CN115184500A - Quality detection method of traditional Chinese medicine composition - Google Patents
Quality detection method of traditional Chinese medicine composition Download PDFInfo
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- CN115184500A CN115184500A CN202210847469.3A CN202210847469A CN115184500A CN 115184500 A CN115184500 A CN 115184500A CN 202210847469 A CN202210847469 A CN 202210847469A CN 115184500 A CN115184500 A CN 115184500A
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Abstract
The invention relates to the technical field of medicines, in particular to a quality detection method of a traditional Chinese medicine composition. The quality detection method comprises the following steps: taking a sample of a Chinese medicinal composition to be tested, wherein the theoretical preparation raw materials of the Chinese medicinal composition comprise radix et rhizoma Rhei, radix astragali, cortex Mori, radix Sophorae Flavescentis, radix Codonopsis, atractylodis rhizoma, poria, radix Polygoni Multiflori Preparata, radix Paeoniae alba, saviae Miltiorrhizae radix, rhizoma Ligustici Chuanxiong, flos Chrysanthemi, ginger processed pinellia Tuber, herba plantaginis, bupleuri radix and Glycyrrhrizae radix; detecting whether the sample of the Chinese medicinal composition to be detected contains radix et rhizoma Rhei, radix Polygoni Multiflori Preparata, radix astragali, saviae Miltiorrhizae radix, radix Sophorae Flavescentis, bupleuri radix, cortex Mori, poria, radix Codonopsis, glycyrrhrizae radix and radix Paeoniae alba by thin layer chromatography; detecting the contents of paeoniflorin, salvianolic acid B, astragaloside IV, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside and matrine in the Chinese medicinal composition sample to be detected by high performance liquid chromatography; and (4) carrying out character detection on the traditional Chinese medicine composition sample to be detected. The invention can ensure that the traditional Chinese medicine composition can obtain effective product quality guarantee in production and use, thereby ensuring the curative effect of the medicine.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a quality detection method of a traditional Chinese medicine composition.
Background
A Chinese medicinal composition is prepared from radix et rhizoma Rhei, radix astragali, cortex Mori, radix Sophorae Flavescentis, radix Codonopsis, atractylodis rhizoma, poria, radix Polygoni Multiflori Preparata, radix Paeoniae alba, saviae Miltiorrhizae radix, rhizoma Ligustici Chuanxiong, flos Chrysanthemi, ginger processed pinellia Tuber, herba plantaginis, bupleuri radix and Glycyrrhrizae radix, has effects of clearing hollow viscera, eliminating turbid pathogen, invigorating spleen, promoting diuresis, promoting blood circulation and removing blood stasis, and can be used for treating chronic renal failure, azotemia and early stage uremia, and spleen deficiency and blood stasis syndrome.
In the Chinese medicinal composition, bupleuri radix, poria, saviae Miltiorrhizae radix, etc. have outstanding effects of resisting inflammation, scavenging free radicals, promoting urination, reducing blood sugar, relieving or preventing further deterioration of nephropathy, reducing urine protein and microalbumin, and eliminatingAGES and erythrocyte number in blood are obviously increased, renal anemia is improved, and inflammatory factors such as CRP, interleukin, tumor necrosis factor and the like in plasma of a patient undergoing Maintenance Hemodialysis (MHD) are eliminated. Guo Liujin, by continuously taking the medicine for the patient in 4 weeks, it was found that the indexes of urine protein, blood fat, renal function and the like in the treatment group are significantly improved compared with those in the control group, which indicates that the Niaoduqing granules can improve renal damage caused by hypertension of the patient. Miao Xugong, etc. induces rat to form chronic renal failure by gastric adenine infusion, detects rat renal cortex transforming growth factor-beta 1 (Transfer Growth Factor-β 1 ,TGF-β 1 ) And the expression change of mRNA of a downstream gene thereof, and the result shows that the kidney shape of a rat is improved after the preparation is used for treating; a decrease in blood creatinine, urea nitrogen levels; the mRNA expression of the TGF-beta 1 and the downstream gene of the TGF-beta 1 is reduced, and the expression of the TGF-beta 1 protein is reduced. The pharmacological action research of the salvianolic acid B is well documented at home and abroad, and comprises Sun Rendi and the like which carry out detailed research on various pharmacological activities of the salvianolic acid B, such as cardiovascular and cerebrovascular protection, liver and kidney protection, fibrosis resistance and the like. The salvianolic acid B has the effects of neuroprotection, antioxidation and the like, and also has the protective effects on the activation of a mitochondrial pathway and the in-vitro apoptosis of Schwann Cells (SC) under the induction of oxidative stress induced by High Glucose (HG).
Therefore, the clinical curative effect of the traditional Chinese medicine composition is reliable. However, the quality research on the traditional Chinese medicine composition is not perfect at present, and comprehensive quality control is necessary.
Disclosure of Invention
Based on the above, the invention provides a quality detection method of the traditional Chinese medicine composition, which can ensure that the traditional Chinese medicine composition can obtain effective product quality guarantee in production and use, and further ensure the curative effect of the medicine.
The specific technical scheme is as follows:
a quality detection method of a traditional Chinese medicine composition comprises the following steps:
taking a sample of a Chinese medicinal composition to be tested, wherein the theoretical preparation raw materials of the Chinese medicinal composition comprise radix et rhizoma Rhei, radix astragali, cortex Mori, radix Sophorae Flavescentis, radix Codonopsis, atractylodis rhizoma, poria, radix Polygoni Multiflori Preparata, radix Paeoniae alba, saviae Miltiorrhizae radix, rhizoma Ligustici Chuanxiong, flos Chrysanthemi, ginger processed pinellia Tuber, herba plantaginis, bupleuri radix and Glycyrrhrizae radix;
detecting whether the sample of the Chinese medicinal composition to be detected contains radix et rhizoma Rhei, radix Polygoni Multiflori Preparata, radix astragali, saviae Miltiorrhizae radix, radix Sophorae Flavescentis, bupleuri radix, cortex Mori, poria, radix Codonopsis, glycyrrhrizae radix and radix Paeoniae alba by thin layer chromatography;
detecting the contents of paeoniflorin, salvianolic acid B, astragaloside IV, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside and matrine in the Chinese medicinal composition sample to be detected respectively by adopting a high performance liquid chromatography, and determining the quality of the Chinese medicinal composition sample to be detected by combining the contents;
and (4) carrying out character detection on the traditional Chinese medicine composition sample to be detected.
Optionally, when the content simultaneously satisfies the following conditions, determining that the quality of the Chinese medicinal composition sample to be tested is qualified:
a) The content of paeoniflorin is not less than 0.7mg/g;
b) The content of the salvianolic acid B is not less than 0.25mg/g;
c) The content of the astragaloside is not less than 0.1mg/g;
d) The 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside content is not less than 0.1mg/g;
e) The content of matrine is not less than 0.3mg/g.
Optionally, the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains rhubarb and prepared fleece flower root by adopting thin-layer chromatography comprises the following steps:
taking a sample of the traditional Chinese medicine composition to be tested, adding a sulfuric acid solution, heating and refluxing, cooling, adding chloroform, heating and refluxing, taking a chloroform solution, drying, and dissolving residues in chloroform to prepare a test solution;
preparing a rhubarb reference medicinal material solution and a prepared fleece-flower root reference medicinal material solution; preparing a mixed reference solution containing rhein, emodin and physcion;
respectively sucking the test solution, the rhubarb reference medicinal material solution, the prepared fleece-flower root reference medicinal material solution and the mixed reference solution, and dropping the solutions on the same silica gel G or H thin-layer plate according to the volume ratio of (10-20): (3-7): (0.5-2) taking the upper solution of petroleum ether, ethyl formate and formic acid as a developing agent, developing, airing and observing.
Preferably, 20 mL-50 mL of sulfuric acid solution with the concentration of 2 mol/L-5 mol/L is added into each 3 g-7 g of the traditional Chinese medicine composition to be detected. More preferably, 20 mL-40 mL of sulfuric acid solution with the concentration of 3mol/L is added into every 5g of the traditional Chinese medicine composition sample to be detected.
Preferably, the time for heating reflux by adding the sulfuric acid solution is 0.5 to 2 hours. More preferably, the time of heating reflux with the sulfuric acid solution is 0.5 to 1.5 hours.
Preferably, 20 mL-50 mL of chloroform is added into each 3 g-7 g sample of the traditional Chinese medicine composition to be detected. More preferably, 20mL to 40mL of chloroform is added to each 5g of the Chinese medicinal composition sample to be tested.
Preferably, the time of heating reflux by adding chloroform is 0.5 to 2 hours. More preferably, the time of heating and refluxing with chloroform is 0.5 to 1.5 hours.
Preferably, the method for preparing the rhubarb reference drug solution comprises the following steps: taking a rhubarb reference medicinal material, and preparing a rhubarb reference medicinal material solution according to the method for preparing the test solution. More preferably, 20 mL-50 mL of sulfuric acid solution with the concentration of 2 mol/L-5 mol/L is added into every 0.5 g-2 g of the rhubarb reference drug. 20mL to 50mL of chloroform was added correspondingly.
Preferably, the method for preparing the radix polygoni multiflori preparata reference medicine solution comprises the following steps: taking the prepared fleece-flower root reference medicinal material, and preparing the prepared fleece-flower root reference medicinal material solution according to the method for preparing the test solution. More preferably, 20 mL-50 mL of sulfuric acid solution with the concentration of 2 mol/L-5 mol/L is added into 0.5 g-2 g of the polygonum multiflorum reference medicinal material. 20mL to 50mL of chloroform was added correspondingly.
Preferably, the solvent for preparing the mixed reference solution containing the rhein reference, the emodin reference and the physcion reference is methanol. More preferably, in the mixed reference solution, the concentration of the rhein reference substance is 0.5 mg/mL-2 mg/mL, the concentration of the emodin reference substance is 0.5 mg/mL-2 mg/mL, and the concentration of the physcion reference substance is 0.5 mg/mL-2 mg/mL.
Preferably, the volume ratio of (14-16): (4-6): (0.5-1.5) using petroleum ether, ethyl formate and formic acid as upper solution as developing agent.
Optionally, the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains astragalus membranaceus by adopting thin-layer chromatography comprises the following steps:
taking a sample of the Chinese medicinal composition to be tested, adding n-butanol, heating and refluxing, filtering, taking filtrate, carrying out alkali washing, removing alkali liquor, adding water-saturated n-butanol, washing to neutrality, taking n-butanol extract, drying, and dissolving residue with methanol to prepare a test solution;
preparing an astragaloside IV reference substance solution;
respectively sucking the test solution and the astragaloside IV reference solution, and dropping the solutions on the same silica gel G or H thin layer plate according to the volume ratio of (12-14): (6-8): (1-4) developing with a lower layer solution of chloroform, methanol and water as a developing agent, and observing after the developing agent is developed and dried.
Preferably, 10 mL-50 mL of n-butanol is added into each 3 g-7 g of the traditional Chinese medicine composition sample to be detected correspondingly. More preferably, 10mL to 30mL of n-butanol is added into every 5g of the traditional Chinese medicine composition sample to be detected.
Preferably, the time for heating and refluxing the added n-butanol is 1 to 3 hours.
Preferably, the alkali liquor is sodium hydroxide solution. More preferably, the mass fraction of the sodium hydroxide solution is 0.5% to 2%. More preferably, the mass fraction of the sodium hydroxide solution is 0.5% to 1.5%.
Preferably, the number of alkaline washes is 2 to 5. More preferably, the number of alkali washes is 2 to 4.
More preferably, 10 mL-50 mL of sodium hydroxide solution with the mass fraction of 0.5% -2% is correspondingly added into each 3 g-7 g of the traditional Chinese medicine composition sample to be detected during each alkaline washing. More preferably, 10mL to 20mL of sodium hydroxide solution with the mass fraction of 0.5 percent to 1.5 percent is correspondingly added into every 5g of the traditional Chinese medicine composition sample to be detected during each alkaline washing.
Preferably, the solvent for preparing the astragaloside control solution is methanol. More preferably, the concentration of the astragaloside IV control in the control solution is 0.5 mg/mL-2 mg/mL.
Preferably, the volume ratio of (12-14): (6-8): (1-3) the lower layer solution of chloroform, methanol and water is a developing solvent.
Preferably, after drying, the method further comprises the step of spraying an ethanol solution of sulfuric acid with the volume fraction of 5-20% and heating until the spots are clearly developed. Preferably, after air drying, the method further comprises the step of spraying an ethanol solution of sulfuric acid with the mass fraction of 5% -15%, and heating until the spots are clearly developed.
Optionally, the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains the salvia miltiorrhiza by adopting the thin-layer chromatography comprises the following steps:
dissolving the Chinese medicinal composition sample in water, filtering, extracting the filtrate with diethyl ether, drying diethyl ether phase, dissolving the residue with ethanol, and preparing test solution;
preparing a salvia miltiorrhiza reference medicinal material solution and a protocatechuic aldehyde reference substance solution;
respectively sucking the test solution, the salvia miltiorrhiza reference medicinal material solution and the protocatechuic aldehyde reference solution, and dropping the solutions on the same silica gel G or H thin layer plate according to the volume ratio of (1-3): (3-5): (3-5): (0.3-0.7) taking hexane, benzene, ethyl acetate and formic acid as developing agents, developing, airing and observing.
Preferably, 20mL to 100mL of water is added into each 3g to 7g of the traditional Chinese medicine composition sample to be detected correspondingly. More preferably, 40mL to 60mL of water is added to every 5g of the traditional Chinese medicine composition sample to be detected.
Preferably, the number of times of extraction with diethyl ether is 1 to 3. More preferably, 10mL to 50mL of diethyl ether is added to each 3g to 7g of the traditional Chinese medicine composition sample to be detected in each extraction. More preferably, 10mL to 20mL of diethyl ether is added to each 5g of the traditional Chinese medicine composition sample to be detected in each extraction.
Preferably, the method for preparing the salvia miltiorrhiza control medicinal material solution comprises the following steps: decocting Saviae Miltiorrhizae radix control in water, concentrating the decoction, and preparing Saviae Miltiorrhizae radix control solution with the above method for preparing test solution.
More preferably, the number of times of decocting with water is 1 to 3 times. More preferably, the time for decocting with water is 5 minutes to 60 minutes. More preferably, the time for decocting with water is 5 minutes to 15 minutes. More preferably, each 3g to 7g of the salvia miltiorrhiza contrast medicinal material is correspondingly concentrated to 5mL to 15mL. More preferably, 20mL to 100mL of water and 10mL to 50mL of ether are added to 4mL to 6mL of the concentrate.
Preferably, the solvent for preparing the protocatechuic aldehyde control solution is ethanol. More preferably, the concentration of the protocatechuic aldehyde control in the control solution is 0.2 mg/mL-5 mg/mL.
Preferably, after being dried, the method also comprises the step of spraying an ethanol solution of the 2, 4-dinitrophenylhydrazine with the mass fraction of 0.05-0.5%. More preferably, the method also comprises the step of spraying an ethanol solution of the 2, 4-dinitrophenylhydrazine with the mass fraction of 0.05-0.15 percent after air drying.
Optionally, the method for detecting whether the sample of the Chinese medicinal composition to be detected contains the sophora flavescens by adopting the thin-layer chromatography comprises the following steps:
taking a sample of the traditional Chinese medicine composition to be tested, adding chloroform and a concentrated ammonia solution, standing, filtering, taking a filtrate for drying, and dissolving residues in chloroform to prepare a test solution;
preparing a matrine reference solution;
respectively sucking the test solution and the matrine reference solution, dropping the test solution and the matrine reference solution on the same silica gel G or H thin layer plate, and mixing the two solutions according to the volume ratio of (10-30): (10-30): (2-4): (0.5-2) developing with toluene, ethyl acetate, methanol and concentrated ammonia solution as developing agent, air drying and observing.
Preferably, 30-80 mL of chloroform and 0.1-0.5 mL of concentrated ammonia solution are added into each 3-7 g of the traditional Chinese medicine composition sample to be detected. More preferably, 40mL to 60mL of chloroform and 0.2mL to 0.4mL of concentrated ammonia solution are added into each 5g of the sample of the traditional Chinese medicine composition to be detected.
Preferably, the residue corresponding to each 3g to 7g of the Chinese medicinal composition sample to be tested is dissolved in 0.3mL to 1mL of chloroform. More preferably, the residue corresponding to 5g of the Chinese medicinal composition sample is dissolved in 0.4-0.6 mL of chloroform.
Preferably, the solvent for preparing the matrine control solution is ethanol. More preferably, the concentration of the matrine control in the control solution is 0.05 mg/mL-0.5 mg/mL.
Preferably, the volume ratio is (15-25): (15 to 25)): (2-4): (0.5-1.5) toluene, ethyl acetate, methanol and concentrated ammonia solution as developing agent.
Preferably, after drying, the method further comprises the step of spraying dilute bismuth iodide test solution.
Optionally, the method for detecting whether the sample of the Chinese medicinal composition to be detected contains the bupleurum root by adopting the thin-layer chromatography comprises the following steps:
taking a sample of the Chinese medicinal composition to be detected, adding water, carrying out ultrasonic extraction, filtering, taking filtrate, passing the filtrate through an AB-8 type macroporous adsorption resin column, eluting by using an ethanol solution with the volume fraction of 40-60%, discarding an eluent, continuously eluting by using an ethanol solution with the volume fraction of 80-100%, taking the eluent, drying, adding methanol into residues for dissolving, and preparing a sample solution;
preparing a radix bupleuri reference medicinal material solution;
respectively sucking the test solution and the radix bupleuri reference medicinal material solution, dropping the test solution and the radix bupleuri reference medicinal material solution on the same silica gel G or H thin-layer plate, and mixing the two solutions according to the volume ratio of (9-13): (1-3): (0.5-2) using ethyl acetate, ethanol and water as developing agents, developing, airing and observing.
Preferably, 20mL to 50mL of water is added into each 3g to 7g of the traditional Chinese medicine composition sample to be detected correspondingly. Preferably, 20 mL-40 mL of water is added into every 5g of the traditional Chinese medicine composition sample to be detected.
Preferably, the time for ultrasonic extraction by adding water is 10 minutes to 30 minutes.
Preferably, 30 mL-70 mL of 40-60% ethanol solution and 30 mL-70 mL of 80-100% ethanol solution are added correspondingly to each 3-7 g of the Chinese medicinal composition sample to be detected. More preferably, 40 mL-60 mL of 45-55% ethanol solution and 4 mL-6 mL of 85-95% ethanol solution are added to each 5g of the sample of the traditional Chinese medicine composition to be detected.
Preferably, the method for preparing the bupleurum root reference medicinal material solution comprises the following steps: taking radix bupleuri as reference material, adding appropriate amount of methanol, ultrasonic extracting, filtering, and concentrating the filtrate to appropriate amount.
More preferably, 15mL to 25mL of methanol is added into each 0.1g to 0.5g of the radix bupleuri reference drug. More preferably, the time of ultrasonic extraction is 5min to 15min. More preferably, the bupleurum root reference medicinal material is correspondingly concentrated to 4 mL-6 mL per 0.1 g-0.5 g.
Preferably, the volume ratio of (10-13): (1-3): (0.5-1.5) ethyl acetate, ethanol and water as developing agents.
Preferably, after drying, spraying 1-3% by mass of aqueous solution of dimethyl formaldehyde and heating until the spots are clearly developed. More preferably, the mass fraction of the sulfuric acid aqueous solution is 30% to 50%.
Optionally, the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains the white mulberry root-bark or not by adopting the thin-layer chromatography comprises the following steps:
adding saturated sodium carbonate solution into the sample of the traditional Chinese medicine composition to be detected for ultrasonic extraction, filtering, taking filtrate, adding hydrochloric acid to adjust the pH value to 1-2, standing and filtering, taking filtrate, adding ethyl acetate for shaking extraction, taking ethyl acetate phase, drying, and adding methanol into residues for dissolving to prepare a sample solution;
preparing a white mulberry root-bark reference medicinal material solution;
respectively sucking the test solution and the radix bupleuri reference solution, dropping on the same polyamide thin layer plate, developing with acetic acid as developing agent, air drying, and observing.
Preferably, 10mL to 50mL of saturated sodium carbonate solution is added to each 0.5g to 5g of the traditional Chinese medicine composition sample to be detected. More preferably, 10mL to 30mL of saturated sodium carbonate solution is added to each 2g of the traditional Chinese medicine composition sample to be detected.
Preferably, the time for adding saturated sodium carbonate solution for ultrasonic extraction is 10 minutes to 30 minutes. More preferably, the time for adding saturated sodium carbonate solution for ultrasonic extraction is 15 minutes to 25 minutes.
Preferably, the standing time is 20 to 50 minutes. More preferably, the time of standing is 25 to 35 minutes.
Preferably, the number of times of adding ethyl acetate and extracting with shaking is 1 to 5 times. Preferably, the number of times of adding ethyl acetate and extracting with shaking is 1 to 3 times.
More preferably, 10mL to 50mL of ethyl acetate is added to each 0.5g to 5g of the traditional Chinese medicine composition sample to be detected in each extraction. More preferably, 15mL to 25mL of ethyl acetate is added to each 2g of the traditional Chinese medicine composition sample to be detected in each extraction.
Preferably, the method for preparing the cortex mori reference drug solution comprises the following steps: taking cortex Mori as reference material, and preparing cortex Mori reference material solution by the above method. More preferably, 10mL to 50mL of saturated sodium carbonate solution is added to 0.5g to 5g of the cortex mori radicis reference drug. 10mL to 50mL of ethyl acetate is added correspondingly.
Optionally, the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains poria cocos by adopting thin-layer chromatography comprises the following steps:
taking a sample of the Chinese medicinal composition to be detected, adding water for ultrasonic extraction, filtering, taking a filtrate, adding water-saturated n-butanol for extraction, taking a n-butanol phase, drying, and adding methanol to dissolve residues to prepare a test solution;
preparing a poria cocos contrast medicinal material solution;
respectively sucking the test solution and the tuckahoe contrast medicinal material solution, dropping the solutions on the same silica gel G or H thin-layer plate, and mixing the solutions according to the volume ratio of (15-25): (3-7): (0.3-0.7) taking toluene, ethyl acetate and formic acid as developing agents, developing, airing and observing.
Preferably, 20mL to 60mL of water is added into each 3g to 7g of the traditional Chinese medicine composition sample to be detected. More preferably, 30mL to 50mL of water is added to each 5g of the Chinese medicinal composition sample to be tested.
Preferably, the time for adding water and carrying out ultrasonic extraction is 20 minutes to 50 minutes. More preferably, the time for ultrasonic extraction with water is 25 to 35 minutes.
Preferably, the number of extractions with water-saturated n-butanol is 2 to 5. More preferably, the number of extractions with water-saturated n-butanol is 2 to 4.
More preferably, 10mL to 30mL of water-saturated n-butanol is correspondingly added into each 3g to 7g of the traditional Chinese medicine composition sample to be detected during each extraction. More preferably, 15 mL-25 mL of water-saturated n-butanol is added into every 5g of the traditional Chinese medicine composition sample to be detected during each extraction.
Preferably, the method for preparing the tuckahoe control medicinal material solution comprises the following steps: taking Poria cocos reference medicinal material, and preparing the Poria cocos reference medicinal material solution according to the method for preparing the test solution. More preferably, 20mL to 60mL of water is added into each 0.5g to 2g of the tuckahoe contrast medicinal material. Correspondingly adding 10 mL-30 mL of water saturated n-butanol.
Preferably, the ratio by volume (15-25): (4-6): toluene, ethyl acetate and formic acid of (0.4-0.6) are used as developing agents.
Preferably, after air drying, the method further comprises the step of spraying a sulfuric acid solution-ethanol (3-5.
Optionally, the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains the codonopsis pilosula or not by adopting the thin-layer chromatography comprises the following steps:
taking a sample of the Chinese medicinal composition to be detected, adding water, carrying out ultrasonic extraction, filtering, taking filtrate, passing the filtrate through an AB-8 type macroporous adsorption resin column, eluting by using an ethanol solution with the volume fraction of 40-60%, discarding an eluent, continuously eluting by using an ethanol solution with the volume fraction of 80-100%, taking the eluent, drying, adding methanol into residues for dissolving, and preparing a sample solution;
preparing radix Codonopsis reference medicinal material solution and radix Codonopsis alkyne glycoside reference substance solution;
respectively sucking the test sample solution, the codonopsis pilosula reference medicinal material solution and the codonopsis pilosula alkyne glycoside reference substance solution, and dropping the solutions on the same silica gel G or H thin layer plate according to the volume ratio of (6-8): (0.5-2): (0.3-0.7) taking n-butanol, glacial acetic acid and water as developing agents, developing, airing and observing.
Preferably, 20mL to 50mL of water is added into each 3g to 7g of the traditional Chinese medicine composition sample to be detected correspondingly. More preferably, 20mL to 40mL of water is added to each 5g of the Chinese medicinal composition sample to be tested.
Preferably, the time for ultrasonic extraction by adding water is 10 minutes to 30 minutes. More preferably, the time for ultrasonic extraction with water is 15 minutes to 25 minutes.
Preferably, 30 mL-70 mL of 40-60% ethanol solution and 30 mL-70 mL of 80-100% ethanol solution are added to each 3-7 g of the Chinese medicinal composition sample to be detected correspondingly. More preferably, 40 mL-60 mL of ethanol solution with volume fraction of 45% -55% is added correspondingly to every 5g of the traditional Chinese medicine composition sample to be detected, and 40 mL-60 mL of ethanol solution with volume fraction of 85% -95% is added correspondingly.
Preferably, the method for preparing the codonopsis pilosula reference medicinal material solution comprises the following steps: taking radix Codonopsis reference medicinal material, and preparing radix Codonopsis reference medicinal material solution according to the above method for preparing test solution. More preferably, 20mL to 50mL of water is added into each 0.5g to 2g of the codonopsis pilosula reference medicine. Correspondingly adding 30 mL-70 mL of ethanol solution with the volume fraction of 40% -60%, and adding 30 mL-70 mL of ethanol solution with the volume fraction of 80% -100%.
Preferably, the solvent for preparing the lobetyolin control solution is methanol. More preferably, the concentration of the lobetyolin control in the control solution is 0.5 mg/mL-2 mg/mL.
Preferably, the volume ratio is (6-8): (0.5-1.5): (0.4-0.6) n-butanol, glacial acetic acid and water as developing agents.
Preferably, after airing, the method further comprises the step of spraying an ethanol solution of sulfuric acid with the mass fraction of 5% -15%, and heating until spots are clearly developed.
Optionally, the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains liquorice and white paeony root by adopting thin-layer chromatography comprises the following steps:
dissolving the Chinese medicinal composition sample in water, extracting with water-saturated n-butanol, collecting n-butanol phase, drying, dissolving the residue in water, and passing through C 18 Eluting with water and methanol, collecting methanol eluate, drying, dissolving the residue in methanol, and making into sample solution;
preparing a liquorice reference medicinal material solution and a paeoniflorin reference substance solution;
respectively sucking the test solution, the liquorice contrast medicinal material solution and the paeoniflorin contrast solution, and dropping the solutions on the same silica gel G or H thin-layer plate according to the volume ratio of (13-17): (0.5-2): (0.5-2): and (1) taking ethyl acetate, formic acid, glacial acetic acid and water as developing agents, developing, airing and observing.
Preferably, 50mL to 150mL of water is added into each 10g to 20g of the traditional Chinese medicine composition sample to be detected correspondingly. More preferably, 90mL to 110mL of water is added to each 15g of the Chinese medicinal composition sample to be tested.
Preferably, the extraction time of the water-saturated n-butanol is 1 to 3 times, and more preferably, 30mL to 50mL of the water-saturated n-butanol is correspondingly added into each 10g to 20g of the sample of the traditional Chinese medicine composition to be detected during each extraction. More preferably, 35 mL-45 mL of water-saturated n-butanol is added into each 15g of the traditional Chinese medicine composition sample to be detected during each extraction.
Preferably, when the test solution is eluted by water, 10mL to 30mL of water is added to each 10g to 20g of the sample of the traditional Chinese medicine composition to be tested. More preferably, when the test solution is eluted by water, 15mL to 25mL of water is added for every 15g of the sample of the traditional Chinese medicine composition to be tested.
Preferably, when methanol is used for elution, 5mL to 15mL of methanol is added to each 10g to 20g of the traditional Chinese medicine composition sample to be detected. More preferably, when methanol is used for elution, 8mL to 12mL of methanol is added for every 15g of the traditional Chinese medicine composition sample to be detected.
Preferably, the residue corresponding to each 10g to 20g of the Chinese medicinal composition sample to be tested is dissolved in 0.5mL to 1.5mL of methanol. More preferably, the residue corresponding to each 15g of the Chinese medicinal composition sample to be tested is dissolved in 0.5 mL-1.5 mL of methanol.
Preferably, the method for preparing the licorice control solution comprises: taking a licorice reference medicinal material, and preparing a licorice reference medicinal material solution according to the method for preparing the test solution. More preferably, 50mL to 150mL of water is added into each 0.1g to 1g of the liquorice control drug. Adding 30-50 mL of water saturated n-butanol. When the liquorice is eluted by water, 10mL to 30mL of water is added to each 0.1g to 1g of liquorice contrast medicine. When methanol is used for elution, 5mL to 15mL of methanol is added into each 0.1g to 1g of the liquorice contrast medicine.
Preferably, the solvent for preparing the paeoniflorin control solution is methanol. More preferably, the concentration of the paeoniflorin control in the control solution is 0.1 mg/mL-1 mg/mL.
Preferably, the volume ratio of (14-16): (0.5-1.5): (0.5-1.5): ethyl acetate, formic acid, glacial acetic acid and water of (1-3) are used as developing agents.
Preferably, after air drying, the method further comprises the steps of spraying an ethanol solution of sulfuric acid with the volume fraction of 5-15%, and heating until spots are clearly developed
Optionally, the method for detecting the content of paeoniflorin in the Chinese medicinal composition sample to be detected by using high performance liquid chromatography comprises the following steps:
preparing a paeoniflorin reference solution;
mixing a sample of the Chinese medicinal composition to be detected with an extraction solvent, carrying out ultrasonic extraction, taking supernatant, passing through a polyamide column, eluting with water, collecting eluent, and preparing a solution of the Chinese medicinal composition sample to be detected;
performing high performance liquid chromatography detection on the paeoniflorin reference solution and the Chinese medicinal composition sample solution to be detected, and calculating the content of paeoniflorin according to the obtained chromatogram;
the chromatographic conditions for detecting by the high performance liquid chromatography comprise: the mobile phase comprises (13-17) by volume: (83-87) and isocratic elution with acetonitrile and potassium dihydrogen phosphate solution.
Preferably, the extraction solvent is water.
Preferably, the dosage of the extraction solvent is 10 mL-50 mL for each 1 g-5 g of the traditional Chinese medicine composition sample to be detected.
Preferably, the time of ultrasonic extraction is 20 min-40 min
Preferably, the concentration of the potassium dihydrogen phosphate in the potassium dihydrogen phosphate solution is 0.01mol/L to 0.1mol/L.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: octadecylsilane chemically bonded silica is used as a filler.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the column temperature is 25-40 ℃.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the detection wavelength is 210-250 nm.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the number of theoretical plates is not less than 3000 calculated according to paeoniflorin peak. More preferably not less than 4000.
Preferably, the solvent of the paeoniflorin control solution is water. More preferably, the concentration of the paeoniflorin control in the paeoniflorin control solution is 0.01 mg/mL-0.1 mg/mL.
Optionally, the method for detecting the content of salvianolic acid B in the Chinese medicinal composition sample to be detected by using the high performance liquid chromatography comprises the following steps:
preparing salvianolic acid B reference solution;
mixing a sample of the Chinese medicinal composition to be detected with an extraction solvent, carrying out ultrasonic extraction, taking an extracting solution, filtering, and preparing a sample solution of the Chinese medicinal composition to be detected;
performing high performance liquid chromatography detection on the salvianolic acid B reference solution and the traditional Chinese medicine composition sample solution to be detected, and calculating the content of salvianolic acid B according to the obtained chromatogram;
the chromatographic conditions of the high performance liquid chromatography detection comprise: the mobile phase comprises the following components in a volume ratio of (18-25): (75-82) and isocratic elution with acetonitrile and formic acid solution.
Preferably, the extraction solvent is a methanol solution. More preferably, the volume fraction of methanol in the methanol solution is 70% to 80%.
Preferably, the dosage of the extraction solvent is 10 mL-50 mL for each 0.1 g-1 g of the traditional Chinese medicine composition sample to be detected.
Preferably, the time of ultrasonic extraction is 20 min-40 min
Preferably, the volume fraction of formic acid in the formic acid solution in the mobile phase is 1% to 2%.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: octadecylsilane chemically bonded silica is used as a filler.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the column temperature is 25-40 ℃.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the detection wavelength is 260-300 nm.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the number of theoretical plates should not be less than 3000 calculated according to salvianolic acid B peak. More preferably not less than 4000.
Preferably, the solvent of the salvianolic acid B control solution is methanol solution. More preferably, the volume fraction of methanol in the methanol solution is 70% to 80%. More preferably, the concentration of the salvianolic acid B control in the salvianolic acid B control solution is 0.05 mg/mL-0.5 mg/mL.
Optionally, the method for detecting the content of astragaloside in the traditional Chinese medicine composition sample to be detected by adopting the high performance liquid chromatography comprises the following steps:
preparing an astragaloside IV reference substance solution;
mixing a sample of the Chinese medicinal composition to be detected with an extraction solvent, heating and refluxing for extraction, taking an extracting solution, drying, dissolving residues in water, shaking and extracting by adding water-saturated n-butanol, taking a n-butanol phase, carrying out alkali washing, removing alkali liquor, drying, and adding the extraction solvent into residues to prepare a sample solution of the Chinese medicinal composition to be detected;
detecting the astragaloside control solution and the traditional Chinese medicine composition sample solution to be detected by high performance liquid chromatography, and calculating the content of astragaloside according to the obtained chromatogram;
the chromatographic conditions for detecting by the high performance liquid chromatography comprise: the mobile phase comprises the following components in a volume ratio of (25-35): (65-75) and carrying out isocratic elution on acetonitrile and water.
Preferably, the extraction solvent is methanol.
Preferably, the dosage of the extraction solvent added into each 5g to 20g of the traditional Chinese medicine composition sample to be detected is 50mL to 200mL. More preferably, the amount of the extraction solvent added is 95 mL-105 mL for every 5 g-20 g of the Chinese medicinal composition sample to be tested.
Preferably, the time for heating reflux extraction is 3h to 5h.
Preferably, the number of times of shaking extraction of water-saturated n-butanol is 3 to 5 times.
Preferably, 30-50 mL of water-saturated n-butanol is added into every 5-20 g of the traditional Chinese medicine composition sample to be detected during shaking extraction.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: octadecylsilane chemically bonded silica is used as a filler.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the number of theoretical plates is not less than 3000 calculated according to astragaloside IV peak by evaporative light scattering detector. More preferably not less than 4000.
More preferably, the evaporative light scattering detector parameters are set as follows: drift tube temperature 105 ℃, carrier gas flow: 2.8L/min; the column temperature is 25-40 ℃.
Preferably, the solvent of the astragaloside control solution is methanol. More preferably, the concentration of the astragaloside IV reference substance in the astragaloside IV reference substance solution is 0.05 mg/mL-0.5 mg/mL.
Optionally, the method for detecting the content of 2,3,5,4' -tetrahydroxystilbene-2-O- β -D glucoside in the to-be-detected traditional Chinese medicine composition sample by using high performance liquid chromatography comprises the following steps:
preparing 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside reference solution;
mixing a sample of the Chinese medicinal composition to be detected with an extraction solvent, carrying out ultrasonic extraction, taking an extracting solution, filtering, and preparing a sample solution of the Chinese medicinal composition to be detected;
performing high performance liquid chromatography detection on the 2,3,5,4 '-tetrahydroxystilbene-2-O-beta-D glucoside reference solution and the traditional Chinese medicine composition sample solution to be detected, and calculating the content of 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside according to the obtained chromatogram;
the chromatographic conditions for detecting by the high performance liquid chromatography comprise: the mobile phase comprises (13-18) by volume: and (82-87) taking acetonitrile and water as mobile phases, and carrying out isocratic elution.
Preferably, the extraction solvent is an ethanol solution. More preferably, the volume fraction of ethanol in the ethanol solution is between 20% and 50%.
Preferably, the dosage of the extraction solvent is 25 mL-100 mL for each 3 g-8 g of the traditional Chinese medicine composition sample to be detected.
Preferably, the time of ultrasonic extraction is 20min to 40min.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: octadecylsilane chemically bonded silica is used as a filler.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the column temperature is 25-40 ℃.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the detection wavelength is 310-330 nm.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the number of theoretical plates is not less than 3000 calculated according to 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside peak. More preferably not less than 4000.
Preferably, the solvent of the 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside peak control solution is an ethanol solution. More preferably, the volume fraction of ethanol in the ethanol solution is between 20% and 50%. More preferably, in the 2,3,5,4 '-tetrahydroxystilbene-2-O-beta-D glucoside peak reference solution, the concentration of the 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside peak reference is 0.5 mg/mL-2.5 mg/mL.
Optionally, the method for detecting the content of matrine in the Chinese medicinal composition sample to be detected by using the high performance liquid chromatography comprises the following steps:
preparing a matrine reference solution;
mixing a sample of the Chinese medicinal composition to be detected, water and ammonia water, performing ultrasonic treatment until the mixture is dissolved, adding a trichloromethane solution for extraction, taking a trichloromethane phase, removing a solvent, dissolving residues in a mobile phase, and filtering to prepare a sample solution of the Chinese medicinal composition to be detected;
performing high performance liquid chromatography detection on the matrine reference substance solution and the Chinese medicinal composition sample solution to be detected, and calculating matrine content according to the obtained chromatogram;
the chromatographic conditions for detecting by the high performance liquid chromatography comprise: the mobile phase comprises acetonitrile, a phosphoric acid solution and triethylamine, wherein the volume ratio of the acetonitrile to the phosphoric acid solution is (18-22): (78-82), adjusting the pH value of the flowing direction to be 8.0 +/-0.1 by triethylamine, and isocratic eluting.
Preferably, the volume fraction of phosphoric acid in the phosphoric acid solution in the mobile phase is 0.05-0.15%.
Preferably, the amount of water added is 10mL to 50mL for each 0.5g to 5g sample of the Chinese medicinal composition to be tested.
Preferably, the dosage of the ammonia water added into each 0.5g to 5g of the traditional Chinese medicine composition sample to be detected is 0.1mL to 0.5mL.
Preferably, the chloroform solution is added for extraction for 2 to 5 times, and the chloroform phases are combined.
Preferably, the dosage of the chloroform is 10mL to 20mL for each 0.5g to 5g sample of the traditional Chinese medicine composition to be detected during each extraction.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: octadecylsilane chemically bonded silica is used as a filler.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the detection wavelength is 210-230 nm.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the column temperature is 25-30 ℃.
Preferably, the chromatographic conditions for the detection by high performance liquid chromatography further comprise: the number of theoretical plates is not less than 4000 calculated according to matrine peak.
Preferably, the solvent of the matrine control solution is a mobile phase. More preferably, the volume fraction of phosphoric acid in the phosphoric acid solution in the mobile phase is 0.05% to 0.15%. More preferably, the concentration of the matrine control in the matrine control solution is 0.05 mg/mL-0.5 mg/mL.
Optionally, in theoretical preparation raw materials of the traditional Chinese medicine composition, the weight ratio of rhubarb, astragalus, white mulberry root-bark, kuh-seng, dangshen, largehead atractylodes rhizome, tuckahoe, prepared fleece-flower root, white paeony root, salvia miltiorrhiza, szechuan lovage rhizome, chrysanthemum, ginger processed pinellia tuber, plantain herb, radix bupleuri and liquorice is (0.5-1.5): (3-5): (2-4): (1-3): (2-4): (4-6): (4-6): (4-6): (2-4): (4-6): (2-4): (1.5-3.5): (1-3): (4-6): (1-2): (0.5-1.5).
Preferably, in the theoretical preparation raw materials of the traditional Chinese medicine composition, the weight ratio of rhubarb, astragalus, white mulberry root-bark, kuh-seng, dangshen, largehead atractylodes rhizome, tuckahoe, prepared fleece-flower root, white paeony root, salvia miltiorrhiza, szechuan lovage rhizome, chrysanthemum, ginger processed pinellia tuber, plantain herb, radix bupleuri and liquorice is 1:4:3:2:3:5:5:5:3:5:3:2.5:2:5:1.5:0.9.
optionally, the theoretical preparation method of the traditional Chinese medicine composition comprises the following steps:
extracting radix et rhizoma Rhei, radix astragali, cortex Mori, radix Sophorae Flavescentis, radix Codonopsis, atractylodis rhizoma, poria, radix Polygoni Multiflori Preparata, radix Paeoniae alba, saviae Miltiorrhizae radix, rhizoma Ligustici Chuanxiong, flos Chrysanthemi, ginger processed pinellia Tuber, herba plantaginis, bupleuri radix and Glycyrrhrizae radix with water, collecting filtrate, adding medicinal adjuvants, and granulating.
Compared with the traditional scheme, the invention has the following beneficial effects:
the invention establishes a quality detection method aiming at a specific traditional Chinese medicine composition through a great deal of research. The detection method has strong specificity, good reproducibility, stability and precision, and can effectively control the quality of the traditional Chinese medicine composition, so that the quality of the traditional Chinese medicine composition provided by the invention is stable, controllable, efficient and safe, and the comprehensive quality control of the traditional Chinese medicine composition is facilitated.
Drawings
FIG. 1 is a chromatogram of TLC identification results of rhubarb and prepared fleece flower root;
FIG. 2 is a chromatogram of TLC identification result of radix astragali;
FIG. 3 is a chromatogram of TLC identification result of Salvia miltiorrhiza;
FIG. 4 is a chart of the TLC identification result of Sophora flavescens ait;
FIG. 5 is a chromatogram of identification result of bupleuri radix by TLC;
FIG. 6 is a spectrum of TLC identification result of cortex Mori;
FIG. 7 is a chromatogram of TLC identification result of Poria cocos;
FIG. 8 is a TLC identification result spectrum of Codonopsis pilosula;
FIG. 9 is TLC identification result spectrum of licorice and white peony root.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Unless otherwise specified, reagents and instruments used in the embodiments of the present invention are all commercially available products.
The solvent of the "solution" in the present invention is water unless otherwise specified. For example, the sulfuric acid solution is an aqueous solution of sulfuric acid and the sodium hydroxide solution is an aqueous solution of sodium hydroxide.
1 reagent
The traditional Chinese medicine composition is uremia clearing granules, is provided by Kang Chenyao industry (inner Mongolia) responsibility limited company, and the preparation method comprises the following steps:
1) According to the weight ratio of rhubarb, astragalus, white mulberry root-bark, lightyellow sophora root, pilose asiabell root, largehead atractylodes rhizome, indian buead, prepared tuber fleeceflower root, white paeony root, danshen root, szechuan lovage rhizome, chrysanthemum, ginger processed pinellia tuber, plantain herb, chinese thorowax root and liquoric root of 1:4:3:2:3:5:5:5:3:5:3:2.5:2:5:1.5:0.9 preparing the above medicinal materials.
2) Extracting the mixture of the above sixteen medicinal materials with water twice, filtering, mixing filtrates, concentrating to obtain fluid extract, and collecting the extract;
3) Taking the above fluid extract, adding appropriate amount of medicinal adjuvants, and spray granulating to obtain granule; drying, sieving to obtain qualified granule, quality testing, and packaging to obtain granule product.
2 detection by thin layer chromatography
2.1 instruments, reagents
The instrument comprises the following steps: mortars, measuring cylinders, round-bottomed flasks, condensers, evaporation pans, chromatography cylinders, etc. were purchased from eastern certified glass instruments ltd, guangzhou, electric mantles (yuanyi yuehua instruments ltd), BS224S analytical balances (beijing siduris instruments ltd), linomat5 semi-automatic spotter (swiss chara), silica G thin layer plates (Qingdao haiyanghua factories ltd), reprostar3 imaging systems (swiss chara), GZX-GFC-01-2-BS ovens (shanghai bosi instruments ltd), TH-II heaters (shanghai kochen biochemistry technologies ltd).
Reagent: methanol, hydrochloric acid, ethyl acetate, trichloromethane, water, petroleum ether (boiling range of 60-90 ℃), sodium hydroxide, sulfuric acid, acetic acid, n-butanol, chloroform and sulfuric acid ethanol test solution.
2.2 reference medicinal materials and reference substances
Reference medicinal materials: rhubarb as a reference drug, radix polygoni multiflori preparata as a reference drug, radix salviae miltiorrhizae as a reference drug, radix bupleuri as a reference drug, cortex mori radicis as a reference drug, poria cocos as a reference drug, radix codonopsis pilosulae as a reference drug and liquorice as a reference drug, which are purchased from the institute for food and drug inspection of China.
Comparison products: rhein, emodin methyl ether, astragaloside IV, protocatechuic aldehyde, matrine, lobetyolin and paeoniflorin, which are purchased from China food and drug testing research institute.
2.3 identification of rhubarb and prepared Polygonum multiflorum
2.3.1 preparation of test solutions
Taking 5g of the traditional Chinese medicine composition under the item 1, adding 30mL of 3mol/L sulfuric acid solution, heating and refluxing for 1 hour, and cooling; then, 30mL of chloroform (chloroform) was added, the mixture was refluxed for 1 hour, cooled, and the chloroform solution was separated and evaporated to dryness, and the residue was dissolved in 1mL of chloroform (chloroform) to prepare a sample solution.
2.3.2 preparation of reference drug solution and reference mixture solution
1g of rhubarb reference medicinal material and 1g of prepared fleece-flower root reference medicinal material are respectively added with 30mL of 3mol/L sulfuric acid solution, heated and refluxed for 1 hour, and cooled; adding chloroform (chloroform) 30mL, heating and refluxing for 1 hr, cooling, collecting chloroform solution, evaporating, and dissolving the residue with chloroform (chloroform) 1mL to obtain radix et rhizoma Rhei reference solution and radix Polygoni Multiflori Preparata reference solution.
Taking appropriate amount of rhein, emodin and physcion reference substances, adding methanol to obtain mixed solution containing 1mg/mL rhein reference substance, 1mg/mL emodin reference substance and 1mg/mL physcion reference substance as reference substance mixed solution.
2.3.3 according to the test of thin-layer chromatography (China pharmacopoeia 2020 edition four-part general rules), respectively absorbing the test solution, the rhubarb reference medicinal material solution, the radix polygoni multiflori preparata reference medicinal material solution and the reference substance mixed solution, dropping the solution on the same silica gel H thin-layer plate, developing by using petroleum ether (boiling point of 30-60 ℃) and ethyl formate and formic acid upper-layer solution as developing agents in a volume ratio of 15. The results are shown in FIG. 1, and 1 to 6 in FIG. 1 are test solution; 7 is radix et rhizoma Rhei reference medicinal solution; 8 is prepared radix Polygoni Multiflori Preparata reference medicinal solution; and 9 is a reference substance mixed solution, wherein A is inspected under UV365nm, and B is inspected under visible light.
2.4 identification of Astragalus
2.4.1 preparation of test solutions
Taking 5g of the traditional Chinese medicine composition under the item 1, adding 20mL of n-butyl alcohol, heating and refluxing for 2 hours, cooling, filtering, washing the filtrate with 1% sodium hydroxide solution by mass fraction for 3 times, 15mL each time, discarding the alkali liquor, washing with water saturated n-butyl alcohol to neutrality, discarding the water layer, evaporating the n-butyl alcohol extract on a water bath, and dissolving the residue with 0.2mL of methanol to obtain a sample solution.
2.4.2 preparation of control solutions
Taking proper amount of astragaloside IV reference substance, adding methanol to make into 1mg/mL solution as reference substance solution.
2.4.3 according to the test of thin-layer chromatography (China pharmacopoeia 2020 edition four ministry of general rules), respectively absorbing the test solution and the reference solution, dropping the test solution and the reference solution on the same silica gel G thin-layer plate, and mixing the two solutions according to the volume ratio of 13:7: and 2, taking the lower layer solution of chloroform (trichloromethane), methanol and water as a developing agent, developing, taking out, airing, spraying an ethanol solution of sulfuric acid with the mass fraction of 10%, heating until spots are clearly developed, and respectively inspecting under visible light and ultraviolet light. The results are shown in FIG. 2, and 1 to 6 in FIG. 2 are test solution; 7 is astragaloside IV reference solution, wherein A is inspected under UV365nm, and B is inspected under visible light.
2.5 identification of Salvia miltiorrhiza
2.5.1 preparation of test solutions
Dissolving 5g of the Chinese medicinal composition under '1' in 50mL of water, filtering, extracting the filtrate with diethyl ether for 2 times, 15mL each time, mixing diethyl ether phases, evaporating to dryness, and dissolving the residue with 2mL of ethanol to obtain a sample solution.
2.5.2 preparation of reference drug solution and reference solution
Decocting Saviae Miltiorrhizae radix with 5g water for 10 min for 2 times, mixing decoctions, concentrating to 10mL fluid extract, dissolving 5mL extract in 50mL water, filtering, extracting the filtrate with diethyl ether for 2 times, 15mL each time, mixing diethyl ether phases, evaporating to dry, and dissolving the residue with 2mL ethanol to obtain control solution.
Taking a proper amount of protocatechualdehyde reference substance, and adding ethanol to prepare a 0.3mg/mL solution as a reference substance solution.
2.5.3 according to the test of thin-layer chromatography (China pharmacopoeia 2020 edition general rules of the four departments), respectively absorbing the test solution, the reference medicinal material solution and the reference solution, dropping the solutions on the same silica gel G thin-layer plate, and mixing the solutions according to the volume ratio of 2:4:4:0.5 of hexane, benzene, ethyl acetate and formic acid are used as developing agents, the developing agents are taken out and dried, ethanol solution of 2, 4-dinitrophenylhydrazine with the mass fraction of 0.1 percent is sprayed, and the mixture is observed under sunlight. The results are shown in FIG. 3, and 1 to 6 in FIG. 3 are test solution; 7 is protocatechuic aldehyde reference substance solution; 8 is the control solution of radix Salviae Miltiorrhizae.
2.6 identification of Sophora flavescens
2.6.1 preparation of test solutions
Taking 5g of the traditional Chinese medicine composition under the item 1, adding 50mL of chloroform (trichloromethane) and 0.3mL of concentrated ammonia solution, mixing uniformly, standing overnight, filtering, evaporating the filtrate to dryness, and dissolving the residue in 0.2mL of chloroform (trichloromethane) to obtain a test solution.
2.6.2 preparation of control solutions
Taking appropriate amount of matrine as reference substance, adding ethanol to make into 0.2mg/mL solution as reference substance solution.
2.6.3 according to thin layer chromatography (China pharmacopoeia 2020 edition four general rules), respectively absorbing the sample solution and the reference solution, dropping on the same silica gel G thin layer plate, and mixing at a volume ratio of 20:20:3:1, developing with toluene, ethyl acetate, methanol and concentrated ammonia solution as developing agent, taking out, air drying, spraying diluted bismuth iodide solution, and inspecting under visible light. The results are shown in FIG. 4, and 1 to 6 in FIG. 4 are test solution; and 7 is matrine reference solution.
2.7 identification of Bupleurum root
2.7.1 preparation of test solutions
Taking 5g of the traditional Chinese medicine composition under the item 1, adding 30mL of water, carrying out ultrasonic extraction for 20 minutes, cooling, filtering, passing the filtrate through an AB-8 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 10 cm), eluting with 50mL of 50% ethanol solution, discarding the eluent, eluting with 50mL of 90% ethanol solution, collecting the eluent, evaporating in a water bath, and dissolving the residue with 1mL of methanol to obtain a sample solution.
2.7.2 preparation of reference drug solution
Taking 0.2g of radix bupleuri as a reference medicinal material, adding a proper amount of methanol, carrying out ultrasonic extraction, filtering, and concentrating the filtrate to a proper amount to serve as a reference medicinal material solution.
2.7.3 according to thin layer chromatography (China pharmacopoeia 2020 edition four general rules), respectively absorbing the test solution and the reference solution, dropping on the same silica gel G thin layer plate, and mixing at a volume ratio of 11:2:1, developing by using ethyl acetate, ethanol and water as developing agents, taking out and airing, spraying a 2 mass percent sulfuric acid aqueous solution of p-dimethylformaldehyde, wherein the mass percent of sulfuric acid in the sulfuric acid aqueous solution is 40%, heating until spots are clearly developed, and respectively inspecting under visible light and ultraviolet light. The results are shown in FIG. 5, and 1 to 3 in FIG. 5 are test solution; 4 is radix bupleuri reference medicinal solution. Wherein, A is inspected under UV365nm, and B is inspected under visible light.
2.8 identification of cortex Mori
2.8.1 preparation of test solutions
Taking 2g of the traditional Chinese medicine composition under the item 1, adding 20mL of saturated sodium carbonate solution, carrying out ultrasonic extraction for 20 minutes, cooling, filtering, adding dilute hydrochloric acid into filtrate to adjust the pH value to 1-2, standing for 30 minutes, filtering, shaking and extracting the filtrate for 2 times by using ethyl acetate, wherein 20mL of ethyl acetate phase is used for each time, combining ethyl acetate phases, evaporating in a water bath to dryness, and dissolving residues by adding 1mL of methanol to obtain a sample solution.
2.8.2 reference drug solution
Taking 2g of cortex mori radicis as a reference medicinal material, adding 20mL of saturated sodium carbonate solution, carrying out ultrasonic extraction for 20 minutes, cooling, filtering, adding dilute hydrochloric acid into filtrate to adjust the pH value to 1-2, standing for 30 minutes, filtering, shaking and extracting the filtrate for 2 times by using ethyl acetate, wherein 20mL of the filtrate is obtained each time, combining ethyl acetate phases, evaporating in a water bath, and dissolving residues by adding 1mL of methanol to prepare a reference medicinal material solution.
2.8.3 according to thin-layer chromatography (China pharmacopoeia 2020 edition four-department general rules), respectively absorbing the test solution and the reference solution, dropping on the same polyamide thin-layer plate, developing with acetic acid as developing agent, taking out, air drying, and inspecting under ultraviolet lamp. The results are shown in FIG. 6, and 1 to 3 in FIG. 6 are test solution; 4-5 is the solution of cortex mori radicis reference medicinal materials.
2.9 identification of Poria
2.9.1 preparation of test solutions
Taking 5g of the traditional Chinese medicine composition under the item '1', adding 40mL of water, carrying out ultrasonic extraction for 30 minutes, cooling, filtering, extracting the filtrate for 3 times with water-saturated n-butanol, 20mL each time, combining the n-butanol phases, evaporating to dryness, and dissolving the residue with 2mL of methanol to obtain a sample solution.
2.9.2 reference drug solution
Collecting Poria contrast medicinal material 1g, adding water 40mL, ultrasonic extracting for 30min, cooling, filtering, extracting filtrate with water saturated n-butanol for 3 times, each time 20mL, mixing n-butanol phases, evaporating to dryness, and dissolving residue with 2mL methanol to obtain contrast medicinal solution.
2.9.3 according to the test of thin-layer chromatography (China pharmacopoeia 2020 edition four ministry of general rules), respectively absorbing the test solution and the reference medicinal material solution, dropping the test solution and the reference medicinal material solution on the same silica gel G thin-layer plate, and mixing the two solutions according to the volume ratio of 20:5:0.5 of toluene, ethyl acetate and formic acid as developing agents, developing, taking out and airing, spraying a sulfuric acid solution-ethanol (4, v/v) mixed solution of vanillin with the mass fraction of 2%, heating until spots are clearly developed, and inspecting in the sunlight. Respectively inspecting under visible light and ultraviolet light. The results are shown in FIG. 7, and 1 to 3 in FIG. 7 are test solution; 4-5 is tuckahoe contrast medicinal solution. Wherein, A is inspected under UV365nm, and B is inspected under visible light.
2.10 identification of Codonopsis pilosula
2.10.1 preparation of test solutions
Taking 5g of the traditional Chinese medicine composition under the item '1', adding 30mL of water, carrying out ultrasonic extraction for 20 minutes, cooling, filtering, passing the filtrate through an AB-8 type macroporous adsorption resin column (the inner diameter is 1.5cm, and the column height is 10 cm), eluting with 50mL of 50% ethanol solution, discarding the eluent, eluting with 50mL of 90% ethanol solution, collecting the eluent, evaporating to dryness in water bath, and dissolving the residue with methanol to obtain a sample solution.
2.10.2 preparation of reference drug solution and reference mixture solution
Taking 1g of radix codonopsitis contrast medicinal material, adding 30mL of water, carrying out ultrasonic extraction for 20 minutes, cooling, filtering, passing the filtrate through an AB-8 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 10 cm), eluting with 50mL of 50% ethanol, discarding the eluent, eluting with 50mL of 90% ethanol, collecting the eluent, evaporating in a water bath to dryness, and dissolving the residue with methanol to prepare a contrast medicinal material solution.
Taking appropriate amount of radix Codonopsis alkynes reference substance, adding methanol to obtain 1mg/mL solution as reference substance solution.
2.10.3 according to thin layer chromatography (China pharmacopoeia 2020 edition four general rules), respectively absorbing the test solution, the reference solution and the reference solution, dropping on the same silica gel G thin layer plate, and mixing the two solutions in a volume ratio of 7:1: developing with 0.5% n-butanol, glacial acetic acid and water as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating until the color of spots is clear, and respectively inspecting under visible light and ultraviolet light. The results are shown in FIG. 8, and 1 to 3 in FIG. 8 are test solution; 4 is radix Codonopsis reference medicinal solution; 5 is radix Codonopsis alkynil reference substance solution.
2.11 differentiation of Licorice and Paeonia lactiflora
2.11.1 preparation of test solutions
Dissolving 15g of the Chinese medicinal composition under '1' in 100mL of water, extracting with water saturated n-butanol for 2 times (40 mL each time), mixing n-butanol phases, evaporating to dryness, dissolving the residue in 3mL of water, adding into the treated C 18 The column (first with a small amount of methanol immersion, then with water washing methanol for use) on, respectively with water 20mL, methanol 10mL elution, methanol eluent collected, evaporation to dryness, the residue with 1mL methanol dissolved, as the sample solution.
2.11.2 preparation of control solutions and control solutions
Dissolving Glycyrrhrizae radix control 0.5g in 100mL of water, extracting with water saturated n-butanol for 2 times (40 mL each time), mixing n-butanol phases, evaporating to dryness, dissolving the residue in 3mL of water, adding into the treated C 18 Eluting with 20mL of water and 10mL of methanol respectively on a small column (first soaking with a small amount of methanol, and then washing with water to remove methanol for later use), collecting methanol eluate, evaporating to dryness, and dissolving the residue with 1mL of methanol to obtain a reference medicinal material solution.
Taking appropriate amount of penoniflorin as control, adding methanol to obtain 0.5mg/mL solution as control solution.
2.11.3 according to the thin-layer chromatography (China pharmacopoeia 2020 edition four-part general rules), the test solution, the reference drug and the reference solution are respectively absorbed and spotted on the same alkaline silica gel G-containing plate (the volume ratio of the silica gel G plate is soaked with 0.3 percent NaOH ethanol solution) according to the volume ratio of 15:1:1:2, developing with ethyl acetate, formic acid, glacial acetic acid and water as developing agents, taking out and airing, spraying an ethanol solution of sulfuric acid with the volume fraction of 10%, heating until spots are clearly developed, and respectively inspecting under visible light and ultraviolet light. The results are shown in FIG. 9, where 1 to 10 in FIG. 9 are test solution; 11 is paeoniflorin control solution; 12 is Glycyrrhrizae radix control solution, A is inspected under UV365nm, and B is inspected under visible light.
3 high performance liquid chromatography detection
3.1 measurement of Paeoniflorin content
3.1.1 instruments, reagents
The instrument comprises the following steps: agilent1260 high performance liquid chromatograph; a DAD detector; a chromatographic column: phenomenex C 18 (250×4.6mm，5μm)；KQ3200DBA numerical control ultrasonic instrument (Chengyi Hua instruments, limited liability company); electric jackets (department city yuehua instruments, llc), BS224S analytical balance (beijing sidolis instruments systems, llc); mortars, graduated cylinders, round-bottomed flasks, condenser tubes, evaporating dishes and the like are all purchased from Dongzhou glass instruments ltd; electric heating jacket (Chengxihua instruments, inc.).
Reagent testing: acetonitrile (chromatographically pure); ultrapure water; formic acid (chromatographic purity), ethanol, methanol, paeoniflorin reference products and traditional Chinese medicine composition samples to be tested (batch numbers: 20140801, 20140802 and 20140803, which are provided by Kang Chenyao company, inner Mongolia responsibility Limited).
3.1.2 preparation of control solution, test solution, negative sample solution
Control solution: drying at 80 deg.C to constant weight of penoniflorin reference substance 10mg, precisely weighing, placing in 50mL measuring flask, adding water to dissolve to constant volume to scale, precisely weighing 5mL, placing in 25mL measuring flask, adding water to scale, and shaking to obtain reference substance solution.
Test solution: precisely weighing 3g of the traditional Chinese medicine composition under the item 1, placing the traditional Chinese medicine composition in a conical flask with a plug, precisely adding 25mL of water, shaking up, carrying out ultrasonic treatment for 30min (power 120W and frequency 40 kHz), cooling, weighing again, complementing the reduced weight with water, shaking up, filtering, precisely absorbing 5mL of supernatant, adding the supernatant onto a polyamide column (30-60 meshes, 1cm multiplied by 20 cm), eluting with water, accurately collecting 25mL of eluent, and shaking up to obtain the traditional Chinese medicine composition.
Negative sample solution: taking the medicinal materials with the prescription amount without the white paeony root, and preparing a negative sample solution according to the preparation process of the traditional Chinese medicine composition under the item 1 and the preparation method of the test solution under the item 3.1.2.
3.1.3 chromatographic conditions
A chromatographic column: phenomenex C 18 (250×4.6mm，5μm)
Mobile phase: acetonitrile: 0.05mol/L potassium dihydrogen phosphate solution =15 (v/v), isocratic elution
Detection wavelength: 230nm
Flow rate: 1.0mL/min
Column temperature: 25 deg.C
Sample introduction amount: 10 μ L
3.1.4 examination of method for preparing test solution
Examination of 3.1.4.1 solvent dosage
Taking 3g of the traditional Chinese medicine composition under the item '1', precisely weighing, and preparing the test solution according to the preparation method of the test solution under the item '3.1.2' under the conditions except that the dosage of water is 20mL,25mL and 30mL respectively. The obtained test solution and the reference solution under item "3.1.2" are respectively injected into a high performance liquid chromatograph under the chromatographic condition under item "3.1.3" for determination, and the content of paeoniflorin is calculated according to the obtained chromatogram, with the results shown in Table 1.
TABLE 1
The results show that: when 25mL of water is extracted, the content of paeoniflorin is the highest, so 25mL of water is selected as the dosage of the extraction solvent.
Examination of 3.1.4.2 ultrasound time
Taking 3g of the Chinese medicinal composition under the item "1", precisely weighing, and preparing the test solution under the conditions of 20min, 30min and 40min except for ultrasonic time, according to the preparation method of the test solution under the item "3.1.2". The obtained test solution and the reference solution under item "3.1.2" are respectively injected into a high performance liquid chromatograph under the chromatographic condition under item "3.1.3" for determination, and the content of paeoniflorin is calculated according to the obtained chromatogram, with the results shown in Table 2.
TABLE 2
The results show that: when the ultrasonic extraction is carried out for 30min, the content of paeoniflorin is the highest, so that 30min is selected as the ultrasonic extraction time.
3.1.4.3 confirmation of preparation method of test sample solution
Taking 3g of the product, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of water, shaking up, carrying out ultrasonic treatment for 30min (power 120W and frequency 40 kHz), cooling, weighing again, supplementing the reduced weight with water, shaking up, filtering, precisely absorbing 5mL of supernatant, adding the supernatant onto a polyamide column (30-60 meshes, 1cm multiplied by 20 cm), eluting with water, accurately collecting 25mL of eluent, and shaking up to obtain a test solution.
3.1.5 examination of chromatographic conditions
Determination of 3.1.5.1 wavelength
According to the paeoniflorin content measuring method in the first part of China pharmacopoeia 2020 edition, the detection wavelength of the paeoniflorin is determined to be 230nm.
5363 investigation of a column 3.1.5.2
Taking 3g of the traditional Chinese medicine composition under the item '1', precisely weighing, preparing a sample solution according to a sample solution preparation method under the item '3.1.4.3', feeding the obtained sample solution into a high performance liquid chromatograph for determination, wherein except chromatographic columns of Phenomenex, agilent and Altima (the specification is shown in table 3), other conditions are carried out according to chromatographic conditions under the item '3.1.3', and the result is shown in table 3 by observing the separation degree and the number of theoretical plates according to the obtained chromatogram.
TABLE 3
The results show that: phenomenex chromatographic column has the best separation effect.
Examination of 3.1.5.3 Mobile phase
Taking 3g of the traditional Chinese medicine composition under the item '1', precisely weighing, preparing a sample solution according to a sample solution preparation method under the item '3.1.4.3', and feeding the obtained sample solution into a high performance liquid chromatograph for measurement, wherein the method is carried out under the chromatographic conditions under the item '3.1.3' except that mobile phases are acetonitrile-0.05 mol/L potassium dihydrogen phosphate solution with the volume ratio of 15 to 85, acetonitrile-0.05 mol/L potassium dihydrogen phosphate solution with the volume ratio of 14 to 86 and acetonitrile-0.05 mol/L potassium dihydrogen phosphate solution with the volume ratio of 16.
The results show that: acetonitrile at a volume ratio of 15.
Examination of column temperature 3.1.5.4
Taking 3g of the traditional Chinese medicine composition under the item '1', precisely weighing, preparing a sample solution according to a sample solution preparation method under the item '3.1.4.3', injecting the obtained sample solution into a high performance liquid chromatograph for determination, wherein the other conditions are carried out according to chromatographic conditions under the item '3.1.3' except that the column temperature is respectively 25 ℃, 30 ℃ and 35 ℃, and the separation degree is observed according to the obtained chromatogram.
The results show that: the column temperature has no influence on the separation degree, and the column temperature is selected to be 25 ℃ in the method.
8978 confirmation of Zxft 8978 chromatographic conditions
A chromatographic column: phenomenex C 18 (250×4.6mm，5μm)
Mobile phase: acetonitrile: 0.05mol/L potassium dihydrogen phosphate solution =15 (v/v), isocratic elution
Detection wavelength: 230nm
Flow rate: 1.0mL/min
Column temperature: 25 deg.C
Sample introduction amount: 10 μ L
3.1.6 systematic Adaptation studies and methodological validation
3.1.6.1 System adaptability study
And (3) chromatographic separation: under the item of 3.1.5.5, the chromatographic peak retention time of paeoniflorin is about 12min, the separation from other peaks is good, the separation degree is more than 1.5, the symmetry is 0.98, and the standard is met.
Theoretical plate number: according to the formula n =5.54 (t) R /W h/2 ) The theoretical plate number of paeoniflorin was calculated to be 13744, taking into account different chromatographic column conditions: the column length, the carrier performance, the filling condition, the mobile phase, the service time and the like, and the theoretical plate number of the tentative paeoniflorin chromatographic peak is not less than 4000.
3.1.6.2 specificity test
10 mu L of each of an extraction solvent (a blank water solvent), a reference substance solution under the item of '3.1.2', a test substance solution under the item of '3.1.4.3' and a negative sample solution under the item of '3.1.2' is respectively taken, and sample introduction is carried out in a high performance liquid chromatograph for determination according to the chromatographic conditions under the item of '3.1.5.5'.
The results show that: negative without interference.
3.1.6.3 Linear Range inspection
Taking a paeoniflorin reference substance, adding water to prepare a proper amount of a paeoniflorin reference substance stock solution, precisely measuring the paeoniflorin reference substance stock solution, and diluting the paeoniflorin reference substance stock solution into reference substance solutions with the concentrations of 101.00, 50.500, 25.250, 12.625 and 6.313 mu g/mL in a double-ratio manner. Introducing the obtained reference solution of each concentration into high performance liquid chromatograph under the chromatography condition of "3.1.5.5" for determination, recording peak area according to the obtained chromatogram, and taking the concentration as abscissa and peak area (A) 1 、A 2 、A 3 ) As an ordinate, a standard curve was plotted, and the results are shown in Table 4, with the regression equation: y =22.549x +56.772 (R) 2 ＝0.9999)。
TABLE 4
The results showed that the linear relationship was good in the range of 0.06313 to 1.01000. Mu.g.
3.1.6.4 precision test
Taking a paeoniflorin reference solution with the concentration of 40.400 mug/mL, continuously feeding the paeoniflorin reference solution into a high performance liquid chromatograph for determination for 5 times according to the chromatographic condition under the item of 3.1.5.5, recording peak areas according to the obtained chromatogram, calculating the content of the paeoniflorin, and calculating RSD, wherein the results are shown in Table 5.
TABLE 5
The result shows that the method has good precision.
3.1.6.5 stability test
Taking 3g of the traditional Chinese medicine composition under the item '1', precisely weighing, preparing a sample solution according to a preparation method of the sample solution under the item '3.1.4.3', injecting samples into a high performance liquid chromatograph for determination at 0, 2, 4, 8, 24, 48 and 96 hours according to the chromatographic condition under the item '3.1.5.5', recording peak area according to the obtained chromatogram, calculating the content of paeoniflorin, and calculating RSD, wherein the result is shown in Table 6.
TABLE 6
The results show that the method is stable within 96 hours.
3.1.6.6 repeatability test
Taking 3g of the traditional Chinese medicine composition under the item "1", precisely weighing, preparing 6 parts of the test solution according to the preparation method of the test solution under the item "3.1.4.3", injecting the sample into a high performance liquid chromatograph for determination according to the chromatographic condition under the item "3.1.5.5", recording the peak area according to the obtained chromatogram, calculating the content of paeoniflorin, calculating RSD, and obtaining the result shown in Table 7.
TABLE 7
The results show that the method has good repeatability.
3.1.6.7 sample application and recovery test
Taking 1.5g of the traditional Chinese medicine composition under the item '1', paralleling 6 parts, precisely weighing, precisely adding 1mL of paeoniflorin reference substance with the concentration of 0.4040mg/mL respectively, volatilizing, preparing 6 parts of test solution according to the preparation method of the test solution under the item '3.1.4.3', injecting sample into a high performance liquid chromatograph under the chromatographic condition of the item '3.1.5.5', measuring, and calculating the content, the recovery rate and the RSD value of the paeoniflorin according to the obtained chromatogram, wherein the results are shown in Table 8.
TABLE 8
3.1.7 determination of the content of three batches of samples
Taking 3 batches (batch number shown in table 9) of Chinese medicinal composition samples to be detected, 3g, precisely weighing and paralleling 2 batches, preparing a Chinese medicinal composition sample solution to be detected according to a preparation method of a test solution under the item 3.1.4.3, respectively injecting the obtained Chinese medicinal composition sample solution to be detected and a reference solution under the item 3.1.2 into a high performance liquid chromatograph under the chromatographic condition of 3.1.5.5 for determination, and calculating the content of paeoniflorin according to the obtained chromatogram, wherein the results are shown in table 9.
TABLE 9
The results show that the content of paeoniflorin in the three samples is more than 0.7mg/g.
3.2 determination of Salvianolic acid B content
3.2.1 instruments, reagents
The instrument comprises: agilent1260 high performance liquid chromatograph; a DAD detector; a chromatographic column: agilent extended-C 18 (250X 4.6mm,5 μm); KQ3200DB type digital control ultrasonic instrument (gu yi shi hua instrument, llc); electric jackets (Chengxihua instruments, inc., of Ouchi city), BS224S analytical balance (Beijing Saedodus instruments systems, inc.); mortars, graduated cylinders, round-bottomed flasks, condenser tubes, evaporating dishes and the like are all purchased from Dongzhou glass instruments ltd; electric heating jackets (Steve City Zealand Prov instruments, inc.).
Reagent testing: acetonitrile (chromatographically pure); ultrapure water; formic acid (chromatographic purity), ethanol, methanol, salvianolic acid B reference products and traditional Chinese medicine composition samples to be tested (batch numbers: 20140801, 20140802 and 20140803, which are provided by Kang Chenyao industry (inner Mongolia) responsibility Co., ltd.).
3.2.2 preparation of control solution, test solution, negative sample solution
Control solution: precisely weighing 10.6mg of salvianolic acid B reference substance in a 25mL volumetric flask, adding 75% methanol solution in volume fraction to constant volume to scale, preparing a reference substance reserve solution with the concentration of 0.424mg/mL, and taking 3mL to constant volume to 10mL to obtain a reference substance solution with the concentration of 0.1272 mg/mL.
Test solution: taking a proper amount of the traditional Chinese medicine composition under the item 1, grinding the mixture by using a mortar, sieving the ground mixture, precisely weighing 0.5g of the ground mixture in a 25mL volumetric flask, adding 75% of methanol solution in volume fraction to a constant volume to reach a scale, carrying out ultrasonic treatment for 30min, taking out the mixture, cooling the mixture, complementing the reduced amount by using 75% of methanol solution in volume fraction, and filtering the mixture by using a 0.45-micrometer filter membrane to obtain a test solution.
Negative sample solution: taking the medicinal materials with the prescription amount without the salvia miltiorrhiza, and preparing a negative sample solution according to the preparation process of the traditional Chinese medicine composition under the item 1 and the preparation method of the test solution under the item 3.2.2.
3.2.3 chromatographic conditions
A chromatographic column: agilent extended-C 18 (250×4.6mm，5μm)
Mobile phase: acetonitrile-formic acid solution with volume fraction of 1.5% =21 (v/v), isocratic elution
Detection wavelength: 286nm
Flow rate: 1.0mL/min
Column temperature: 30 deg.C
Sample introduction amount: 10 μ L
3.2.4 examination of chromatographic conditions
3.2.4.1 determination of wavelength
According to the method for measuring the content of salvianolic acid B in the first part of the China pharmacopoeia 2020 edition, the detection wavelength of the salvianolic acid B is determined to be 286nm.
3.2.4.2 investigation of the column
Taking a proper amount of the traditional Chinese medicine composition under the item '1', preparing a sample solution according to a sample solution preparation method under the item '3.2.2', feeding the obtained sample solution into a high performance liquid chromatograph for determination, wherein except for Phenomenex, agilent and Alltima (specifications shown in table 10) of chromatographic columns, other conditions are carried out according to chromatographic conditions under the item '3.2.3', and the separation degree and the number of theoretical plates are observed according to the obtained chromatogram, and the results are shown in table 10.
The results show that: the Agilent chromatographic column has the best separation effect.
Examination of 3.2.4.3 Mobile phase
Taking a proper amount of the traditional Chinese medicine composition under the item '1', preparing a test solution according to a test solution preparation method under the item '3.2.2', injecting the obtained test solution into a high performance liquid chromatograph for measurement, wherein the other conditions are carried out according to chromatographic conditions under the item '3.2.3' except that mobile phases are respectively 21% acetonitrile-formic acid solution with volume fraction of 1.5%, 20% acetonitrile-formic acid solution with volume fraction of 1.5%, and 22% acetonitrile-formic acid solution with volume fraction of 78, and the separation degree is observed according to the obtained chromatogram.
The results show that: acetonitrile at a volume ratio of 21.
Examination of column temperature 3.2.4.4
Taking a proper amount of the traditional Chinese medicine composition under the item '1', preparing a sample solution according to a sample solution preparation method under the item '3.2.2', injecting the obtained sample solution into a high performance liquid chromatograph for determination, wherein the other conditions are carried out according to chromatographic conditions under the item '3.2.3' except that the column temperature is respectively 25 ℃, 30 ℃ and 35 ℃, and the separation degree is observed according to the obtained chromatogram.
The results show that: the column temperature has no influence on the separation degree, and the column temperature is selected to be 30 ℃ in the method.
8978 confirmation of chromatographic conditions for zxft 8978
And (3) chromatographic column: agilent extended-C 18 (250×4.6mm，5μm)
Mobile phase: acetonitrile-formic acid solution with volume fraction of 1.5% =21 (v/v), isocratic elution
Detection wavelength: 286nm
Flow rate: 1.0mL/min
Column temperature: 30 deg.C
Sample introduction amount: 10 μ L
3.2.5 System Adaptation study and methodological validation
3.2.5.1 System Adaptation study
And (3) chromatographic separation: under the '3.2.4.5' term, the chromatographic peak retention time of the salvianolic acid B is about 16min, the separation from other peaks is good, the separation degree is more than 1.5, the symmetry is 0.99, and the standard is met.
Theoretical plate number: according to the formula n =5.54 (t) R /W h/2 ) The theoretical plate number of the salvianolic acid B peak was calculated to be 12557, taking into account different chromatographic column conditions: the column length, the carrier performance, the filling condition, the mobile phase, the service time and the like, and the theoretical plate number of the tentative salvianolic acid B chromatographic peak is not less than 4000.
3.2.5.2 specificity test
10 μ L of each of the extraction solvent, the control solution under the item "3.2.2", the test solution under the item "3.2.2", and the negative sample solution under the item "3.2.2" was sampled into a high performance liquid chromatograph under the chromatographic conditions of "3.2.4.5" for measurement.
The results show that: and negative without interference.
3.2.5.3 Linear Range inspection
Accurately weighing 72.4mg of salvianolic acid B reference substance, adding an appropriate amount of methanol, dissolving in a 50mL volumetric flask, and performing ultrasonic treatment for 1h to obtain a concentration of 0.144mg/mL. Respectively sucking 5, 10, 15, 20, 25 and 30 mu L of the sample, injecting the sample into a high performance liquid chromatograph for measurement according to the chromatographic conditions under the item of 3.2.4.5, recording peak areas according to the obtained chromatogram, drawing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, and obtaining the result shown in the table 11 and the regression equation as follows: y =989225x +7436.1 (R) 2 ＝0.9999)。
TABLE 11
As a result, the linear relationship was good in the range of 0.72 to 4.32. Mu.g.
3.2.5.4 precision test
Taking a salvianolic acid B reference solution with the concentration of 0.1272mg/mL, continuously feeding the sample into a high performance liquid chromatograph according to the chromatographic condition under the item of 3.2.4.5, determining for 5 times, recording peak areas according to the obtained chromatogram, calculating the content of salvianolic acid B, and calculating RSD, wherein the RSD is 0.31%.
The results show that the method is accurate.
3.2.5.5 stability test
Taking a proper amount of the traditional Chinese medicine composition under the item '1', preparing a test solution according to a preparation method of the test solution under the item '3.2.2', injecting samples into a high performance liquid chromatograph for measurement at 0, 2, 4, 8 and 24 hours according to the chromatographic conditions under the item '3.2.4.5', recording peak areas according to the obtained chromatogram, calculating the content of salvianolic acid B, calculating RSD, and obtaining the result shown in the table 12.
TABLE 12
The results show that the process is stable within 24 hours.
3.2.5.6 repeatability test
Taking a proper amount of the traditional Chinese medicine composition under the item '1', preparing 6 parts of the test solution according to the preparation method of the test solution under the item '3.2.2', injecting the test solution into a high performance liquid chromatograph for determination according to the chromatographic conditions under the item '3.2.4.5', recording the peak area according to the obtained chromatogram, calculating the content of salvianolic acid B, and calculating RSD, wherein the results are shown in Table 13.
Watch 13
The results show that the method has good repeatability.
3.2.5.7 sample application and recovery test
Taking 0.5g of the traditional Chinese medicine composition under the item '1', paralleling 6 parts, precisely weighing, precisely adding 2, 4, 8 and 8mL of reference substance solution respectively, adding 75% methanol solution to scale, carrying out ultrasonic treatment for 30min, taking out, standing and cooling, adding 75% methanol solution to complement lost volume, taking 1mL, filtering with a 0.45-micrometer microporous filter head, preparing 6 parts of test substance solution, injecting into a high performance liquid chromatograph for measurement according to the chromatographic condition under the item '3.2.4.5', recording peak area according to the obtained chromatogram, and calculating the content, recovery rate and RSD value of salvianolic acid B. The results are shown in Table 14.
TABLE 14
3.2.6 determination of the content of three batches of samples
Taking 0.5g of 3 batches (batch numbers are shown in table 15) of traditional Chinese medicine composition samples to be detected, precisely weighing and paralleling 2 batches, preparing a traditional Chinese medicine composition sample solution to be detected according to the preparation method of the test solution under the item 3.2.2, injecting sample into a high performance liquid chromatograph under the chromatographic condition under the item 3.2.4.5, measuring, and calculating the content of salvianolic acid B according to the obtained chromatogram, wherein the result is shown in table 15.
Watch 15
The results show that the salvianolic acid B content in the three samples is more than 0.25mg/g.
3.3 determination of Astragaloside IV content
3.3.1 instruments, reagents
The instrument comprises the following steps: agilent1260 high performance liquid chromatograph; evaporative light detector (ALLTEACH, 2000 ES); and (3) chromatographic column: thermo ODS-2Hypersil (150 mm. Times.4.6 mm,5 μm); mortars, graduated cylinders, round-bottomed flasks, condenser tubes, evaporating dishes and the like are all purchased from Dongzhou glass instruments ltd; electric heating jackets (Chengyi Yunhua instruments, limited liability company); electric mantles (department of Instrument, inc., yuanhua, ouchi), BS224S analytical balance (Instrument systems, inc., sadoris, beijing).
Reagent testing: acetonitrile (chromatographically pure); ultrapure water; n-butanol; methanol; ammonia water; astragaloside IV reference substance, chinese medicinal composition sample to be tested (batch number: 20140801, 20140802, 20140803, 20051211, all provided by Kang Chenyao (inner Mongolia) responsibility Co., ltd.).
3.3.2 preparation of control solution, test solution, negative sample solution
Control solution: accurately weighing appropriate amount of astragaloside IV reference substance, adding methanol to obtain solution containing astragaloside IV 0.15mg per 1mL to obtain reference substance solution.
Test solution: grinding a proper amount of the traditional Chinese medicine composition under the item 1, precisely weighing about 10g of the traditional Chinese medicine composition, placing the mixture in a Soxhlet extractor, adding 100mL of methanol, heating and refluxing for extraction for 4 hours, recovering a solvent from an extracting solution, concentrating to dryness, adding 20mL of water into residues, slightly heating to dissolve the residues, shaking and extracting for 4 times with water-saturated n-butyl alcohol, 40mL each time, combining n-butyl alcohol phases, fully washing for 2 times with an ammonia test solution, 40mL each time, discarding the ammonia solution, evaporating the n-butyl alcohol phases to dryness, dissolving the residues with methanol, transferring to a 5mL measuring flask, adding methanol to the scale, and shaking uniformly to obtain a sample solution.
Negative sample solution: taking the medicinal materials with the prescription amount without the astragalus root, and preparing a negative sample solution according to the preparation process of the traditional Chinese medicine composition under the item 1 and the preparation method of the test solution under the item 3.3.2.
3.3.3 chromatographic conditions
A chromatographic column: thermo ODS-2Hypersil (150 mm. Times.4.6 mm,5 μm)
Mobile phase: acetonitrile-water solution =30 (v/v), isocratic elution
Detector parameters: n is a radical of 2 The flow rate was 2.8mL/min and the temperature was 105 deg.C
Flow rate: 1mL/min
Column temperature: 30 deg.C
Sample introduction amount: 10 μ L
3.3.4 examination of chromatographic conditions
5363 investigation of a column 3.3.4.1
Referring to the determination of astragaloside IV in the first part of the Chinese pharmacopoeia 2020 edition, octadecylsilane chemically bonded silica is used as a filler in a chromatographic column. Taking a proper amount of the traditional Chinese medicine composition under the item ' 1 ', preparing a sample solution according to the preparation method of the sample solution under the item ' 3.3.2 ', feeding the obtained sample solution into a high performance liquid chromatograph for measurement, wherein the other conditions are carried out according to the chromatographic conditions under the item ' 3.3.3 except that chromatographic columns are Thermo ODS-2Hypersil, agilent and Alltima (the specification is shown in table 16), and the separation degree and the number of theoretical plates are observed according to the obtained chromatogram, and the results are shown in table 16.
TABLE 16
The results show that: the three chromatographic columns all achieve good separation effect, and the method selects a Thermo ODS-2Hypersil chromatographic column.
Examination of 3.3.4.2 Mobile phase
Based on a method for measuring the content of astragaloside IV in the first part of China pharmacopoeia 2020 edition, taking a proper amount of a traditional Chinese medicine composition under the item '1', preparing a test solution according to a test solution preparation method under the item '3.3.2', injecting the obtained test solution into a high performance liquid chromatograph for measurement, wherein the method comprises the following steps of (1) performing the following steps of (1) measuring the content of astragaloside IV in the chromatogram obtained by using the following conditions except that mobile phases are acetonitrile-water with a volume ratio of 30.
The results show that: the difference between the three is not obvious. The method selects a mobile phase of acetonitrile-water with a volume ratio of 30.
Examination of 3.3.4.3 column temperature
Taking a proper amount of the traditional Chinese medicine composition under the item '1', preparing a sample solution according to a sample solution preparation method under the item '3.3.2', injecting the obtained sample solution into a high performance liquid chromatograph for determination, wherein the other conditions are carried out according to chromatographic conditions under the item '3.3.3' except that the column temperature is respectively 25 ℃, 30 ℃ and 40 ℃, and the separation degree is observed according to the obtained chromatogram.
The results show that: the best effect is achieved when the column temperature is 30 ℃, and the column temperature is selected to be 30 ℃.
8978 confirmation of chromatographic conditions for zxft 8978
A chromatographic column: thermo ODS-2Hypersil (150 mm. Times.4.6 mm,5 μm)
Mobile phase: acetonitrile-water solution =30 (v/v), isocratic elution
Detector parameters: n is a radical of 2 The flow rate was 2.8mL/min and the temperature was 105 deg.C
Flow rate: 1mL/min
Column temperature: 30 deg.C
Sample introduction amount: 10 μ L
3.3.5 systems Adaptation Studies and methodological validation
3.3.5.1 System Adaptation study
And (3) chromatographic separation: under the '3.3.4.4' term, the retention time of the chromatographic peak of astragaloside is about 11min, the separation from other chromatographic peaks is good, the separation degree is more than 1.5, the symmetry is 0.97, and the standard is met.
Theoretical plate number: according to the formula n =5.54 (t) R /W h/2 ) The theoretical plate number of the astragaloside IV peak is calculated to be 14294, and is tentatively not less than 4000 in consideration of the difference of different chromatographic column conditions (column length, carrier performance, filling condition, mobile phase proportion, service time and the like).
3.3.5.2 specificity test
10. Mu.L of each of a methanol blank solvent, a control solution under the item "3.3.2", a test solution under the item "3.3.2" and a negative sample solution under the item "3.3.2" was sampled into a high performance liquid chromatograph and measured under the chromatographic conditions under the item "3.3.4.4".
The results show that: negative without interference.
3.3.5.3 Linear Range inspection
Taking the reference substance solution under the item "3.3.2", respectively injecting 4, 6, 10, 20 and 25 μ L, injecting the sample into a high performance liquid chromatograph for determination according to the chromatographic condition under the item "3.3.4.4", recording the peak area according to the obtained chromatogram, drawing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, and obtaining the result shown in the table 17 and the regression equation as follows: y =0.6667x-4.259 (R) 2 ＝0.9986)。
TABLE 17
As a result, the linear relationship was good in the range of 0.63 to 3.9. Mu.g.
3.3.5.4 precision test
Taking the reference solution under the item "3.3.2", continuously feeding the sample into a high performance liquid chromatograph according to the chromatographic conditions under the item "3.3.4.4" to measure for 5 times, recording the peak area according to the obtained chromatogram, calculating the content of astragaloside IV, and calculating RSD, wherein the results are shown in Table 18.
Watch 18
The results show that the method is accurate.
3.3.5.5 stability test
Taking a proper amount of the traditional Chinese medicine composition under the item '1', preparing a test solution according to a preparation method of the test solution under the item '3.3.2', injecting samples into a high performance liquid chromatograph for measurement at 0, 12, 15, 18 and 30 hours according to the chromatographic conditions under the item '3.3.4.4', recording peak areas according to the obtained chromatogram, calculating the content of astragaloside, calculating RSD, and obtaining the result shown in the table 19.
Watch 19
The results show that the process is stable within 30 hours.
3.3.5.6 repeatability test
Taking a proper amount of the traditional Chinese medicine composition under the item '1', preparing 6 parts of test solution according to the preparation method of the test solution under the item '3.3.2', injecting the test solution into a high performance liquid chromatograph for determination according to the chromatographic conditions under the item '3.3.4.4', recording the peak area according to the obtained chromatogram, calculating the content of astragaloside IV, and calculating RSD, wherein the results are shown in a table 20.
Watch 20
The results show that the method has good repeatability.
3.3.5.7 sample application and recovery test
Taking 6 parts of the traditional Chinese medicine composition (batch number 20051211), each part of 6 parts of the traditional Chinese medicine composition is about 0.5g, precisely weighing, respectively adding 5mL of an astragaloside IV reference substance solution (0.1569 mg/mL), preparing 6 parts of test substance solution according to the preparation method of the test substance solution under the item '3.3.2', injecting the prepared test substance solution into a high performance liquid chromatograph for determination according to the chromatographic condition under the item '3.3.4.4', recording peak areas according to the obtained chromatogram, and calculating the content, recovery rate and RSD value of the astragaloside IV. The results are shown in Table 21.
TABLE 21
The results show that: the recovery rate is between 94 and 103 percent, and the RSD is less than or equal to 3 percent.
3.3.6 determination of the content of three batches of samples
Taking 10g of each of 3 batches (batch number shown in table 22) of Chinese medicinal composition samples to be detected, precisely weighing and paralleling 2 parts, preparing a Chinese medicinal composition sample solution to be detected according to a preparation method of a test solution under the item 3.3.2, injecting a sample into a high performance liquid chromatograph for determination according to a chromatographic condition under the item 3.3.4.4, and calculating the content of astragaloside according to the obtained chromatogram, wherein the result is shown in table 22.
TABLE 22
The results show that the content of astragaloside in the three samples is more than 0.1mg/g.
3.4 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside content determination
3.4.1 instruments, reagents
The instrument comprises the following steps: waters2695/2487 high performance liquid chromatograph, TU-1901 ultraviolet spectrophotometer; a chromatographic column: phenomenex C 18 (250X 4.6mm,5 μm); KQ3200DB type digital control ultrasonic instrument (gu yi shi hua instrument, llc); electric jackets (Chengxihua instruments, inc., of Ouchi city), BS224S analytical balance (Beijing Saedodus instruments systems, inc.); mortars, graduated cylinders, round-bottomed flasks, condenser tubes, evaporating dishes and the like are all purchased from Dongzhou glass instruments ltd; electric heating jacket (Chengxihua instruments, inc.).
Reagent testing: acetonitrile (chromatographically pure); ultrapure water; formic acid (chromatographically pure), ethanol, methanol, 2,3,5,4' -tetrahydroxystilbene-2-O- β -D glucoside control, samples (lot nos. 20140801, 20140802, 20140803, all provided by Kang Chenyao company, inner mongolia responsibility ltd).
3.4.2 preparation of control solution, test solution and negative sample solution
Control solution: weighing a proper amount of 2,3,5,4 '-tetrahydroxystilbene-2-O-beta-D glucoside reference substance, adding diluted ethanol (30% ethanol solution by volume fraction) to fix the volume to a 20mL measuring flask, performing ultrasonic treatment to completely dissolve the diluted solution to prepare 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside reference substance stock solution (the concentration is 0.6000 mg/mL), and diluting to prepare a reference substance solution.
Test solution: precisely weighing 6g of the Chinese medicinal composition under item 1, placing in a conical flask with a stopper, precisely adding diluted ethanol, sealing the stopper, weighing, treating with ultrasound for 30min, cooling, weighing again, supplementing the reduced weight with diluted ethanol, shaking, and filtering to obtain the test solution.
Negative sample solution: taking the medicinal materials with the prescription amount without the prepared fleece-flower root, and preparing a negative sample solution according to the preparation process of the traditional Chinese medicine composition under the item 1 and the preparation method of the test solution under the item 3.4.2.
3.4.3 chromatographic conditions
A chromatographic column: dikma C 18 (200×4.6mm，5μm)
Mobile phase: acetonitrile-water =15 (v/v), isocratic elution
Detection wavelength: 320nm
Flow rate: 1.0mL/min
Column temperature: 25 deg.C
Sample introduction amount: 10 μ L
3.4.4 examination of method for preparing test solution
Examination of 3.4.4.1 extraction solvent
Taking 6g of the Chinese medicinal preparation under the item "1", precisely weighing, and preparing the test solution under the conditions of the preparation method of the test solution under the item "3.4.2" except that the extraction solvents are respectively diluted ethanol, ethanol solution with volume fraction of 70% and methanol. The obtained sample solution and the control solution under the item "3.4.2" were respectively injected into a high performance liquid chromatograph under the chromatographic condition under the item "3.4.3" for measurement, and the content of 2,3,5,4' -tetrahydroxystilbene-2-O- β -D glucoside was calculated from the obtained chromatogram, with the results shown in table 23.
TABLE 23
The results show that: the difference between the dilute ethanol and the ethanol solution with the volume fraction of 70 percent is not great, and the extraction solvent is selected to be the dilute ethanol in consideration of the cost.
Examination of extraction time 3.4.4.2
Taking 6g of the traditional Chinese medicine composition under the item '1', precisely weighing, and preparing the test solution under the conditions of 15min, 30min and 60min of ultrasonic extraction and the preparation method of the test solution under the item '3.4.2'. The obtained sample solution and the reference solution under the item "3.4.2" were respectively injected into a high performance liquid chromatograph under the chromatographic condition under the item "3.4.3" for measurement, and the content of 2,3,5,4' -tetrahydroxystilbene-2-O- β -D glucoside was calculated from the obtained chromatogram, with the results shown in table 24.
TABLE 24
The results show that: the extraction efficiency of ultrasonic extraction for 30min is the maximum, so the time of ultrasonic extraction is selected to be 30min.
8978 confirmation of preparation method of test solution zxft 8978
Weighing 6g of the product, precisely weighing, placing in a conical flask with a stopper, precisely adding diluted ethanol, sealing the stopper, weighing, treating with ultrasound for 30min, cooling, weighing again, supplementing the reduced weight with diluted ethanol, shaking, and filtering to obtain the test solution.
3.4.5 examination of chromatographic conditions
Determination of 3.4.5.1 wavelength
Taking 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside reference substance about 10mg, adding diluted ethanol to fix the volume to 20mL, precisely absorbing 2mL, adding diluted ethanol to fix the volume to 10mL, and scanning and measuring at 210-450 nm.
The results show that: 2,3,5,4' -tetrahydroxystilbene-2-O- β -D glucoside has a maximum absorption at 320 nm.
Examination of 3.4.5.2 column
Taking 6g of the traditional Chinese medicine composition under the item "1", precisely weighing, preparing the test solution according to the preparation method of the test solution under the item "3.4.4.3", introducing the obtained test solution into a high performance liquid chromatograph for determination, wherein the other conditions are carried out according to the chromatographic conditions under the item "3.4.3" except that chromatographic columns are Dikma, agilent and Kromil (the specifications are shown in table 25), and the results are shown in table 25 by observing the separation degree and the theoretical plate number according to the obtained chromatogram.
TABLE 25
The results show that: the Dikma chromatographic column has the best separation effect.
Examination of 3.4.5.3 Mobile phase
Taking 6g of the traditional Chinese medicine composition under the item '1', precisely weighing, preparing a sample solution according to a sample solution preparation method under the item '3.4.4.3', and injecting the obtained sample solution into a high performance liquid chromatograph for determination, wherein the method is carried out under the chromatographic conditions under the item '3.4.3' except that mobile phases are acetonitrile-water with a volume ratio of 15.
The results show that: finally, acetonitrile-water with a mobile phase volume ratio of 15.
Examination of column temperature 3.4.5.4
In the research, different column temperatures have certain influence on the separation degree of the component to be detected and the impurity peak. Taking 6g of the traditional Chinese medicine composition under the item '1', precisely weighing, preparing a sample solution according to a sample solution preparation method under the item '3.4.4.3', injecting the obtained sample solution into a high performance liquid chromatograph for determination, wherein the other conditions are carried out according to chromatographic conditions under the item '3.4.3' except that the column temperature is respectively 25 ℃, 30 ℃ and 40 ℃, and observing the separation degree according to the obtained chromatogram.
The results show that: in general, as the column temperature increased, 2,3,5,4' -tetrahydroxystilbene-2-O- β -D glucoside decreased in separation from the impurity peak. In actual test, the proper column temperature is selected within the range of 25-40 ℃ according to the separation condition of the sample. The column temperature of the method is selected to be 25 ℃.
8978 confirmation of chromatographic conditions for zxft 8978
A chromatographic column: dikma C 18 (200×4.6mm，5μm)
Mobile phase: acetonitrile-water =15 (v/v), isocratic elution
Detection wavelength: 320nm
Flow rate: 1.0mL/min
Column temperature: 25 deg.C
Sample introduction amount: 10 μ L
3.4.6 systematic Adaptation study and methodological validation
3.4.6.1 systematic Adaptation study
And (3) chromatographic separation: under the term of 3.4.5.5, the retention time of a chromatographic peak of 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside is about 12min, the separation from other peaks is good, the separation degree is more than 1.5, the symmetry is 0.96, and the standard is met.
Theoretical plate number: by formula n =5.54 (t) R /W h/2 ) The theoretical plate number of 2,3,5,4' -tetrahydroxystilbene-2-O- β -D glucoside was calculated to be 8395, taking into account the different column conditions: the theoretical plate number of 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside chromatographic peak is not less than 4000.
3.4.6.2 specificity test
10 μ L of each of an extraction solvent (blank aqueous solvent), a control solution under the section of "3.4.2", a test solution under the section of "3.4.4.3" and a negative sample solution under the section of "3.4.2" was taken, and introduced into a high performance liquid chromatograph under the chromatographic conditions under the section of "3.4.5.5" for measurement.
The results show that: negative without interference.
3.4.6.3 Linear Range inspection
Taking a reference substance stock solution (with the concentration of 0.6000 mg/mL) under the item of 3.4.2, precisely measuring 0.5, 1, 2,3, 4 and 5mL of the reference substance stock solution, respectively placing the reference substance stock solution into 20mL measuring bottles, adding diluted ethanol to dilute the reference substance stock solution to a scale, shaking the measuring bottles uniformly, respectively and precisely absorbing 10 mu L of the reference substance stock solution, injecting the reference substance stock solution into a high performance liquid chromatograph for measurement according to the chromatographic condition under the item of 3.4.5.5, recording peak areas according to the obtained chromatogram, drawing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, and obtaining a result shown in a table 26, wherein the regression equation is as follows: y =3528919.5251x +16945.7014
Watch 26
The results showed that the linear relationship was good in the range of 0.1500 to 1.5000. Mu.g.
3.4.6.4 precision test
2,3,5,4 '-tetrahydroxystilbene-2-O-beta-D glucoside reference solution with the concentration of 90.000 mug/mL is taken, sample introduction is continuously carried out for 5 times in a high performance liquid chromatograph according to the chromatographic condition under the item of 3.4.5.5, peak areas are recorded according to the obtained chromatogram, the content of 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside is calculated, and RSD is calculated, and the result is shown in Table 27.
Watch 27
The result shows that the method has good precision.
3.4.6.5 stability test
Taking 6g of the traditional Chinese medicine composition under the item ' 1 ', precisely weighing, preparing a test solution according to a preparation method of the test solution under the item ' 3.4.4.3 ', injecting samples into a high performance liquid chromatograph for determination at 0, 1, 2, 4, 8, 16 and 24 hours according to the chromatographic condition under the item ' 3.4.5.5 ', recording peak areas according to the obtained chromatogram, calculating 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside content, and calculating RSD, wherein the results are shown in Table 28.
Watch 28
The results show that the process is stable within 24 hours.
3.4.6.6 repeatability test
Taking 6g of the traditional Chinese medicine composition under the item ' 1 ', precisely weighing, preparing 6 parts of test solution according to the preparation method of the test solution under the item ' 3.4.4.3 ', injecting the sample into a high performance liquid chromatograph for determination according to the chromatographic condition under the item ' 3.4.5.5 ', recording peak areas according to the obtained chromatogram, calculating the content of 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside, and calculating RSD, wherein the results are shown in Table 29.
Watch 29
The results show that the method has good repeatability.
3.4.6.7 sample application and recovery test
The method comprises the steps of taking 3g of a traditional Chinese medicine composition with known 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside content, adding 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside reference solution (the concentration is 0.6360 mg/mL) into 2mL, 2.5mL and 3mL respectively, volatilizing a solvent, preparing 6 parts of test solution according to the preparation method of the test solution under the item ' 3.4.4.3 ', injecting the test solution into a high performance liquid chromatograph according to the chromatographic condition under the item ' 3.4.5.5 ' for determination, and calculating the content, the recovery rate and the RSD value of 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside according to the obtained chromatogram, wherein the results are shown in Table 30.
Watch 30
3.4.7 three-batch determination of sample content
Taking 3 batches (batch number is shown in table 31) of traditional Chinese medicine composition samples to be detected, 6g, precisely weighing and paralleling 2 batches, preparing a traditional Chinese medicine composition sample solution to be detected according to a preparation method of a test solution under the item 3.4.4.3, respectively injecting the obtained traditional Chinese medicine composition sample solution to be detected and a reference solution under the item 3.4.2 into a high performance liquid chromatograph according to the chromatographic condition under the item 3.4.5.5, respectively, measuring, and calculating the content of 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside according to the obtained chromatogram, wherein the results are shown in table 31.
Watch 31
The results show that the 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside content in the three samples is greater than 0.1mg/g.
3.5 matrine content determination
3.5.1 instruments, reagents
The instrument comprises the following steps: agilent1260 high performance liquid chromatograph, DAD detector; a chromatographic column: zorbax eclipseC 18 (250X 4.6mm,5 μm); KQ3200DB type digital controlled ultrasonic instrument (proclaimed instruments, llc); electric jackets (Chengxihua instruments, inc., of Ouchi city), BS224S analytical balance (Beijing Saedodus instruments systems, inc.); mortars, graduated cylinders, round-bottomed flasks, condenser tubes, evaporating dishes and the like are all purchased from Dongzhou glass instruments ltd; electric heating jacket (Chengxihua instruments, inc.).
Reagent testing: acetonitrile (chromatographically pure); ultrapure water; formic acid (chromatographic purity), ethanol, methanol, matrine reference substances and traditional Chinese medicine composition samples to be tested (batch numbers: 20140801, 20140802 and 20140803, which are provided by Kang Chenyao company, inner Mongolia responsibility Co., ltd.).
3.5.2 preparation of control solution, test solution, negative sample solution
Control solution: precisely weighing appropriate amount of matrine control dried by phosphorus pentoxide, adding mobile phase under the item of 3.5.3 for dissolving, and making into solution containing 0.1mg per 1mL to obtain control solution.
Test solution: taking a proper amount of the traditional Chinese medicine composition under the item '1', grinding, taking about 2g, precisely weighing, placing into a conical flask with a plug, adding 25mL of water and 0.1mL of ammonia water, carrying out ultrasonic treatment to completely dissolve the traditional Chinese medicine composition, transferring the solution into a separating funnel, washing the container with a small amount of water, transferring the washing solution into the separating funnel, adding a trichloromethane solution for extraction for 3 times, 15mL each time, combining trichloromethane extraction solutions, decompressing and recovering the solvent, dissolving residues with a mobile phase under the item '3.5.3', transferring the residues into a 10mL measuring flask, diluting the mobile phase under the item '3.5.3' to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a sample solution.
Negative sample solution: taking the medicinal materials with the prescription amount without the lightyellow sophora root, and preparing a negative sample solution according to the preparation process of the traditional Chinese medicine composition under the item 1 and the preparation method of the test solution under the item 3.5.2.
3.5.3 chromatography conditions
A chromatographic column: agilent Zorbax Eclipse C 18 (250×4.6mm，5μm)
Mobile phase: acetonitrile-phosphoric acid solution with volume fraction of 0.1% (pH of mobile phase adjusted to 8.0 ± 0.1 with triethylamine) =20 (v/v), isocratic elution
Detection wavelength: 220nm
Flow rate: 1.0mL/min
Column temperature: 25 deg.C
Sample injection amount: 10 μ L
Examination of preparation method of 3.5.4 test solution
3.5.4.1 examination of the amount of Ammonia used
Taking a proper amount of the traditional Chinese medicine composition under the item '1', grinding the mixture into fine powder, taking about 2g of the mixture, precisely weighing the mixture, and preparing a test solution according to the preparation method of the test solution under the item '3.5.2' except the addition amount of ammonia water of 0.1mL, 0.3mL and 0.5mL respectively under other conditions. The obtained test solution and the control solution under item "3.5.2" were respectively injected into a high performance liquid chromatograph under the chromatographic conditions under item "3.5.3" for determination, and the matrine content was calculated from the obtained chromatogram, with the results shown in table 32.
Watch 32
The result shows that the matrine content is the highest when the adding amount of the ammonia water is 0.1 mL.
Examination of 3.5.4.2 Water usage
Taking a proper amount of the traditional Chinese medicine composition under the item '1', grinding, taking about 2g, precisely weighing, and preparing a test solution according to the preparation method of the test solution under the item '3.5.2' under the conditions except that the adding amount of water is respectively 25mL, 50mL and 100mL. The obtained test solution and the control solution under item "3.5.2" were respectively injected into a high performance liquid chromatograph under the chromatographic conditions under item "3.5.3" for determination, and the matrine content was calculated from the obtained chromatogram, with the results shown in table 33.
Watch 33
The result shows that the matrine content is the highest when the water addition amount is 25mL.
Examination of extraction times of 3.5.4.3 chloroform
Taking 2 parts of a proper amount of the traditional Chinese medicine composition under the item '1', grinding, taking about 2g, precisely weighing, collecting a trichloromethane phase each time after extracting the trichloromethane solution for 3 times, and preparing the test solution according to the preparation method of the test solution under the item '3.5.2' under other conditions. The obtained test solution and the control solution under item "3.5.2" were respectively injected into a high performance liquid chromatograph under the chromatographic conditions under item "3.5.3" for determination, and the matrine content was calculated from the obtained chromatogram, with the results shown in table 34.
Watch 34
The result shows that the matrine content is the highest when the three extractions are summed up.
3.5.4.4 examination of chloroform extraction dosage
Taking appropriate amount of 4 parts of the Chinese medicinal composition under item 1, grinding, taking about 2g, precisely weighing, except that the amount of chloroform solution for three times of extraction is 10mL, 105mL, 15mL, 10mL, 20mL,25mL,25mL, 25mL respectively, other conditions were carried out according to the method for preparing a test solution under the section "3.5.2" to prepare a test solution. The obtained test solution and the reference solution under item "3.5.2" are respectively injected into a high performance liquid chromatograph under the chromatographic condition of item "3.5.3" for determination, and the content of matrine is calculated according to the obtained chromatogram, with the results shown in table 35.
Watch 35
The results show that the content has no obvious difference when the dosage is 15mL,20mL and 25mL, and 15mL is selected for extraction dosage in order to control the inspection cost.
8978 confirmation of preparation method of test solution zxft 8978
2g of the product is precisely weighed and placed in a conical flask with a plug, 25mL of water and 0.1mL of ammonia water are added, ultrasonic treatment is carried out to completely dissolve the product, the solution is transferred to a separating funnel, a small amount of water is used for washing a container, the washing solution is transferred to the separating funnel together, a trichloromethane solution is added for extraction for 3 times, 15mL of the solution is added each time, trichloromethane extraction liquid is combined, the solvent is recovered under reduced pressure, the residue is dissolved by a mobile phase under the item '3.5.3', the residue is transferred to a 10mL measuring flask, the mobile phase under the item '3.5.3' is used for dilution to the scale, shaking is carried out uniformly, filtration is carried out, and a subsequent filtrate is taken, thus obtaining the test solution.
3.5.5 examination of chromatographic conditions
3.5.5.1 determination of wavelength
Referring to 'Chinese pharmacopoeia' of 2020 edition, the method selects the detection wavelength to be 220nm.
3.5.5.2 inspection of chromatography columns
Taking a proper amount of the traditional Chinese medicine composition under the item '1', grinding the traditional Chinese medicine composition into fine powder, taking about 2g of the traditional Chinese medicine composition, precisely weighing, preparing a sample solution according to a sample solution preparation method under the item '3.5.4.5', injecting the obtained sample solution into a high performance liquid chromatograph for determination, wherein the conditions are carried out according to the chromatographic conditions under the item '3.5.3' except that chromatographic columns are Agilent, ultimate and Agilent (the specification is shown in a table 36), and observing the separation degree and the number of theoretical plates according to the obtained chromatogram, and the result is shown in a table 36.
Watch 36
The results show that: when the condition of the matrine chromatographic peak front peak is noticed, the Ultimate XB-C18 does not reach the separation requirement because the matrine chromatographic peak contains an impurity peak, the measurement result is higher, and the Agilent ZORBAX Eclipse chromatographic column can meet the matrine content measurement requirement.
Examination of 3.5.5.3 Mobile phase
Taking a proper amount of the traditional Chinese medicine composition under the item '1', grinding, taking about 2g of the traditional Chinese medicine composition, precisely weighing, preparing a sample solution according to a sample solution preparation method under the item '3.5.4.5', injecting the obtained sample solution into a high performance liquid chromatograph for determination, wherein except that the pH values of mobile phases are respectively 7.8, 8.0 and 8.2, the other conditions are carried out according to chromatographic conditions under the item '3.5.3', recording peak areas according to the obtained chromatogram, and calculating the matrine content, wherein the result is shown in a table 37.
Watch 37
The results show that small changes in the pH of the mobile phase have no effect on matrine content determination. When the pH value is 7.8, the symmetry of a chromatographic peak is obviously reduced, the pH value is increased, the separation of the chromatographic peak is facilitated, but the damage to a chromatographic column is large, so that the pH value of the mobile phase is determined to be 8.0 +/-0.1.
3.5.5.4 examination of column temperature
Taking a proper amount of the traditional Chinese medicine composition under the item '1', grinding, taking about 2g, precisely weighing, preparing a sample solution according to a sample solution preparation method under the item '3.5.4.5', injecting the obtained sample solution into a high performance liquid chromatograph for determination, wherein the other conditions are carried out according to chromatographic conditions under the item '3.5.3' except column temperatures of 20 ℃,25 ℃ and 30 ℃, recording peak areas according to the obtained chromatogram, and calculating the matrine content, wherein the results are shown in table 38.
Watch 38
The results show that small changes in column temperature have no effect on matrine content determination. The column temperature of the method is selected to be 25 ℃.
3.5.5.5 investigation of flow Rate
Taking a proper amount of the traditional Chinese medicine composition under the item '1', grinding, taking about 2g, precisely weighing, preparing a sample solution according to a sample solution preparation method under the item '3.5.4.5', injecting the obtained sample solution into a high performance liquid chromatograph for determination, wherein the other conditions are carried out according to chromatographic conditions under the item '3.5.3' except that the flow rates are respectively 0.8mL/min, 1.0mL/min and 1.2mL/min, recording peak areas according to the obtained chromatogram, and calculating the matrine content, wherein the results are shown in a table 39.
Watch 39
The results show that small changes in flow rate have no effect on matrine content determination. The flow rate of the method is selected to be 1.0mL/min.
3.5.5.6 confirmation of chromatographic conditions
A chromatographic column: agilent Zorbax Eclipse C 18 (250×4.6mm，5μm)
Mobile phase: acetonitrile-phosphoric acid solution with volume fraction of 0.1% (pH of mobile phase adjusted to 8.0 ± 0.1 with triethylamine) =20 (v/v), isocratic elution
Detection wavelength: 220nm
Flow rate: 1.0mL/min
Column temperature: 25 deg.C
Sample introduction amount: 10 μ L
3.5.6 System Adaptation study and methodological validation
3.5.6.1 systematic Adaptation study
And (3) chromatographic separation: under the item of '3.5.5.6', the retention time of the chromatographic peak of the matrine is about 15min, the matrine is well separated from other peaks, the separation degree is more than 1.5, the symmetry is 1.06, and the standard is met.
Theoretical plate number: according to the formula n =5.54 (t) R /W h/2 ) The theoretical plate number of matrine was calculated to be 11849, taking into account different chromatographic column conditions: the difference in column length, carrier properties, packing conditions, mobile phase, service time, etc., temporarily bitterThe theoretical plate number of the peak of the reference alkali chromatographic spectrum is not less than 4000.
3.5.6.2 specificity test
10 mul of extraction solvent (mixed solvent of ammonia water and water), the reference solution under the item of '3.5.2', the sample solution under the item of '3.5.4.5' and the negative sample solution under the item of '3.5.2' are respectively taken, and sample introduction is carried out in a high performance liquid chromatograph for determination according to the chromatographic condition under the item of '3.5.5.6'.
The results show that: negative without interference.
3.5.6.3 Linear Range inspection
Precisely absorbing 2, 4, 8, 12, 16 and 20 mu L of the matrine reference substance solution (with the concentration of 0.1096 mg/mL), feeding the matrine reference substance solution into a high performance liquid chromatograph for determination according to the chromatographic condition under the item of 3.5.5.6, recording the peak area according to the obtained chromatogram, drawing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, and obtaining the result shown in the table 40 and the regression equation as follows: y =807.42x-2.2416 2 ＝1。
Watch 40
The results showed that the linear relationship was good in the range of 0.2192 to 2.192. Mu.g.
3.5.6.4 intermediate precision test
Taking a proper amount of the traditional Chinese medicine composition under the item '1', grinding, taking about 2g, precisely weighing, preparing 6 parts of the test solution according to the preparation method of the test solution under the item '3.5.4.5', respectively injecting samples of different personnel A, B and C into a high performance liquid chromatograph for determination according to the chromatographic conditions under the item '3.5.5.6' on different instruments on different dates, recording peak areas according to the obtained chromatogram, calculating the content of matrine, and calculating RSD, wherein the results are shown in a table 41.
Table 41
The results show that the method has good intermediate precision.
3.5.6.5 stability test
Taking a proper amount of the traditional Chinese medicine composition under the item '1', grinding, taking about 2g, precisely weighing, preparing a test solution according to a preparation method of the test solution under the item '3.5.4.5', injecting samples into a high performance liquid chromatograph for measurement at 0, 2, 4, 6 and 8 hours according to chromatographic conditions under the item '3.5.5.6', recording peak areas according to the obtained chromatogram, calculating the content of matrine, and calculating RSD, wherein the results are shown in a table 42.
Watch 42
The results show that the process is stable within 8 hours.
3.5.6.6 repeatability test
Taking a proper amount of the traditional Chinese medicine composition under the item '1', grinding, taking about 2g of the ground traditional Chinese medicine composition, precisely weighing, preparing 6 parts of a test sample solution according to the preparation method of the test sample solution under the item '3.5.4.5', injecting the sample into a high performance liquid chromatograph for determination according to the chromatographic condition under the item '3.5.5.6', recording the peak area according to the obtained chromatogram, calculating the content of the matrine, and calculating the RSD, wherein the result is shown in a table 43.
Watch 43
The results show that the method has good repeatability.
3.5.6.7 sample application and recovery test
Taking a proper amount of the traditional Chinese medicine composition under the item '1' (batch number 20140616, the content is calculated according to 0.663mg/g, sampling 6 parts), grinding, taking about 1g, precisely adding 0.9mL of a reference substance solution (the concentration is 0.769 mg/mL), preparing 6 parts of a test substance solution according to the preparation method of the test substance solution under the item '3.5.4.5', injecting a sample into a high performance liquid chromatograph for determination according to the chromatographic condition under the item '3.5.5.6', recording peak areas according to the obtained chromatogram, and calculating the content, the recovery rate and the RSD value of the matrine, wherein the results are shown in a table 44.
Watch 44
The results show that the recovery of 6 samples is between 97.36 and 100.69% with an RSD% of 1.426, indicating good recovery results.
3.5.7 determination of the content of three batches of samples
Taking 2g of each of 3 batches (batch numbers are shown in Table 45) of Chinese medicinal composition samples to be detected, preparing a Chinese medicinal composition sample solution to be detected according to the preparation method of a test solution under the item of 3.5.4.5, injecting the sample solution into a high performance liquid chromatograph for determination according to the chromatographic condition under the item of 3.5.5.6, and calculating the content of matrine according to the obtained chromatogram, wherein the results are shown in Table 45.
TABLE 45
The results show that the matrine content in the three samples is more than 0.3mg/g.
4 trait detection
4.1 Properties: visual inspection, nasal smell and oral taste are adopted, and the quality indexes are as follows: the product is brown or dark brown granule; sweet and slightly bitter.
4.2 referring to the requirement examination under the item of the granule in the general rules of the four divisions in the 2020 edition of Chinese pharmacopoeia, the quality index is in accordance with the relevant regulations under the item of the granule in the general rules of the four divisions in the 2020 edition of Chinese pharmacopoeia.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (19)
1. A quality detection method of a traditional Chinese medicine composition is characterized by comprising the following steps:
taking a sample of a Chinese medicinal composition to be tested, wherein the theoretical preparation raw materials of the Chinese medicinal composition comprise radix et rhizoma Rhei, radix astragali, cortex Mori, radix Sophorae Flavescentis, radix Codonopsis, atractylodis rhizoma, poria, radix Polygoni Multiflori Preparata, radix Paeoniae alba, saviae Miltiorrhizae radix, rhizoma Ligustici Chuanxiong, flos Chrysanthemi, ginger processed pinellia Tuber, herba plantaginis, bupleuri radix and Glycyrrhrizae radix;
detecting whether the sample of the Chinese medicinal composition to be detected contains radix et rhizoma Rhei, radix Polygoni Multiflori Preparata, radix astragali, saviae Miltiorrhizae radix, radix Sophorae Flavescentis, bupleuri radix, cortex Mori, poria, radix Codonopsis, glycyrrhrizae radix and radix Paeoniae alba by thin layer chromatography;
detecting the contents of paeoniflorin, salvianolic acid B, astragaloside IV, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside and matrine in the Chinese medicinal composition sample to be detected respectively by adopting a high performance liquid chromatography, and determining the quality of the Chinese medicinal composition sample to be detected by combining the contents;
and (4) carrying out character detection on the traditional Chinese medicine composition sample to be detected.
2. The method for detecting the quality of the traditional Chinese medicine composition according to claim 1, wherein the quality of a sample of the traditional Chinese medicine composition to be detected is determined to be qualified when the content simultaneously satisfies the following conditions:
a) The content of paeoniflorin is not less than 0.7mg/g;
b) The content of the salvianolic acid B is not less than 0.25mg/g;
c) The content of the astragaloside is not less than 0.1mg/g;
d) The 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside content is not less than 0.1mg/g;
e) The matrine content is not less than 0.3mg/g.
3. The quality detection method of the traditional Chinese medicine composition as claimed in claim 1, wherein the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains rhubarb and prepared fleece-flower root by adopting thin layer chromatography comprises the following steps:
taking a sample of the traditional Chinese medicine composition to be tested, adding a sulfuric acid solution, heating and refluxing, cooling, adding chloroform, heating and refluxing, taking a chloroform solution, drying, and dissolving residues in chloroform to prepare a test solution;
preparing a rhubarb reference medicinal material solution and a prepared fleece-flower root reference medicinal material solution; preparing a mixed reference solution containing a rhein reference substance, an emodin reference substance and an physcion reference substance;
respectively sucking the test solution, the rhubarb reference medicinal material solution, the prepared fleece-flower root reference medicinal material solution and the mixed reference solution, and dropping the solutions on the same silica gel G or H thin-layer plate according to the volume ratio of (10-20): (3-7): (0.5-2) taking the upper solution of petroleum ether, ethyl formate and formic acid as a developing agent, developing, airing and observing.
4. The quality detection method of the traditional Chinese medicine composition as claimed in claim 1, wherein the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains astragalus membranaceus by adopting thin-layer chromatography comprises the following steps:
taking a sample of the Chinese medicinal composition to be tested, adding n-butanol, heating and refluxing, filtering, taking filtrate, performing alkali washing, removing alkali liquor, adding water saturated n-butanol, washing to neutrality, taking n-butanol extract, drying, and dissolving residue with methanol to prepare a test solution;
preparing an astragaloside IV reference substance solution;
respectively sucking the test solution and the astragaloside IV reference solution, and dropping the solutions on the same silica gel G or H thin layer plate according to the volume ratio of (12-14): (6-8): (1-4) developing with a lower layer solution of chloroform, methanol and water as a developing agent, and observing after the developing agent is developed and dried.
5. The quality detection method of the traditional Chinese medicine composition according to claim 1, wherein the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains the salvia miltiorrhiza bunge by adopting the thin-layer chromatography comprises the following steps:
dissolving the Chinese medicinal composition sample in water, filtering, extracting the filtrate with diethyl ether, drying diethyl ether phase, and dissolving the residue with ethanol to obtain sample solution;
preparing a salvia miltiorrhiza reference medicinal material solution and a protocatechuic aldehyde reference substance solution;
respectively sucking the test solution, the salvia miltiorrhiza reference medicinal material solution and the protocatechuic aldehyde reference substance solution, and spotting on the same silica gel G or H thin-layer plate, wherein the volume ratio of the reference medicinal material solution to the protocatechuic aldehyde reference substance solution is (1-3): (3-5): (3-5): (0.3-0.7) taking hexane, benzene, ethyl acetate and formic acid as developing agents, developing, airing and observing.
6. The quality detection method of the traditional Chinese medicine composition according to claim 1, wherein the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains the radix sophorae flavescentis by adopting the thin-layer chromatography comprises the following steps:
taking a sample of the traditional Chinese medicine composition to be tested, adding chloroform and a concentrated ammonia solution, standing, filtering, taking a filtrate for drying, and dissolving residues in chloroform to prepare a test solution;
preparing a matrine reference solution;
respectively sucking the test solution and the matrine reference solution, dropping the test solution and the matrine reference solution on the same silica gel G or H thin layer plate, and mixing the two solutions according to the volume ratio of (10-30): (10-30): (2-4): (0.5-2) developing with toluene, ethyl acetate, methanol and concentrated ammonia solution as developing agent, air drying and observing.
7. The quality detection method of the traditional Chinese medicine composition as claimed in claim 1, wherein the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains the radix bupleuri or not by adopting the thin-layer chromatography comprises the following steps:
taking a sample of the Chinese medicinal composition to be detected, adding water, carrying out ultrasonic extraction, filtering, taking filtrate, passing the filtrate through an AB-8 type macroporous adsorption resin column, eluting by using an ethanol solution with the volume fraction of 40-60%, discarding an eluent, continuously eluting by using an ethanol solution with the volume fraction of 80-100%, taking the eluent, drying, adding methanol into residues for dissolving, and preparing a sample solution;
preparing a radix bupleuri reference medicinal material solution;
respectively sucking the test solution and the radix bupleuri reference medicinal material solution, dropping the test solution and the radix bupleuri reference medicinal material solution on the same silica gel G or H thin-layer plate, and mixing the two solutions according to the volume ratio of (9-13): (1-3): (0.5-2) using ethyl acetate, ethanol and water as developing agents, developing, airing and observing.
8. The quality detection method of the traditional Chinese medicine composition as claimed in claim 1, wherein the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains the white mulberry root-bark or not by adopting the thin layer chromatography comprises the following steps:
adding saturated sodium carbonate solution into the sample of the traditional Chinese medicine composition to be detected for ultrasonic extraction, filtering, taking filtrate, adding hydrochloric acid to adjust the pH value to 1-2, standing and filtering, taking filtrate, adding ethyl acetate for shaking extraction, taking ethyl acetate phase, drying, and adding methanol into residues for dissolving to prepare a sample solution;
preparing a white mulberry root-bark reference medicinal material solution;
respectively sucking the test solution and the radix bupleuri reference solution, dropping on the same polyamide thin layer plate, developing with acetic acid as developing agent, air drying, and observing.
9. The quality detection method of the traditional Chinese medicine composition as claimed in claim 1, wherein the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains the poria cocos by adopting the thin-layer chromatography comprises the following steps:
taking a sample of the Chinese medicinal composition to be detected, adding water for ultrasonic extraction, filtering, taking a filtrate, adding water-saturated n-butanol for extraction, taking a n-butanol phase, drying, and adding methanol to dissolve residues to prepare a test solution;
preparing a poria cocos contrast medicinal material solution;
respectively sucking the test solution and the tuckahoe contrast medicinal material solution, dropping the solutions on the same silica gel G or H thin-layer plate, and mixing the solutions according to the volume ratio of (15-25): (3-7): (0.3-0.7) taking toluene, ethyl acetate and formic acid as developing agents, developing, airing and observing.
10. The quality detection method of the traditional Chinese medicine composition as claimed in claim 1, wherein the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains the codonopsis pilosula by adopting the thin-layer chromatography comprises the following steps:
taking a sample of the Chinese medicinal composition to be detected, adding water, carrying out ultrasonic extraction, filtering, taking filtrate, passing the filtrate through an AB-8 type macroporous adsorption resin column, eluting by using an ethanol solution with the volume fraction of 40-60%, discarding an eluent, continuously eluting by using an ethanol solution with the volume fraction of 80-100%, taking the eluent, drying, adding methanol into residues for dissolving, and preparing a sample solution;
preparing radix Codonopsis reference medicinal material solution and radix Codonopsis alkyne glycoside reference substance solution;
respectively sucking the test sample solution, the codonopsis pilosula reference medicinal material solution and the codonopsis pilosula alkyne glycoside reference substance solution, and dropping the solutions on the same silica gel G or H thin layer plate according to the volume ratio of (6-8): (0.5-2): (0.3-0.7) taking n-butanol, glacial acetic acid and water as developing agents, developing, airing and observing.
11. The quality detection method of the traditional Chinese medicine composition according to claim 1, wherein the method for detecting whether the sample of the traditional Chinese medicine composition to be detected contains liquorice and white paeony root by adopting the thin-layer chromatography comprises the following steps:
dissolving the Chinese medicinal composition sample in water, extracting with water-saturated n-butanol, collecting n-butanol phase, drying, dissolving the residue in water, and passing through C 18 Eluting with water and methanol, collecting methanol eluate, drying, dissolving the residue in methanol, and making into sample solution;
preparing a liquorice reference medicinal material solution and a paeoniflorin reference substance solution;
respectively sucking the test solution, the liquorice contrast medicinal material solution and the paeoniflorin contrast solution, and dropping the solutions on the same silica gel G or H thin-layer plate according to the volume ratio of (13-17): (0.5-2): (0.5-2): and (1) taking ethyl acetate, formic acid, glacial acetic acid and water as developing agents, developing, airing and observing.
12. The method for detecting the quality of the traditional Chinese medicine composition according to any one of claims 1 to 11, wherein the method for detecting the content of paeoniflorin in the sample of the traditional Chinese medicine composition to be detected by using the high performance liquid chromatography comprises the following steps:
preparing a paeoniflorin reference solution;
mixing a sample of the Chinese medicinal composition to be detected with an extraction solvent, carrying out ultrasonic extraction, taking supernatant, passing through a polyamide column, eluting with water, collecting eluent, and preparing a solution of the Chinese medicinal composition sample to be detected;
performing high performance liquid chromatography detection on the paeoniflorin control solution and the traditional Chinese medicine composition sample solution to be detected, and calculating the content of paeoniflorin according to the obtained chromatogram;
the chromatographic conditions for detecting by the high performance liquid chromatography comprise: the mobile phase comprises (13-17) by volume: (83-87) and isocratic elution with acetonitrile and potassium dihydrogen phosphate solution.
13. The method for detecting the quality of the traditional Chinese medicine composition according to any one of claims 1 to 11, wherein the method for detecting the content of the salvianolic acid B in the sample of the traditional Chinese medicine composition to be detected by adopting the high performance liquid chromatography comprises the following steps:
preparing salvianolic acid B reference solution;
mixing a sample of the Chinese medicinal composition to be detected with an extraction solvent, carrying out ultrasonic extraction, taking an extracting solution, filtering, and preparing a sample solution of the Chinese medicinal composition to be detected;
performing high performance liquid chromatography detection on the salvianolic acid B reference solution and the traditional Chinese medicine composition sample solution to be detected, and calculating the content of salvianolic acid B according to the obtained chromatogram;
the chromatographic conditions for detecting by the high performance liquid chromatography comprise: the mobile phase comprises the following components in a volume ratio of (18-25): (75-82) and isocratic elution with acetonitrile and formic acid solution.
14. The quality detection method of the traditional Chinese medicine composition according to any one of claims 1 to 11, wherein the method for detecting the content of astragaloside in the traditional Chinese medicine composition sample to be detected by adopting the high performance liquid chromatography comprises the following steps:
preparing an astragaloside IV reference substance solution;
mixing a sample of the Chinese medicinal composition to be detected with an extraction solvent, heating and refluxing for extraction, taking an extracting solution, drying, dissolving residues in water, shaking and extracting by adding water-saturated n-butanol, taking a n-butanol phase, carrying out alkali washing, removing alkali liquor, drying, and adding the extraction solvent into residues to prepare a sample solution of the Chinese medicinal composition to be detected;
detecting the astragaloside control solution and the traditional Chinese medicine composition sample solution to be detected by high performance liquid chromatography, and calculating the content of astragaloside according to the obtained chromatogram;
the chromatographic conditions of the high performance liquid chromatography detection comprise: the mobile phase comprises the following components in a volume ratio of (25-35): (65-75) acetonitrile and water, and isocratic elution.
15. The method for detecting the quality of the traditional Chinese medicine composition according to any one of claims 1 to 11, wherein the method for detecting the content of 2,3,5,4' -tetrahydroxystilbene-2-O- β -D glucoside in the sample of the traditional Chinese medicine composition to be detected by adopting the high performance liquid chromatography comprises the following steps:
preparing 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside reference solution;
mixing a sample of the Chinese medicinal composition to be detected with an extraction solvent, carrying out ultrasonic extraction, taking an extracting solution, filtering, and preparing a sample solution of the Chinese medicinal composition to be detected;
performing high performance liquid chromatography detection on the 2,3,5,4 '-tetrahydroxystilbene-2-O-beta-D glucoside reference solution and the traditional Chinese medicine composition sample solution to be detected, and calculating the content of 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D glucoside according to the obtained chromatogram;
the chromatographic conditions of the high performance liquid chromatography detection comprise: the mobile phase comprises (13-18) by volume: and (82-87) taking acetonitrile and water as mobile phases, and carrying out isocratic elution.
16. The method for detecting the quality of the traditional Chinese medicine composition according to any one of claims 1 to 11, wherein the method for detecting the content of the matrine in the sample of the traditional Chinese medicine composition to be detected by adopting the high performance liquid chromatography comprises the following steps:
preparing a matrine reference solution;
mixing a sample of the Chinese medicinal composition to be detected, water and ammonia water, performing ultrasonic treatment until the mixture is dissolved, adding a trichloromethane solution for extraction, taking a trichloromethane phase, removing a solvent, dissolving residues in a mobile phase, and filtering to prepare a sample solution of the Chinese medicinal composition to be detected;
performing high performance liquid chromatography detection on the matrine reference substance solution and the Chinese medicinal composition sample solution to be detected, and calculating matrine content according to the obtained chromatogram;
the chromatographic conditions for detecting by the high performance liquid chromatography comprise: the mobile phase comprises acetonitrile, phosphoric acid solution and triethylamine, wherein the volume ratio of the acetonitrile to the phosphoric acid solution is (18-22): (78-82), adjusting the pH value of the flowing direction to be 8.0 +/-0.1 by triethylamine, and isocratic eluting.
17. The method for detecting the quality of the traditional Chinese medicine composition according to any one of claims 1 to 11, wherein the property detection comprises detecting whether the sample of the traditional Chinese medicine composition to be detected conforms to brown or tan granules or whether the sample is sweet and slightly bitter by visual inspection, nasal smell and oral taste.
18. The method for detecting the quality of the traditional Chinese medicine composition according to any one of claims 1 to 11, wherein in the theoretical preparation raw materials of the traditional Chinese medicine composition, the weight ratio of rhubarb, astragalus root, white mulberry root-bark, lightyellow sophora root, pilose asiabell root, largehead atractylodes rhizome, indian buead, prepared tuber fleeceflower root, white paeony root, danshen root, szechuan lovage rhizome, chrysanthemum, ginger processed pinellia tuber, plantain herb, chinese thorowax root and liquoric root is (0.5-1.5): (3-5): (2-4): (1-3): (2-4): (4-6): (4-6): (4-6): (2-4): (4-6): (2-4): (1.5-3.5): (1-3): (4-6): (1-2): (0.5-1.5).
19. The method for detecting the quality of the traditional Chinese medicine composition according to any one of claims 1 to 11, wherein the theoretical preparation method of the traditional Chinese medicine composition comprises the following steps:
extracting radix et rhizoma Rhei, radix astragali, cortex Mori, radix Sophorae Flavescentis, radix Codonopsis, atractylodis rhizoma, poria, radix Polygoni Multiflori Preparata, radix Paeoniae alba, saviae Miltiorrhizae radix, rhizoma Ligustici Chuanxiong, flos Chrysanthemi, ginger processed pinellia Tuber, herba plantaginis, bupleuri radix and Glycyrrhrizae radix with water, collecting filtrate, adding medicinal adjuvants, and granulating.
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