorescent spots appear at the corresponding positions of the chromatogram of the control medicinal material.

[0050] 2) Thin layer identification of Panax notoginseng: Take 4g of the content of this product, add 15mL of petroleum ether, ultrasonically treat for 20min, filter, add 15mL of n-butanol to the residue, ultrasonically treat for 20min, filter, wash the filtrate once with a saturated aqueous solution of n-butanol, take the n-butanol layer, evaporate to dryness, and make up the volume of the residue with methanol as the test solution; take another 0.5g of Panax notoginseng reference medicinal material, add 10mL of petroleum ether, and prepare the reference medicinal material solution in the same way; then take Panax notoginseng saponin R1 and ginsenoside Rg1 reference substances, add methanol to make A mixed solution containing 2 mg of each was prepared in 1 mL as the reference solution; according to the thin layer chromatography test, 4 μL of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and the lower layer solution of dichloromethane-n-butanol-water-ammonia water (15:6:2:1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 5% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored; in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference medicinal material and the reference substance.

[0051] 3) Determination of the content of lupine ketone, psoralen and isopsoralen:

[0052] Preparation of test solution: accurately weigh 5 g of the product, place in a stoppered conical flask, add 50 mL of methanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of methanol and transfer to a 10 mL volumetric flask, make up to volume, shake well, filter, and obtain;

[0053] Preparation of reference solution: accurately weigh 0.02 g of lupin ketone reference substance, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to obtain a lupin ketone reference substance mother solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of psoralen and 0.02 g of isopsoralea corylifolia, place them in a 10 mL volumetric flask, accurately transfer 1 mL of lupin ketone reference substance mother solution, add methanol to dilute to the mark, shake well, and obtain a mixed reference substance solution;

[0054] Chromatographic conditions: Chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 35℃; flow rate: 0.8mL / min; detection wavelength: 225nm; mobile phase A: a mixture of methanol and acetonitrile in a volume ratio of 45:55, mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution, the gradient elution program is as follows:

[0055] 0-14min, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0056] At 14-16 min, the volume ratio of mobile phase A to mobile phase B was changed from 30:70 to 98:2;

[0057] 16-25min, the volume ratio of mobile phase A to mobile phase B was 98:2;

[0058] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 98:2 to 30:70;

[0059] Determination method: Accurately pipette 5 μL of the mixed reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0060] Example 3

[0061] The quality detection method of the pharmaceutical composition for osteoarthritis includes thin layer identification of banana root, thin layer identification of Panax notoginseng, and determination of the contents of lupine ketone, psoralen and isopsoralen, as follows:

[0062] 1) Thin layer chromatography identification of banana root: Take 5.5g of the content of this product, add 35mL of methanol, ultrasonically treat for 40min, filter, and concentrate the filtrate to 1mL as the test solution; take another 1.2g of banana root control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 6μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate (20:1) as the developing agent, develop, take out, dry, spray with 15% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine them under daylight and ultraviolet light at 365nm respectively. In the chromatogram of the test product, spots of the same color or fluorescent spots appear at the corresponding positions of the chromatogram of the control medicinal material.

[0063] 2) Thin layer identification of Panax notoginseng: Take 6g of the content of this product, add 25mL of petroleum ether, ultrasonically treat for 40min, filter, add 25mL of n-butanol to the residue, ultrasonically treat for 40min, filter, wash the filtrate with a saturated aqueous solution of n-butanol for 3 times, take the n-butanol layer, evaporate to dryness, and make up the volume of the residue with methanol as the test solution; take another 1.5g of Panax notoginseng reference medicinal material, add 10mL of petroleum ether, and make a reference medicinal material solution in the same way; then take Panax notoginseng saponin R1 and ginsenoside Rg1 reference substances, add methanol to make A mixed solution containing 2 mg of each substance per 1 mL was used as the reference substance solution. According to the thin layer chromatography test, 10 μL of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and a lower layer solution of dichloromethane-n-butanol-water-ammonia water (15:6:2:1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 15% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test substance, spots of the same color appeared at the corresponding positions of the chromatograms of the reference medicinal material and the reference substance.

[0064] 3) Determination of the content of lupine ketone, psoralen and isopsoralen:

[0065] Preparation of test solution: accurately weigh 5 g of the product, place in a stoppered conical flask, add 50 mL of methanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of methanol and transfer to a 10 mL volumetric flask, make up to volume, shake well, filter, and obtain;

[0066] Preparation of reference solution: accurately weigh 0.02 g of lupin ketone reference substance, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to obtain a lupin ketone reference substance mother solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of psoralen and 0.02 g of isopsoralea corylifolia, place them in a 10 mL volumetric flask, accurately transfer 1 mL of lupin ketone reference substance mother solution, add methanol to dilute to the mark, shake well, and obtain a mixed reference substance solution;

[0067] Chromatographic conditions: Chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 45℃; flow rate: 1.2mL / min; detection wavelength: 225nm; using a mixed solution of methanol and acetonitrile with a volume ratio of 55:45 as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution was performed, and the gradient elution program was as follows:

[0068] 0-14min, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0069] At 14-16 min, the volume ratio of mobile phase A to mobile phase B was changed from 30:70 to 98:2;

[0070] 16-25min, the volume ratio of mobile phase A to mobile phase B was 98:2;

[0071] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 98:2 to 30:70;

[0072] Determination method: Accurately pipette 5 μL of the mixed reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0073] Example 4

[0074] The quality detection method of the pharmaceutical composition for osteoarthritis includes thin layer identification of banana root, thin layer identification of Panax notoginseng, and determination of the contents of lupine ketone, psoralen and isopsoralen, as follows:

[0075] 1) Thin layer chromatography identification of banana root: Take 5.5g of the content of this product, add 35mL of methanol, ultrasonically treat for 40min, filter, and concentrate the filtrate to 1mL as the test solution; take another 1.2g of banana root control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 6μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate (20:1) as the developing agent, develop, take out, dry, spray with 15% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine them under daylight and ultraviolet light at 365nm respectively. In the chromatogram of the test product, spots of the same color or fluorescent spots appear at the corresponding positions of the chromatogram of the control medicinal material.

[0076] 2) Thin layer identification of Panax notoginseng: Take 4.5g of the content of this product, add 25mL of petroleum ether, ultrasonically treat for 35min, filter, add 25mL of n-butanol to the residue, ultrasonically treat for 35min, filter, wash the filtrate twice with a saturated aqueous solution of n-butanol, take the n-butanol layer, evaporate to dryness, and make up the volume of the residue with methanol as the test solution; take another 1g of Panax notoginseng reference medicinal material, add 10mL of petroleum ether, and prepare the reference medicinal material solution in the same way; then take Panax notoginseng saponin R1 and ginsenoside Rg1 reference substances, add methanol to make A mixed solution containing 2 mg of each was prepared in 1 mL as the reference solution; according to the thin layer chromatography test, 8 μL of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and the lower layer solution of dichloromethane-n-butanol-water-ammonia water (15:6:2:1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 12% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored; in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference medicinal material and the reference substance.

[0077] 3) Determination of the content of lupine ketone, psoralen and isopsoralen:

[0078] Preparation of test solution: accurately weigh 5 g of the product, place in a stoppered conical flask, add 50 mL of methanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of methanol and transfer to a 10 mL volumetric flask, make up to volume, shake well, filter, and obtain;

[0079] Preparation of reference solution: accurately weigh 0.02 g of lupin ketone reference substance, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to obtain a lupin ketone reference substance mother solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of psoralen and 0.02 g of isopsoralea corylifolia, place them in a 10 mL volumetric flask, accurately transfer 1 mL of lupin ketone reference substance mother solution, add methanol to dilute to the mark, shake well, and obtain a mixed reference substance solution;

[0080] Chromatographic conditions: Chromatographic column is C 18Column, specification: 250mm×4.6mm, 5μm; column temperature: 40℃; flow rate: 1.0mL / min; detection wavelength: 225nm; mobile phase A: a mixture of methanol and acetonitrile in a volume ratio of 50:50, mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution, the gradient elution program is as follows:

[0081] 0-14min, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0082] At 14-16 min, the volume ratio of mobile phase A to mobile phase B was changed from 30:70 to 98:2;

[0083] 16-25min, the volume ratio of mobile phase A to mobile phase B was 98:2;

[0084] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 98:2 to 30:70;

[0085] Determination method: Accurately pipette 5 μL of the mixed reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0086] Example 5

[0087] The quality detection method of the pharmaceutical composition for osteoarthritis includes thin layer identification of banana root, thin layer identification of Panax notoginseng, and determination of the contents of lupine ketone, psoralen and isopsoralen, as follows:

[0088] 1) Thin layer identification of banana root: Take 5g of the content of this product, add 30mL of methanol, ultrasonically treat for 30min, filter, and concentrate the filtrate to 1mL as the test solution; take another 1.2g of banana root control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 6μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate (20:1) as the developing agent, develop, take out, dry, spray with 15% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine them under daylight and ultraviolet light at 365nm respectively. In the chromatogram of the test product, spots of the same color or fluorescent spots appear at the corresponding positions of the chromatogram of the control medicinal material.

[0089] 2) Thin layer identification of Panax notoginseng: Take 5.5g of the content of this product, add 35mL of petroleum ether, ultrasonically treat for 35min, filter, add 25mL of n-butanol to the residue, ultrasonically treat for 35min, filter, wash the filtrate twice with a saturated aqueous solution of n-butanol, take the n-butanol layer, evaporate to dryness, and make up the volume of the residue with methanol as the test solution; take another 1g of Panax notoginseng reference medicinal material, add 10mL of petroleum ether, and prepare the reference medicinal material solution in the same way; then take Panax notoginseng saponin R1 and ginsenoside Rg1 reference substances, add methanol to make A mixed solution containing 2 mg of each was prepared in 1 mL as the reference solution; according to the thin layer chromatography test, 6 μL of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and the lower layer solution of dichloromethane-n-butanol-water-ammonia water (15:6:2:1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 12% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored; in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference medicinal material and the reference substance.

[0090] 3) Determination of the content of lupine ketone, psoralen and isopsoralen:

[0091] Preparation of test solution: accurately weigh 5 g of the product, place in a stoppered conical flask, add 50 mL of methanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of methanol and transfer to a 10 mL volumetric flask, make up to volume, shake well, filter, and obtain;

[0092] Preparation of reference solution: accurately weigh 0.02 g of lupin ketone reference substance, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to obtain a lupin ketone reference substance mother solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of psoralen and 0.02 g of isopsoralea corylifolia, place them in a 10 mL volumetric flask, accurately transfer 1 mL of lupin ketone reference substance mother solution, add methanol to dilute to the mark, shake well, and obtain a mixed reference substance solution;

[0093] Chromatographic conditions: Chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 40℃; flow rate: 1.0mL / min; detection wavelength: 225nm; mobile phase A: a mixture of methanol and acetonitrile in a volume ratio of 50:50, mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution, the gradient elution program is as follows:

[0094] 0-14min, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0095] At 14-16 min, the volume ratio of mobile phase A to mobile phase B was changed from 30:70 to 98:2;

[0096] 16-25min, the volume ratio of mobile phase A to mobile phase B was 98:2;

[0097] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 98:2 to 30:70;

[0098] Determination method: Accurately pipette 5 μL of the mixed reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0099] In order to further verify the reliability of the present invention and screen out the best solution, the inventor conducted a series of experiments, as follows:

[0100] 1. Experimental Materials

[0101] 1.1 Main Reagents

[0102] Methanol, chromatographic grade; acetonitrile, chromatographic grade; phosphoric acid, analytical grade; petroleum ether, analytical grade; ethyl acetate, analytical grade; ethanolic sulfate, analytical grade; n-butanol, chromatographic grade; phosphoric acid, analytical grade; water is laboratory-made ultrapure water; banana root reference medicinal material; Panax notoginseng reference medicinal material; Panax notoginseng saponin R1 reference substance; ginsenoside Rg1 reference substance; lupine ketone reference substance; psoralen reference substance; isopsoralen reference substance.

[0103] Reference medicinal materials for deficiency of Panax notoginseng: Take 3g of banana root and grind it into fine powder; take 57g of banana root, 40g of Oxalis sorrel, 60g of Psoralea corylifolia and 30g of Dipsacus asper, add water and boil three times, each time for 1h, combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.35 at 60℃, add the above fine powder, mix well, dry, add appropriate amount of starch to adjust the total amount to 40g, and grind it into fine powder.

[0104] 1.2 Main instruments

[0105] Electronic balance (Shanghai Mettler-Toledo Instrument Co., Ltd.); high performance liquid chromatograph (Aglent 1260, Agilent Technologies, USA).

[0106] 2. Thin layer identification of Panax notoginseng

[0107] 2.1 Solution preparation

[0108] Accurately weigh 5 g of the content of this product, add 20 mL of petroleum ether, ultrasonically treat for 30 min, filter, add 20 mL of n-butanol to the residue, ultrasonically treat for 30 min, filter, wash the filtrate twice with a saturated aqueous solution of n-butanol, take the n-butanol layer, evaporate to dryness, and make up the volume of the residue with methanol as the test solution; accurately weigh 5 g of the control medicinal material lacking Panax notoginseng, add 20 mL of petroleum ether, and prepare a Panax notoginseng negative medicinal material solution in the same way; accurately weigh 1 g of Panax notoginseng control medicinal material, add 15 mL of petroleum ether, and prepare a control medicinal material solution in the same way.

[0109] Accurately weigh 0.02 g each of notoginsenoside R1 and ginsenoside Rg1 reference substances, place them in a 10 mL volumetric flask, add methanol to dissolve and make up to volume to prepare a mixed reference substance solution.

[0110] 2.2 Determination method

[0111] According to the thin layer chromatography method (Appendix VI B of Part I of the 2010 edition of the Chinese Pharmacopoeia), 7 μL of the test solution, control medicinal material solution and mixed reference solution were respectively spotted on the same silica gel G thin layer plate, and the lower layer solution of dichloromethane-n-butanol-water-ammonia water (15:6:2:1) was used as the developing solvent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored.

[0112] 2.3 Methodology

[0113] 2.3.1 Investigation of extraction solvent

[0114] 5 g of the product was accurately weighed and degreased with petroleum ether. The effects of n-butanol, ethyl acetate and dichloromethane on the chromatographic separation were investigated. The results are shown in Table 1.

[0115] Table 1 Effects of different extraction solvents on TLC identification of Panax notoginseng

[0116]

[0117] The results show that when n-butanol is used as the extraction solvent, the spots are clear, and the separation degree and Rf value meet the requirements. Therefore, n-butanol is selected as the extraction solvent.

[0118] 2.3.2 Investigation of extraction methods

[0119] 5 g of the product was accurately weighed, degreased with petroleum ether, and then extracted with n-butanol. The sample was treated with reflux and ultrasonic extraction methods, respectively, to investigate the effects of different extraction methods on chromatographic separation. The results are shown in Table 2.

[0120] Table 2 Effects of different extraction methods on thin-layer identification of Panax notoginseng

[0121]

[0122] As shown in Table 2, after the sample was treated by ultrasonic extraction, the spots were clearer than those by ultrasonic extraction, and this method was simple to operate. Therefore, ultrasonic extraction was selected for subsequent experiments.

[0123] 2.3.3 Extraction time investigation

[0124] 5 g of the product was accurately weighed, degreased with petroleum ether, and then treated with n-butanol as the extraction solvent by ultrasonic extraction. The effects of extraction times of 20 min, 30 min, and 40 min on the chromatographic separation effect were investigated. The results are shown in Table 3.

[0125] Table 3 Effect of different extraction time on TLC identification of Panax notoginseng

[0126]

[0127] It can be seen from Table 3 that when the extraction time is 30 min and 40 min, the separation effect is good. After comprehensive consideration, the extraction time is selected as 30 min.

[0128] 2.3.4 Development agent investigation

[0129] The solution was prepared according to the method under "2.1" and the effects of different developing agents on the chromatographic separation were investigated. The results are shown in Table 4.

[0130] Table 4 Effects of different developing agents on thin layer identification of Panax notoginseng

[0131]

[0132] It can be seen from Table 4 that when the developing solvent is dichloromethane-n-butanol-water-ammonia water, the chromatographic separation effect is the best, so the developing solvent is selected as dichloromethane-n-butanol-water-ammonia water.

[0133] 2.3.5 Study on the ratio of developing agent

[0134] The solution was prepared according to the method under "2.1", and dichloromethane-n-butanol-water-ammonia water was used as the developing solvent. The effects of different developing solvent ratios on the chromatographic separation effect were investigated. The results are shown in Table 5.

[0135] Table 5 Effect of different developing agent ratios on thin layer identification of Panax notoginseng

[0136]

[0137] In summary, when the ratio of dichloromethane-n-butanol-water-ammonia water is 15:6:2:1, the chromatographic separation effect is the best. Therefore, the optimal ratio of dichloromethane-n-butanol-water-ammonia water is determined to be 15:6:2:1.

[0138] 2.4 Results and Discussion

[0139] From the above results, it can be seen that in the thin layer identification of Panax notoginseng, the best extraction solvent for the preparation of the test solution is n-butanol, the best extraction method is ultrasonic extraction, and the extraction time is 30min; the best parameters of the chromatographic separation conditions are: the developing solvent is dichloromethane-n-butanol-water-ammonia water, and the ratio is 15:6:2:1. Under the above optimal conditions, the thin layer identification of Panax notoginseng was carried out on the pharmaceutical composition for osteoarthritis. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the control medicinal material and the reference substance, and the spots were clear, the negative had no interference, the separation degree met the requirements, the Rf value was moderate, and the operation was simple and fast.

[0140] 3. Determination of the contents of lupine ketone, psoralen and isopsoralen

[0141] 3.1 Preparation of test solution

[0142] Accurately weigh 5 g of the product, place it in a stoppered conical flask, add 50 mL of methanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, add appropriate amount of methanol to dissolve the residue and transfer it to a 10 mL volumetric flask, make up to volume, shake well, filter, and obtain.

[0143] 3.2 Preparation of reference solution

[0144] Accurately weigh 0.02 g of lupine ketone reference substance, place it in a 100 mL volumetric flask, dissolve it in ethanol and make up to the mark to prepare a lupine ketone reference substance mother solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of psoralen and 0.02 g of isopsoralea corylifolia, place them in a 10 mL volumetric flask, accurately transfer 1 mL of lupine ketone reference substance mother solution, add methanol to make up to the mark, shake well, and obtain a mixed reference substance solution.

[0145] 3.3 Chromatographic conditions

[0146] The chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 40℃; flow rate: 1.0mL / min; detection wavelength: 225nm; using a mixed solution of methanol and acetonitrile in a volume ratio of 45:55 as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution was performed, and the gradient elution program was as follows:

[0147] 0-14min, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0148] At 14-16 min, the volume ratio of mobile phase A to mobile phase B was changed from 30:70 to 98:2;

[0149] 16-25min, the volume ratio of mobile phase A to mobile phase B was 98:2;

[0150] From 25 to 30 min, the volume ratio of mobile phase A to mobile phase B was changed from 98:2 to 30:70.

[0151] 3.4 Determination method

[0152] Accurately pipette 5 μL of the mixed reference solution and test solution respectively, inject into high performance liquid chromatography, and measure to obtain the result.

[0153] 3.5 Investigation of chromatographic conditions

[0154] 3.5.1 Column temperature investigation

[0155] Weigh 5g of the product accurately, prepare the test solution according to the method under "3.1", and use the mixed reference solution prepared under "3.2" as the control to investigate the effect of column temperature on chromatographic separation at 35℃, 40℃, and 45℃. The results show that when the column temperature is 40℃, the chromatographic peak shape is good and the separation degree meets the requirements, so the column temperature is selected to be 40℃.

[0156] 3.5.2 Investigation of mobile phase flow rate

[0157] Accurately weigh 5g of the content of this product, prepare the test solution according to the method under "3.1", and use the mixed reference solution prepared under "3.2" as the control to investigate the effect of mobile phase flow rates of 0.8mL / min, 1.0mL / min, and 1.2mL / min on the chromatographic separation effect. The results show that when the flow rate is 1.0mL / min, the separation effect of psoralen and isopsoralen is the best, so the flow rate is selected to be 1.0mL / min.

[0158] 3.5.3 Investigation of mobile phase ratio

[0159] Accurately weigh 5g of the content of this product, prepare the test solution according to the method under "3.1", and use the mixed reference solution prepared under "3.2" as the control to investigate the effect of the ratio of methanol to acetonitrile in mobile phase A of 45:55, 50:50, and 55:45 on the chromatographic separation effect. The results show that different ratios of methanol and acetonitrile in mobile phase A affect the peak time and separation effect of lupetone, psoralen and isopsoralen. When the ratio of methanol to acetonitrile is 45:55, the chromatographic separation effect is the best, so the ratio of methanol to acetonitrile in mobile phase A is selected to be 45:55.

[0160] 3.6 Methodological Validation

[0161] 3.6.1 Precision investigation

[0162] Accurately pipette 5 μL of the mixed reference solution under item "3.2" and repeat the injection 6 times. Record the peak areas of lupeone, psoralea corylifolia and isopsoralea corylifolia respectively. The results are shown in Table 6. The RSD values ​​are 0.12% and 0.13% respectively, indicating that the precision of the instrument is good.

[0163] Table 6 Precision test results

[0164]

[0165] 3.6.2 Stability test

[0166] Take the test solution prepared under "3.2" and inject samples at 0, 2, 4, 8, 12 and 24 hours respectively, and record the peak areas of lupeone, psoralea corylifolia and isopsoralea corylifolia respectively. The results are shown in Table 7. It can be seen from the results that the RSD values ​​are 0.26% and 0.03% respectively, indicating that the precision of the instrument is good.

[0167] Table 7 Stability test results

[0168]

[0169] 3.6.3 Repeatability test

[0170] Accurately weigh 5 g of the content of this product, weigh a total of 6 portions, prepare the test solution according to the method under "3.2", use the mixed reference solution prepared under "3.2" as the control, perform HPLC analysis, calculate the lupeinone content, psoralen and isopsoralen total content, the results are shown in Table 8, it can be seen from the results that the RSD values ​​are 0.62% and 1.14%, respectively, indicating that the method has good repeatability.

[0171] Table 8 Repeatability test results

[0172]

[0173] 3.7 Summary and Discussion

[0174] In this study, a method for simultaneously determining the content of lupine ketone, psoralen and isopsoralen was established, and the optimal HPLC parameters for the determination of the above active ingredients were determined through condition screening experiments: column temperature of 40°C; mobile phase flow rate of 1.0 mL / min; the volume ratio of methanol and acetonitrile in mobile phase A was 45:55. Methodological experiments confirmed that this method has high precision, strong stability, good repeatability, and simple and quick operation. This method further improves the quality inspection standards of pharmaceutical compositions for osteoarthritis, which is conducive to better control of product quality.

[0175] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A quality inspection method for a pharmaceutical composition for osteoarthritis, wherein the prescription of the pharmaceutical composition for osteoarthritis is: 600g of banana root, 400g of oxalis, 600g of psoralea corylifolia, 300g of dipsacus root, and 150g of Panax notoginseng; the preparation method of the pharmaceutical composition for osteoarthritis is: of the above five flavors, 30g of banana root and Panax notoginseng are respectively crushed into fine powder; the remaining banana root and the other three flavors such as psoralea corylifolia are decocted with water for three times, each time for 1 hour, the decoction is combined, filtered, and the filtrate is concentrated to a thick paste with a relative density of 1.31-1.35 at 60°C, the above fine powder is added, mixed, dried, and an appropriate amount of starch is added to adjust the total amount to 400g, crushed into fine powder, mixed, and filled into capsules to make 1000 capsules, that is, It is characterized in that The quality detection method of the pharmaceutical composition for osteoarthritis includes thin layer identification of banana root, thin layer identification of Panax notoginseng, and determination of the contents of lupine ketone, psoralen and isopsoralen, as follows: 1) Thin layer identification of banana root: Take 4.5-5.5g of the content of this product, add 25-35mL of methanol, ultrasonically treat for 20-40min, filter, and concentrate the filtrate to 1mL as the test solution; take 0.8-1.2g of banana root as a control medicinal material, and prepare a control medicinal material solution in the same way; according to the thin layer chromatography test, take 4-6μL of each of the above two solutions, respectively spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate as the developing agent, develop, take out, dry, spray with 5%-15% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and place them under sunlight and ultraviolet light at 365nm for inspection. In the chromatogram of the test product, spots of the same color or fluorescent spots appear at the corresponding positions of the chromatogram of the control medicinal material; 2) Thin layer identification of Panax notoginseng: Take 4-6g of the content of this product, add 15-25mL of petroleum ether, ultrasonically treat for 20-40min, filter, add 15-25mL of n-butanol to the residue, ultrasonically treat for 20-40min, filter, wash the filtrate with a saturated aqueous solution of n-butanol for 1-3 times, take the n-butanol layer, evaporate to dryness, and make up the volume of the residue with methanol as the test solution; take another 0.5-1.5g of Panax notoginseng reference medicinal material, add 10-20mL of petroleum ether, and prepare the reference medicinal material solution in the same way; then take notoginseng saponin R1 and ginseng Saponin Rg1 reference substance, add methanol to prepare a mixed solution containing 2 mg of each in 1 mL, as the reference substance solution; according to the thin layer chromatography test, take 4-10 μL of each of the above three solutions, respectively spot on the same silica gel G thin layer plate, use the lower layer solution of dichloromethane-n-butanol-water-ammonia water as the developing agent, develop, take out, dry, spray with 5%-15% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatograms of the reference medicinal material and the reference substance; 3) Determination of the content of lupine ketone, psoralen and isopsoralen: Preparation of test solution: accurately weigh 5 g of the product, place in a stoppered conical flask, add 50 mL of methanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of methanol and transfer to a 10 mL volumetric flask, make up to volume, shake well, filter, and obtain; Preparation of reference solution: accurately weigh 0.02 g of lupin ketone reference substance, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to obtain a lupin ketone reference substance mother solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of psoralen and 0.02 g of isopsoralea corylifolia, place them in a 10 mL volumetric flask, accurately transfer 1 mL of lupin ketone reference substance mother solution, add methanol to dilute to the mark, shake well, and obtain a mixed reference substance solution; Chromatographic conditions: Chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 35-45℃; flow rate: 0.8-1.2mL / min; detection wavelength: 225nm; using a mixed solution of methanol and acetonitrile in a volume ratio of 45-55:45-55 as mobile phase A and a 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution was performed, and the gradient elution procedure was as follows: 0-14min, the volume ratio of mobile phase A to mobile phase B was 30:70; At 14-16 min, the volume ratio of mobile phase A to mobile phase B was changed from 30:70 to 98:2; 16-25min, the volume ratio of mobile phase A to mobile phase B was 98:2; 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 98:2 to 30:70; Determination method: Accurately pipette 5 μL of the mixed reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

2. The quality detection method of the pharmaceutical composition for osteoarthritis according to claim 1, characterized in that: The thin layer identification of the banana root is specifically as follows: take 5g of the content of the product, add 30mL of methanol, ultrasonically treat for 30min, filter, and concentrate the filtrate to 1mL as the test solution; take 1g of the banana root control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test, take 5μL of the above two solutions respectively, and spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and place them under daylight and ultraviolet light at 365nm for inspection. In the test sample chromatogram, spots of the same color or fluorescent spots appear at the corresponding positions of the control medicinal material chromatogram.

3. The quality detection method of the pharmaceutical composition for osteoarthritis according to any one of claims 1 or 2, characterized in that: The boiling range of petroleum ether in the thin layer identification of banana roots is 60-90° C.; the ratio of petroleum ether to acetate is 20:

1.

4. The quality detection method of the pharmaceutical composition for osteoarthritis according to claim 1, characterized in that: The specific thin-layer identification of Panax notoginseng is as follows: 4.5-5.5 g of the content of the product is taken, 20-25 mL of petroleum ether is added, ultrasonic treatment is performed for 25-35 min, and filtration is performed. 20-25 mL of n-butanol is added to the residue, ultrasonic treatment is performed for 25-35 min, and filtration is performed. The filtrate is washed 2-3 times with an aqueous solution saturated with n-butanol, and the n-butanol layer is taken, evaporated to dryness, and the residue is fixed to volume with methanol as the test solution; 0.8-1.2 g of Panax notoginseng control medicinal material is taken separately, 10-15 mL of petroleum ether is added, and a control medicinal material solution is prepared in the same manner; and Panax notoginseng saponin R is taken.

1. For the reference substance of ginsenoside Rg1, methanol was added to prepare a mixed solution containing 2 mg of each substance per 1 mL, which was used as the reference substance solution. According to the thin layer chromatography test, 6-8 μL of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and the lower layer solution of dichloromethane-n-butanol-water-ammonia was used as the developing agent. The plate was developed, taken out, dried, sprayed with 8%-12% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test substance, spots of the same color appeared at the corresponding positions of the chromatograms of the reference medicinal materials and the reference substance.

5. The quality detection method of the pharmaceutical composition for osteoarthritis according to claim 4, characterized in that: The specific thin-layer identification of Panax notoginseng is as follows: 5 g of the content of the product is taken, 20 mL of petroleum ether is added, ultrasonic treatment is performed for 30 min, and filtration is performed. 20 mL of n-butanol is added to the residue, ultrasonic treatment is performed for 30 min, and filtration is performed. The filtrate is washed twice with a saturated aqueous solution of n-butanol, and the n-butanol layer is taken, evaporated to dryness, and the residue is fixed to volume with methanol as the test solution; 1 g of Panax notoginseng reference medicinal material is taken, 15 mL of petroleum ether is added, and a reference medicinal material solution is prepared in the same manner; then Panax notoginseng saponin R1 and ginsenoside Rg1 reference substances are taken. , add methanol to make a mixed solution containing 2 mg of each in 1 mL, as the reference solution; according to the thin layer chromatography test, take 7 μL of each of the three solutions, spot them on the same silica gel G thin layer plate, use the lower layer solution of dichloromethane-n-butanol-water-ammonia water as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and in the test sample chromatogram, spots of the same color appear at the corresponding positions of the reference medicinal material and the reference substance chromatogram.

6. The quality detection method of the pharmaceutical composition for osteoarthritis according to any one of claims 1, 4 or 5, characterized in that: The ratio of dichloromethane-n-butanol-water-ammonia water in the thin layer identification of Panax notoginseng is 15:6:2:

1.

7. The quality detection method of the pharmaceutical composition for osteoarthritis according to claim 1, characterized in that: The chromatographic conditions for the determination of lupine ketone, psoralen and isopsoralen are as follows: the column temperature is 40-45° C. and the flow rate is 1.0-1.2 mL / min.

8. The quality detection method of the pharmaceutical composition for osteoarthritis according to claim 7, characterized in that: The chromatographic conditions for the determination of lupine ketone, psoralen and isopsoralen are as follows: the column temperature is 40° C. and the flow rate is 1.0 mL / min.

9. The quality detection method of the pharmaceutical composition for osteoarthritis according to claim 1, characterized in that: The volume ratio of methanol to acetonitrile in the mobile phase A for the content determination of lupetone, psoralen and isopsoralen is 45-50:50-55.

10. The quality detection method of the pharmaceutical composition for osteoarthritis according to claim 9, characterized in that: The volume ratio of methanol to acetonitrile in the mobile phase A for the determination of lupetone, psoralen and isopsoralen is 45:55.

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