CN112587642B - Preparation method and detection method of vitality-maintaining pharmaceutical composition - Google Patents
Preparation method and detection method of vitality-maintaining pharmaceutical composition Download PDFInfo
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- CN112587642B CN112587642B CN202011521544.4A CN202011521544A CN112587642B CN 112587642 B CN112587642 B CN 112587642B CN 202011521544 A CN202011521544 A CN 202011521544A CN 112587642 B CN112587642 B CN 112587642B
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Abstract
The invention relates to a preparation method and a detection method of a Baoyuan medicinal composition, which take the cream yield, the total amount of ginsenoside Rg1, Rb1 and Re, the content of astragaloside and a fingerprint spectrum as indexes to determine the optimal relevant parameters of the pretreatment, heating mode, decoction time, filtration, drying and other optimal processes of the medicinal composition, thereby determining the optimal preparation method of the Baoyuan medicinal composition and further improving the content of effective components and the cream yield of products. The best detection method is determined by searching the detection method of the Baoyuan medicinal composition, and the quality of the product is better controlled.
Description
Technical Field
The invention relates to the field of medicine preparation and detection, in particular to a preparation method and a detection method of a vitality-maintaining medicine composition.
Background
Baoyuan Tang comes from brief doctor hub (Ming, Sun)Will you get well), treat primordial qi deficiency, lassitude, muscular softness and slowness, poor appetite and a pale complexion
Bai, sleeping and quiet, … … and miscellaneous syndromes are all weak and should be taken. Is prepared from ginseng, astragalus root, liquorice root, cinnamon bark, etc. The decocting method recorded in the original text comprises the following steps: ginseng, astragalus root, licorice, cinnamon, ginger and water. The data unit in the 'preparation method and usage' in the Baoyuan decoction is converted with the modern data unit according to 'historic standard measurement and scale' and 'Chinese historic standard degree, quantity and scale evolution profile'. One piece of money is 3.69g at present, one piece is 0.37 g, namely the 'preparation method' of the Baoyuan decoction decocting process can be described as taking 3.69g of ginseng, 7.38g of astragalus, 1.85g of liquorice, 0.74g of cinnamon and 1 piece of ginger, adding 1920mL of water, soaking for 30min, heating and decocting, and filtering the dregs to obtain the Baoyuan decoction. According to the method recorded in the list of classical famous prescriptions, the decoction preparation of the standard Baoyuan decoction substance is carried out by combining the Ministry of health and the administration of the national traditional Chinese medicine administration, the management Specification of the traditional Chinese medicine decoction rooms of the medical institutions (No. 2009) 3), and the preparation of the standard Baoyuan decoction substance is close to the tradition and follows the principles of the ancient prescription, the preparation method and the detection method of the invention are further researched, so that the content of the effective components of the Baoyuan composition is further improved, and the scientific detection method is provided.
Based on the reasons, the invention determines the optimal process related parameters of the pretreatment, the heating mode, the decocting time, the filtration, the drying and the like of the pharmaceutical composition by taking the cream yield, the total amount of the ginsenosides Rg1, Rb1 and Re, the astragaloside content and the fingerprint as indexes, thereby determining the optimal preparation method of the Baoyuan pharmaceutical composition and further improving the content of effective components and the cream yield of products; the best detection method is determined by searching the detection method of the Baoyuan medicinal composition, and the quality of the product is better controlled.
Disclosure of Invention
The invention aims to provide a preparation method of a vitality-maintaining pharmaceutical composition.
The invention aims to provide a detection method of a vitality-maintaining pharmaceutical composition.
The preparation method of the vitality-maintaining pharmaceutical composition comprises the following steps: 60-85 parts of ginseng, 120-165 parts of astragalus membranaceus, 20-50 parts of liquorice, 10-20 parts of cinnamon and 15-25 parts of ginger are placed in an electronic decoction pot, 1500-3020 ml of water is added, the decoction is carried out by covering, strong fire boiling is carried out, slow fire boiling is carried out, the decoction is kept for 0.5-2 h, 200-mesh filter cloth is filtered while hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55-60 ℃ until the total feeding amount ratio of the liquid medicine to the decoction pieces is 1: 3, and at the pre-freezing temperature: freeze-drying at-15-50 deg.C, freeze-drying temperature-45-75 deg.C, and vacuum degree less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding pharmaceutically acceptable adjuvants, and making into pharmaceutically acceptable dosage forms.
Preferably, the preparation method of the Baoyuan medicine composition comprises the following steps: 74 parts of ginseng, 148 parts of astragalus, 37 parts of liquorice, 15 parts of cinnamon and 20 parts of ginger are placed in an electronic decoction pot, 1920ml of water is added, the decoction is carried out by covering, the strong fire boiling is changed into the slow fire boiling for keeping the micro boiling, the decoction is carried out for 1h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.06 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the liquid medicine to the total feeding amount of decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze drying at-20-45 deg.c, freeze drying temperature-50-70 deg.c and vacuum degree lower than 300Pa, and collecting dry extract powder, or collecting dry extract powder and adding pharmaceutically acceptable supplementary material to prepare pharmaceutically acceptable preparation.
The pharmaceutically acceptable preparation provided by the invention is a solid preparation or a liquid preparation.
The solid preparation provided by the invention is granules, capsules, tablets, pills, powder and freeze-dried powder injection.
The liquid preparation provided by the invention is an injection preparation and an oral liquid.
The auxiliary materials are not limited and can be accepted in pharmacy.
The ginger tablet is counted by 3 g.
The detection method comprises the following steps:
the characteristics are as follows: the product is brown to tan powder, slightly fragrant, slightly bitter and sweet;
and (3) identification: 1) taking 0.5-1.5 g of the product powder, adding 20-60 ml of chloroform, heating and refluxing for 0.5-2 hours, removing chloroform liquid, volatilizing solvent from decoction dregs, adding 0.5ml of water, stirring and wetting, adding 10ml of saturated n-butyl alcohol, carrying out ultrasonic treatment for 20-40 minutes, absorbing supernate, adding 2-4 times of ammonia test solution, shaking uniformly, standing and layering, taking supernatant liquid, evaporating to dryness, and adding 1ml of methanol to residues for dissolving to obtain a test solution; preparing 1g of Ginseng radix reference material, and making reference material solution by the same method; adding methanol into ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 as reference solutions to obtain mixed solutions each containing 2mg per 1 ml; performing thin-layer chromatography (0502 of the four ministry of the national pharmacopoeia 2015), sucking the sample solution, the reference medicinal material solution and the reference solution 1-2 mu 1 respectively, and respectively dropping the sample solution, the reference medicinal material solution and the reference solution on the same silica gel G thin-layer plate according to the proportion of 15: 40: 22: 10, taking a lower layer solution of chloroform-ethyl acetate-methanol-water placed at the temperature of below 6-15 ℃ as a developing agent, developing, taking out, airing, spraying a 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clearly developed, respectively placing the solution under sunlight and 365nm ultraviolet lamps for inspection, wherein in the chromatogram of a test sample, the same spots are developed on the positions corresponding to the chromatogram of a reference substance, and a negative chromatogram has no spots on the positions corresponding to the chromatogram of the reference substance;
2) taking 1-2 g of the product, adding 15-35 mL of water, carrying out ultrasonic treatment for 20-40 minutes, filtering, shaking and extracting filtrate with water saturated n-butyl alcohol for 2-4 times, 20-30 mL each time, combining n-butyl alcohol extract, washing with ammonia test solution for 2-4 times, 20-40 mL each time, separately taking n-butyl alcohol solution, evaporating to dryness, and adding methanol lmL into residues to dissolve the residues to obtain a test solution; taking 1-2 g of astragalus mongholicus reference medicinal material, adding 40-60 ml of water, decocting for 20-40 minutes, filtering, and preparing a reference medicinal material solution from the filtrate by the same method; adding methanol into astragaloside IV reference substance to obtain solution containing 2mg of astragaloside IV per lmL as reference substance solution; performing thin-layer chromatography (0502 of the four ministry of the national pharmacopoeia 2015), sucking 10 μ l of each of the reference solution, the reference medicinal material solution and the test solution, respectively dropping the solution on a same silica gel G thin-layer plate, developing the solution at the lower layer which is placed at the temperature of 5-15 ℃ in the ratio of 13:7:2 of chloroform-methanol-water as a developing agent, taking out, drying in the air, spraying 10% sulfuric acid ethanol solution, and heating at 105 ℃ until spots are clearly developed; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; observing under 365nm ultraviolet lamp to show fluorescent spots of the same color;
3) taking 0.5-1.5 g of the product and a negative sample without liquorice respectively, adding 20-60 ml of water, performing ultrasonic treatment for 20-40 minutes, filtering, performing shake extraction for 2-4 times by using ether, 10-30 ml each time, discarding ether liquid, performing shake extraction for 2-4 times by using water-saturated n-butyl alcohol for 15-25 ml each time, combining n-butyl alcohol liquid, performing shake extraction for 2-4 times by using ammonia test solution for 20-40 ml each time, combining the ammonia test solutions, and adjusting the pH value to 3-4 by using hydrochloric acid; extracting with ethyl acetate for 2-4 times by shaking, wherein 20-40 ml of ethyl acetate is extracted each time, combining ethyl acetate solutions, evaporating to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a test sample solution and a liquorice-lacking negative sample solution; taking 0.5-1.5 g of honey-fried licorice root decoction pieces, adding 40-60 ml of water, decocting for 20-40 minutes, filtering, and preparing a control medicinal solution by the same method; taking appropriate amount of liquiritin reference substance, adding 70% ethanol to obtain solution containing 0.5mg per 1mL as reference substance solution; performing thin-layer chromatography (0502 of the four ministry of the Chinese pharmacopoeia 2015 edition), sucking 5-10 mul of the test solution, the liquorice-lacking negative sample solution, the control solution and the control solution respectively, and respectively dropping the two solutions on the same silica gel G thin-layer plate according to the proportion of 13:7:2, taking the subnatant of chloroform-methanol-water placed at 5-10 ℃ as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the color of the spots is clear, and observing under sunlight and 365nm ultraviolet lamps, wherein spots or fluorescent spots with the same color appear in the chromatogram of the test solution and at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material;
4) taking 0.5-1.5 g of the product and the cinnamomum japonicum negative freeze-dried powder respectively, adding 5-15 ml of water to dissolve the product, shaking and extracting the product by using ether for 1-3 times, 15-35 ml each time, combining ether solution, volatilizing the ether solution, adding 2ml of ether to dissolve residues to be used as test solution and cinnamomum japonicum negative sample solutionLiquid; taking 0.5-1.5 g of cinnamon decoction pieces, adding 40-60 ml of water, decocting for 20-40 minutes, filtering, and preparing a control medicinal solution by the same method; taking a cinnamic acid reference substance, and preparing 1ml of 0.2mg solution by using 40-65% methanol to serve as a reference substance solution; performing thin layer chromatography (0502 of the four parts of the pharmacopoeia of China 2015), sucking the above sample solution, pulp-deficient Cassia negative sample solution, control solution, and control solution 3 μ l respectively, and dropping on the same silica gel GF254Developing on a thin layer plate with cyclohexane-diethyl ether-glacial acetic acid as developing agent at a ratio of 5:5:0.3, taking out, air drying, inspecting under 254nm ultraviolet lamp, and displaying spots or fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution;
and (4) checking: the water content is not over 12.0 percent according to the second method (0832 in the fourth general rule of the Chinese pharmacopoeia 2015 edition);
extract: the extract content is 18-81.0% by hot dipping method according to alcohol-soluble extract determination method (2201 in the four departments of the pharmacopoeia of China 2015 edition);
fingerprint spectrum: measuring by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia);
octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; a chromatographic column: InertSustain-AQ-C18, acetonitrile is used as a mobile phase A, a 0.1% phosphoric acid solution is used as a mobile phase B, the flow rate is 1mL/min, the detection wavelength is 200-220 nm, and the specific mobile phase gradient elution program is as follows:
mobile phase gradient elution procedure
Preparation of reference substance solution A proper amount of calycosin glucoside reference substance is precisely weighed, and 40-60% methanol is added to prepare 50.4592 mug/ml solution of calycosin glucoside per 1ml, so as to obtain the reference substance;
preparing a sample solution, taking 1.5-3.5 g of the product, precisely weighing, placing the product in a 50mL triangular conical flask, adding 40-60 mL of 40-60% methanol, carrying out ultrasonic treatment for 30-60min, filtering, recovering the solvent from the filtrate under reduced pressure until the solvent is nearly dry, dissolving the residue in 20mL of water, extracting with a water-saturated n-butyl alcohol solution for 2-3 times, 15-35 mL each time, combining the n-butyl alcohol solution, extracting with 20-40 mL of an ammonia test solution for 1 time, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residue in 2mL of water, carrying out column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating the 95% ethanol eluate, dissolving the residue in 40-60% methanol, transferring to a volumetric flask, fixing the volume to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the product;
the determination method comprises precisely sucking reference solution and sample solution 10 μ L respectively, injecting into liquid chromatograph, determining, and recording chromatogram for 70 min;
corresponding chromatographic peaks with same retention time of reference substance chromatographic peaks are respectively presented in the fingerprint of the test sample; calculating according to the similarity evaluation system of traditional Chinese medicine chromatogram fingerprint, wherein the similarity between the sample fingerprint and the comparison fingerprint is not less than 0.85, and the comparison fingerprint is shown in figure 1;
content determination: measuring Ginseng radix by high performance liquid chromatography (0512 in the four ministry of communications in 2015 pharmacopoeia);
in chromatographic conditions and system applicability tests, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, an aqueous solution is used as a mobile phase B, the detection wavelength is 190-210 nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1; performing gradient elution according to a specified gradient elution procedure, wherein the mobile phase gradient elution procedure comprises the following steps:
mobile phase gradient elution procedure
Preparing reference substance solution by accurately weighing appropriate amount of ginsenoside Rg1, Rb1, and Re reference substance, and adding methanol to obtain 0.2mg mixed solution containing ginsenoside Rg1, Rb1, and Re per 1 ml;
preparing a test sample solution, taking 0.5-1.5 g of the product, precisely weighing, adding 12.5ml of water for dissolving, placing the product in a separating funnel, adding chloroform for extracting for 2-4 times, 10-20 ml each time, discarding a chloroform layer solution, extracting the solution for 2-4 times with a water saturated n-butanol solution for 20-40 ml each time, combining n-butanol solutions, washing for 2-4 times with an ammonia test solution for 20-40 ml each time, discarding an ammonia test solution layer, washing n-butanol solution with a water saturated n-butanol solution water layer solution for 10-30 ml, washing for 1 time, discarding a water layer solution, evaporating the n-butanol solution to dryness, adding methanol for dissolving residues, transferring the residues into a 5ml volumetric flask, adding methanol for metering to a scale, and shaking up to obtain the product;
the determination method comprises precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording 110min chromatogram;
the product contains 0.6 mg/g-2.4 mg/g of ginseng in each 1g of dry product, calculated by the total content of ginsenoside Rg1, Rb1 and Re;
measuring content of radix astragali by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia of China);
chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; acetonitrile-water (35: 65) is used as a mobile phase; detecting by an evaporative light scattering detector; the number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak;
preparing reference solution by accurately weighing appropriate amount of astragaloside IV reference, and adding methanol to obtain solution containing 0.5mg per lml;
preparing a test solution, taking 0.5-1.5 g of the product, precisely weighing, precisely adding 40-60 ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 30-60 minutes, weighing again, supplementing the loss weight with water, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, shaking and extracting with water-saturated n-butyl alcohol for 2-6 times (20-40 ml each time), combining n-butyl alcohol extract, washing with ammonia test solution for 2-4 times (20-40 ml each time), discarding ammonia test solution washing liquor, separately taking n-butyl alcohol solution, evaporating to dryness, dissolving residues with methanol, transferring into a 5ml measuring flask, adding methanol to dilute to a scale, and shaking up to obtain the test solution.
The product contains astragaloside IV (C) per 1g of radix astragali41H68O14) The content of the active carbon is 0.10mg/g to 1.40 mg/g.
Preferably, the detection method of the present invention is:
1) taking 1g of the product powder, adding 40ml of trichloromethane, heating and refluxing for 1 hour, removing trichloromethane liquid, volatilizing solvent from medicine residue, adding 0.5ml of water, stirring and moistening, adding 10ml of saturated n-butyl alcohol, performing ultrasonic treatment for 30 minutes, sucking supernatant, adding 3 times of ammonia test solution, shaking uniformly, standing for layering, taking supernatant, evaporating to dryness, and adding 1ml of methanol to dissolve residues to obtain a test solution; preparing 1g of Ginseng radix reference material, and making reference material solution by the same method; adding methanol into ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 as reference solutions to obtain mixed solutions each containing 2mg of ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 per 1 ml; performing thin-layer chromatography (general rule 0502) test, sucking the sample solution, the reference medicinal material solution and the reference solution 1-2 mu 1 respectively, and respectively dropping on the same silica gel G thin-layer plate according to the proportion of 15: 40: 22: 10, taking a lower layer solution of chloroform-ethyl acetate-methanol-water placed below 10 ℃ as a developing agent, developing, taking out, airing, spraying a 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clearly developed, respectively placing the solution under sunlight and 365nm ultraviolet lamps for inspection, wherein in a chromatogram of a test sample, the same spots are developed on a position corresponding to a chromatogram of a reference substance, and a negative chromatogram has no spots on a position corresponding to the control chromatogram;
2) collecting 1.5g of the product, adding 25mL of water, performing ultrasonic treatment for 30min, filtering, extracting the filtrate with 25mL of water-saturated n-butanol under shaking for 2 times, mixing n-butanol extractive solutions, washing with ammonia solution for 2 times, 30mL each time, collecting n-butanol solution, evaporating to dry, and dissolving the residue with methanol lmL to obtain sample solution; adding water 50ml into radix astragali control 1.5g, decocting for 30min, filtering, and making into control solution; adding methanol into astragaloside IV reference substance to obtain solution containing 2mg of astragaloside IV per lmL as reference substance solution; performing thin layer chromatography (0502 of general guidelines of the four parts of the year in the pharmacopoeia of China 2015), sucking 10 μ l of each of a reference solution, a reference medicinal material solution and a test solution, respectively dropping the solution on a same silica gel G thin layer plate, developing the solution at the lower layer which is placed at the temperature of 10 ℃ below the ratio of 13:7:2 of chloroform-methanol-water as a developing agent, taking out, drying in the air, spraying 10% sulfuric acid ethanol solution, and heating at 105 ℃ until spots are clearly developed; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; observing under 365nm ultraviolet lamp to show fluorescent spots of the same color;
3) taking 1g of the product and a negative sample without liquorice respectively, adding 40ml of water, carrying out ultrasonic treatment for 30 minutes, filtering, extracting for 2 times by shaking diethyl ether and 20ml each time, discarding the diethyl ether solution, extracting the water solution by shaking water-saturated n-butyl alcohol for 3 times by shaking and 20ml each time, combining the n-butyl alcohol solutions, extracting for 2 times by shaking and 30ml each time by using an ammonia test solution, combining the ammonia test solutions, and adjusting the pH value to 3-4 by using hydrochloric acid; extracting with ethyl acetate for 2 times (30 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1ml methanol to obtain test solution and Glycyrrhrizae radix-deficient negative sample solution; adding water 50ml into radix Glycyrrhizae Preparata decoction pieces 1g, decocting for 30min, filtering, and making into control medicinal solution by the same method; taking appropriate amount of liquiritin reference substance, adding 70% ethanol to obtain solution containing 0.5mg per 1mL as reference substance solution; performing thin layer chromatography (general rule 0502) test, sucking 10 μ l each of the sample solution, the Glycyrrhrizae radix-deficient negative sample solution, the control solution, and the control solution, respectively dropping on the same silica gel G thin layer plate at a ratio of 13:7:2, taking the subnatant of chloroform-methanol-water placed at 5-10 ℃ as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the color of the spots is clear, and observing under sunlight and 365nm ultraviolet lamps, wherein spots or fluorescent spots with the same color appear in the chromatogram of the test solution and at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material;
4) dissolving the product and the lyophilized powder with water 10ml each 1g, extracting with diethyl ether under shaking for 1 time, 25ml each time, volatilizing the diethyl ether solution, dissolving the residue with diethyl ether 2ml to obtain test solution and pulp-deficient cortex Cinnamomi negative sample solution; another cortex Cinnamomi decoctionAdding water 50ml into 1g of tablet, decocting for 30min, filtering, and making into control medicinal solution by the same method; taking a cinnamic acid reference substance, and preparing 1ml of 0.2mg solution with 50% methanol as a reference substance solution; performing thin layer chromatography (0502 of the four parts of the pharmacopoeia of China 2015 edition), sucking the sample solution, the negative sample solution of the pulp-deficient cinnamon, the reference solution, and 3 μ l of the reference solution, dropping on the same silica gel GF254Developing on a thin layer plate with cyclohexane-ether-glacial acetic acid as developing agent at a ratio of 5:5:0.3, taking out, air drying, inspecting under 254nm ultraviolet lamp, and displaying spots or fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution and the reference solution;
preferably, the fingerprint detection method of the invention comprises the following steps:
fingerprint spectrum: measuring by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia);
octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; a chromatographic column: InertSustain-AQ-C18, acetonitrile is used as a mobile phase A, 0.1% phosphoric acid solution is used as a mobile phase B, the flow rate is 1mL/min, the detection wavelength is 210nm, and the specific mobile phase gradient elution program is as follows:
mobile phase gradient elution procedure
Preparation of reference substance solution A proper amount of calycosin glucoside reference substance is precisely weighed, and 50% methanol is added to prepare 50.4592 μ g/ml solution containing calycosin glucoside per 1ml, so as to obtain the product;
preparing a sample solution, precisely weighing 2.5g of the product, placing the product in a 50mL triangular cone bottle, adding 50% methanol into 50mL of the triangular cone bottle, performing ultrasonic treatment for 45min, filtering, recovering a solvent from a filtrate under reduced pressure till the solvent is nearly dry, dissolving residues in 20mL of water, extracting for 2 times with water-saturated n-butyl alcohol solution, 25mL each time, combining the n-butyl alcohol solution, extracting for 1 time with 30mL of ammonia test solution, removing the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residues in 2mL of water, performing column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating the 95% ethanol eluate to dryness, dissolving the residues in 50% methanol, transferring to a 10mL volumetric flask, fixing the volume to the scale, shaking up, filtering, and taking a continuous filtrate to obtain the product;
the determination method comprises precisely sucking reference solution and sample solution 10 μ L respectively, injecting into liquid chromatograph, determining, and recording chromatogram for 70 min;
corresponding chromatographic peaks with same retention time of reference substance chromatographic peaks are respectively presented in the fingerprint of the test sample; calculating according to the similarity evaluation system of traditional Chinese medicine chromatogram fingerprint, wherein the similarity between the sample fingerprint and the comparison fingerprint is not less than 0.85, and the comparison fingerprint is shown in figure 1;
preferably, the method for measuring the content of the ginseng comprises the following steps:
measuring Ginseng radix by high performance liquid chromatography (0512 in the four ministry of communications in 2015 pharmacopoeia);
in chromatographic conditions and system applicability tests, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, an aqueous solution is used as a mobile phase B, the detection wavelength is 203nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1; performing gradient elution according to a specified gradient elution procedure, wherein the gradient elution procedure comprises the following steps:
gradient elution procedure
Preparing reference solution by accurately weighing appropriate amount of ginsenoside Rg1, Rb1 and Re reference, and adding methanol to obtain 0.2 mg/1 ml mixed solution containing ginsenoside Rg1, Rb1 and Re;
preparing a sample solution, precisely weighing 1g of the product, dissolving the product in 12.5ml of water, placing the solution in a separating funnel, extracting the solution for 3 times by adding chloroform, extracting 15ml of the solution each time, removing a chloroform layer solution, extracting the solution for 3 times by using a water-saturated n-butanol solution, extracting 30ml of the solution each time, combining n-butanol solutions, washing the solution for 2 times by using an ammonia test solution, removing the ammonia test solution layer each time by 30ml, removing the ammonia test solution layer by using a water-saturated n-butanol solution water layer solution 20ml of the n-butanol solution, washing for 1 time, removing the water layer solution, evaporating the n-butanol solution to dryness, dissolving residues by adding methanol, transferring the residues into a 5ml volumetric flask, adding methanol to fix the volume to the scale, and shaking up to obtain the product;
the determination method comprises precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram of 110 min;
the product contains 0.6 mg/g-2.4 mg/g of ginseng in each 1g of dry product, calculated by the total content of ginsenoside Rg1, Rb1 and Re;
preferably, the method for measuring the content of the astragalus membranaceus comprises the following steps:
measuring content of radix astragali by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia of China);
chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; acetonitrile-water (35: 65) is used as a mobile phase; detecting by an evaporative light scattering detector; the number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak;
preparing reference solution by accurately weighing appropriate amount of astragaloside IV reference, and adding methanol to obtain solution containing 0.5mg per lml;
preparing a test solution, precisely weighing 1.0g of the product, precisely adding 50ml of water, weighing, ultrasonically treating for 45 minutes, weighing again, supplementing the lost weight with water, shaking up, filtering, precisely weighing 25ml of a subsequent filtrate, shaking and extracting with water-saturated n-butyl alcohol for 4 times, 25ml each time, combining n-butyl alcohol extract, washing with ammonia test solution for 3 times, 30ml each time, discarding ammonia test solution washing liquor, separating n-butyl alcohol solution, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product.
The product contains astragaloside IV (C) per 1g of radix astragali41H68O14) The content of the active carbon is 0.10mg/g to 1.40 mg/g.
Has the advantages that: compared with the prior art, the invention has the following beneficial effects:
1. the invention takes the cream yield, the total amount of the ginsenosides Rg1, Rb1 and Re, the content of the astragaloside and the fingerprint as the investigation indexes, and determines the optimal process related parameters of the pharmaceutical composition such as pretreatment, heating mode, decoction time, filtration, drying and the like, thereby determining the optimal preparation method of the Baoyuan pharmaceutical composition and further improving the content of the effective components and the cream yield of the product. The best detection method is determined by searching the detection method of the Baoyuan medicinal composition, and the quality of the product is better controlled.
2. According to the invention, three batches of verification results show that the data of the extracting solution and the dry paste powder are basically parallel and the material transfer loss is small in the technical process by analyzing the total amount of the ginsenoside Rg1, Rb1 and Re and the total amount of the astragaloside IV; three batches verify that the dry paste powder has no obvious difference by analyzing extracts and water. When the fingerprint is analyzed, peaks 2, 3, 6 and 10 are licorice chromatographic peaks, and peaks 1, 4, 5, 8, 11 and 12 are astragalus chromatographic peaks. The peak No. 1 is temporarily taken as a reference peak to calculate the relative retention time, and the result shows that the common peak in the chromatogram of the medicinal materials, the decoction pieces, the extracting solution and the dry paste powder has the relative retention time RSD less than 2.0 percent, so that the determined substance standard preparation process is stable and feasible.
3. According to the invention, two heating modes of open fire and an electronic decoction pot are investigated through process research, and the two heating modes are not different from the comprehensive analysis of the decoction time, the paste yield, the total amount of ginsenoside Rg1, Rb1 and Re and the content of astragaloside. Researching the decoction time instead of the volume of the extractive solution as the decoction endpoint, determining the decoction time of the composition as reference, preferably 60min, of 30-60min, and the total content of ginsenoside Rg1, Rb1 and Re in the extractive solution is 0.106 mg/ml; the content of astragaloside in the extract is 0.031 mg/g; the total content of transfer rate ginsenoside Rg1, Rb1 and Re is 27.32%; the highest total content of the transfer rate astragaloside reaches 24.86 percent; the solid content was 0.064 g/mL.
4. The water absorption weight of the mixed medicinal materials tends to be stable after the soaking time of the mixed medicinal materials is 40min, so the optimal extraction soaking time of the Baoyuan decoction medicinal materials is 40 min.
5. As can be seen from the consideration of water addition amount, the water addition amount of the material standard of the Baoyuan medicinal composition is determined to be 1920-3020ml, and the optimal decoction water addition amount is 7 times of water, namely 1920 ml. The total content of the ginsenoside Rg1, Rb1 and Re in the extract is 0.078 mg/g; the content of the extracting solution is 13.61 mg/g; the total content of transfer rate ginsenoside Rg1, Rb1 and Re is 17.70 percent; the highest total content of the transfer rate of the astragaloside reaches 26.25 percent; the solid content was 0.065 g/mL.
6. According to the invention, through a filtration experiment, a 200-mesh filter cloth is preferably selected for a filtration method for filtration, and the clarity are clear.
7. According to the invention, by observing a concentration method, compared with normal-pressure concentration and vacuum concentration, a vacuum method is preferentially adopted for concentration, and the highest total content of the ginsenosides Rg1, Rb1 and Re is 0.351 mg/ml; the content of astragaloside in the extract can reach 0.089mg/g at most; the total content of transfer rate ginsenoside Rg1, Rb1 and Re is 17.07%; the highest content of the astragaloside reaches 23.10 percent; the solid content was 0.235 g/mL. The freeze-dried powder is not foamed and melted, and the texture of the dry paste powder is loose.
8. According to the invention, through the investigation of the concentration vacuum degree, the concentration vacuum degree is determined as follows: concentrating under-0.08 MPa to-0.04 MPa and the best vacuum degree is-0.08 MPa to-0.06 MPa. The total content of ginsenoside Rg1, Rb1 and Re can reach up to 0.390 mg/ml; the content of astragaloside in the extract can reach 0.103mg/g at most; the total content of transfer rate ginsenoside Rg1, Rb1 and Re is 18.98 percent; the highest content of the astragaloside reaches 27.14 percent; the solid content was 0.248%.
9. According to the invention, through the investigation of concentration temperature, the concentration temperature is determined to be 46-60 ℃, the optimal concentration temperature is 55-60 ℃, and the highest total content of the ginsenosides Rg1, Rb1 and Re is 0.487 mg/ml; the content of astragaloside in the extracting solution can reach 0.098mg/g at most; the total content of transfer rate ginsenoside Rg1, Rb1 and Re is 20.35%; the highest content of the transfer rate of the astragaloside reaches 25.06 percent; the solid content was 0.244%.
10. The invention is determined by process verification, the total content of the ginsenoside Rg1, Rb1 and Re is 2.306mg/g, and the content of the astragaloside IV is 0.890 mg/g.
11. Through a groping test of ginseng [ identification ], the sample application amount of the ginsenoside Rg1, Rb1, Re reference substance solution and ginseng decoction pieces is preferably 10 mu l, the sample application amount of the sample solution is 10 mu l, and color spots at corresponding positions are all clear and visible without trailing. And (3) durability inspection, namely, the durability inspection is carried out on the silica gel G thin-layer plates in low-temperature low-humidity, normal-temperature and high-temperature high-humidity environments and different manufacturers, and the durability of the ginseng thin-layer identification condition is good under different influence factors.
12. According to the invention, through a groping test of astragalus mongholicus [ identification ], the sample application amount of an astragaloside IV reference substance solution and astragalus mongholicus decoction pieces is preferably 10 mu l, the sample application amount of a sample solution is 5 mu l, 8 mu l and 10 mu l respectively, color spots at corresponding positions are clear and visible, no tailing phenomenon occurs, durability is inspected, and durability inspection is performed on silica gel G thin layer plates in low-temperature low-humidity, normal-temperature and high-temperature high-humidity environments and different manufacturers, and the durability of the astragalus mongholicus thin layer identification condition is good under different influence factors.
13. In the invention, through a groping test of liquorice [ identification ], spots with the same color appear on the chromatogram of the test sample at the positions corresponding to the chromatograms of the liquorice control drug and the reference drug, no spots are seriously trailing, and therefore, the point sample amount of the test sample solution, the reference drug solution and the reference drug solution is preferably 10 mu l. Through durability investigation of low-temperature low-humidity, normal-temperature and high-temperature environments, the thin layer identification condition has good durability under different influence factors.
14. According to the invention, through a groping test on cinnamon [ identification ], spots with the same color appear on the chromatogram of a test sample at the positions corresponding to the chromatograms of a cinnamon reference medicinal material and a cinnamon reference medicinal material, no spots are seriously trailing, and therefore, the point sample amount of the test sample solution, the cinnamon reference medicinal material solution and the cinnamon reference medicinal material solution is preferably 10 mu l. Through durability investigation of low-temperature low-humidity, normal-temperature and high-temperature environments, the thin layer identification condition has good durability under different influence factors.
15. The invention determines the detection method of the fingerprint by researching the fingerprint, and has good spectrum separation degree and few impurity peaks. Through methodology investigation, comparison of a blank solvent spectrum and a chromatographic peak of a test solution shows that the blank is free of interference, and the instrument has good precision, stability and repeatability.
Drawings
FIG. 1 shows a comparison of fingerprint (Peak 1 (S): calycosin glucoside Peak 11: ammonium glycyrrhizinate).
FIG. 2 shows comparison of finger print of Baoyuan decoction by reference process verification (1, BYT-2019102401T; 2, BYT-2019102402T; 3, BYT-2019102403T; 4, BYT-2019102401N; 5, BYT-2019102402N; 6, BYT-2019102403N; 7, BYT-2019102401D; 8, BYT-2019102402D; 9, BYT-2019102403D; 10, licorice root solution; 11, ammonium glycyrrhizinate reference substance; 12, Astragalus root decoction pieces solution; 13, calycosin glucoside)
FIG. 3 is a transfer superposition view of herbal medicine-decoction pieces-extract liquid-substance standard quantity (from bottom to top: substance standard D-181016-01, concentrated solution N-181016-01, extract liquid T-181016-01, licorice decoction pieces GC-ZY-2017110301Y, radix astragali decoction pieces BS-MC-2017112201Y)
FIG. 4 shows the properties of different batches of corresponding real objects (lyophilized powder)
FIG. 5 fingerprint chromatogram-1
FIG. 6 fingerprint chromatogram-2
FIG. 7 fingerprint chromatogram-3
FIG. 8 fingerprint chromatogram-4
FIG. 9 fingerprint chromatogram-5
FIG. 10 measurement of finger-prints of different solvents (S1: 50% methanol extraction; S2: water-saturated n-butanol solution extraction; S3: ethyl acetate extraction)
FIG. 11 shows the chromatogram of the fingerprint decoction pieces (S1: Ginseng radix; S2: radix astragali decoction pieces; S3: Glycyrrhrizae radix; S4: cortex Cinnamomi; S5: calycosin glucoside; S6: test sample; S7: ammonium glycyrrhizinate; S8: blank solvent)
FIG. 12 chromatogram for the whole examination (S1: test solution; S2: calycosin glucoside control solution; S3: blank solvent)
Figure 13 fingerprint precision measurement chromatogram
FIG. 14 chromatogram for fingerprint stability measurement (S1: BYT-2019091604D-0 h; S2: BYT-2019091604D-3 h; S3: BYT-2019091604D-6 h; S4: BYT-2019091604D-9 h; S5: BYT-2019091604D-12 h; S6: BYT-2019091604D-24h)
FIG. 15 chromatogram obtained by repetitive fingerprint measurement (S1: BYT-2019091604D-1; S2: BYT-2019091604D-2; S3: BYT-2019091604D-3; S4: BYT-2019091604D-4; S5: BYT-2019091604D-5; S6: BYT-2019091604D-6)
FIG. 16 shows a common pattern of substance-based fingerprints of different batches-1 (R, control; S1, BYT-2019091001D; S2, BYT-2019091002 DS; 3, BYT-2019091003D; S4, BYT-2019091004D; S5, BYT-2019091005D; S6, BYT-2019091606D; S7, BYT-2019091807D; S8, BYT-2019091008D; S9, BYT-2019091009D; S10, BYT-2019092310D; S11, BYT-2019092311D; S12, BYT-2019092312D; S13, BYT-2019092413D; S14, BYT-2019092414D; S15, BYT-2019092415D)
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples.
EXAMPLE 1 preparation method
74g of ginseng, 148g of astragalus, 37g of liquorice, 15g of cinnamon and 20 ginger slices are placed in an electronic decoction pot, 1920ml of water is added, the decoction is carried out by covering, the strong fire boiling is changed into the slow fire boiling for keeping the micro boiling, the decoction is carried out for 1h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.06 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the liquid medicine to the total feeding amount of decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-20-45 deg.C, freeze-drying temperature-50-70 deg.C, and vacuum degree of less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), mixing, and granulating to obtain granule.
EXAMPLE 2 preparation method
74g of ginseng, 148g of astragalus, 37g of liquorice, 15g of cinnamon and 20 ginger slices are placed in an electronic decoction pot, 1920ml of water is added, the decoction is carried out by covering, the strong fire boiling is changed into the slow fire boiling for keeping the micro boiling, the decoction is carried out for 1h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.06 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the liquid medicine to the total feeding amount of decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-20-45 deg.C, freeze-drying temperature-50-70 deg.C, and vacuum degree of less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), mixing, and encapsulating to obtain capsule.
Example 3 preparation method
74g of ginseng, 148g of astragalus, 37g of liquorice, 15g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, 1920ml of water is added, the decoction pot is covered with a cover, strong fire boiling is carried out, the decoction is carried out on the mixture with slow fire for keeping micro boiling, the decoction is carried out for 1h, 200-mesh filter cloth is filtered while the mixture is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.06 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the liquid medicine to the total feeding amount of decoction pieces is 1: 3, and the temperature of the extract is pre-freezing: freeze-drying at-20-45 deg.C, freeze-drying at-50-70 deg.C and vacuum degree of less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), mixing, and tabletting to obtain tablet.
EXAMPLE 4 preparation method
74g of ginseng, 148g of astragalus, 37g of liquorice, 15g of cinnamon and 20 ginger slices are placed in an electronic decoction pot, 1920ml of water is added, the decoction is carried out by covering, the strong fire boiling is changed into the slow fire boiling for keeping the micro boiling, the decoction is carried out for 1h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.06 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the liquid medicine to the total feeding amount of decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-20-45 deg.C, freeze-drying at-50-70 deg.C and vacuum degree of less than 300Pa, collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), mixing, drying, and making into pill.
EXAMPLE 5 preparation method
74g of ginseng, 148g of astragalus, 37g of liquorice, 15g of cinnamon and 20 ginger slices are placed in an electronic decoction pot, 1920ml of water is added, the decoction is carried out by covering, the strong fire boiling is changed into the slow fire boiling for keeping the micro boiling, the decoction is carried out for 1h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.06 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the liquid medicine to the total feeding amount of decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-20-45 deg.C and-50-70 deg.C under vacuum degree of less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding 15 times of pure water, mixing, filtering, sterilizing, and bottling to obtain oral liquid.
Example 6 preparation method
74g of ginseng, 148g of astragalus, 37g of liquorice, 15g of cinnamon and 20 ginger slices are placed in an electronic decoction pot, 1920ml of water is added, the decoction is carried out by covering, the strong fire boiling is changed into the slow fire boiling for keeping the micro boiling, the decoction is carried out for 1h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.06 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the liquid medicine to the total feeding amount of decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-20-45 deg.C, freeze-drying temperature-50-70 deg.C, and vacuum degree less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding 28 times of water for injection, mixing, filtering, and sterilizing to obtain injection.
Example 7 preparation method
60g of ginseng, 120g of astragalus, 20g of liquorice, 10g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, 1500ml of water is added, the mixture is covered and decocted, the mixture is boiled with strong fire and is decocted with slow fire to keep micro-boiling, the decoction is carried out for 0.5h, 200-mesh filter cloth is filtered while the mixture is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the total feeding ratio of the liquid medicine to the decoction pieces is 1: 3, and the temperature of the extract is pre-freezing: freeze-drying at-15-50 deg.C, freeze-drying temperature-45-75 deg.C, vacuum degree less than 300Pa, collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), mixing, and granulating to obtain granule.
EXAMPLE 8 preparation method
60g of ginseng, 120g of astragalus, 20g of liquorice, 10g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, 1500ml of water is added, the mixture is covered and decocted, the mixture is boiled with strong fire and is decocted with slow fire to keep micro-boiling, the decoction is carried out for 0.5h, 200-mesh filter cloth is filtered while the mixture is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the total feeding ratio of the liquid medicine to the decoction pieces is 1: 3, and the temperature of the extract is pre-freezing: freeze-drying at-15-50 deg.C, freeze-drying temperature-45-75 deg.C, vacuum degree less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), and mixing well to obtain capsule.
Example 9 preparation method
60g of ginseng, 120g of astragalus, 20g of liquorice, 10g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, 1500ml of water is added, the mixture is covered and decocted, the mixture is boiled with strong fire and is decocted with slow fire to keep micro-boiling, the decoction is carried out for 0.5h, 200-mesh filter cloth is filtered while the mixture is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the total feeding ratio of the liquid medicine to the decoction pieces is 1: 3, and the temperature of the extract is pre-freezing: freeze-drying at-15-50 deg.C, freeze-drying temperature-45-75 deg.C, vacuum degree less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), and mixing well to obtain tablet.
EXAMPLE 10 preparation method
60g of ginseng, 120g of astragalus, 20g of liquorice, 10g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, 1500ml of water is added, the mixture is covered and decocted, the mixture is boiled with strong fire and is decocted with slow fire to keep micro-boiling, the decoction is carried out for 0.5h, 200-mesh filter cloth is filtered while the mixture is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the total feeding ratio of the liquid medicine to the decoction pieces is 1: 3, and the temperature of the extract is pre-freezing: freeze-drying at-15-50 deg.C, freeze-drying temperature-45-75 deg.C, and vacuum degree less than 300Pa, collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), mixing, drying, and making into pill.
EXAMPLE 11 preparation method
60g of ginseng, 120g of astragalus, 20g of liquorice, 10g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, 1500-3020 ml of water is added, the mixture is covered and decocted, the mixture is boiled with strong fire and is decocted with slow fire to keep micro-boiling, the decoction is carried out for 0.5-2 h, 200-mesh filter cloth is filtered while hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55-60 ℃, the concentration is carried out until the ratio of the liquid medicine to the total feeding amount of decoction pieces is 1: 3, and the temperature of the extract is pre-freezing: freeze-drying at-15-50 deg.C and-45-75 deg.C under vacuum degree of less than 300Pa, and collecting dry extract powder, or adding 15 times of pure water, mixing, filtering, sterilizing, and bottling to obtain oral liquid.
EXAMPLE 12 preparation method
60g of ginseng, 120g of astragalus, 20g of liquorice, 10g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, 1500ml of water is added, the mixture is covered and decocted, the mixture is boiled with strong fire and is decocted with slow fire to keep micro-boiling, the decoction is carried out for 0.5h, 200-mesh filter cloth is filtered while the mixture is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the total feeding ratio of the liquid medicine to the decoction pieces is 1: 3, and the temperature of the extract is pre-freezing: freeze-drying at-15-50 deg.C and-45-75 deg.C under vacuum degree of less than 300Pa, and collecting dry extract powder, or adding 28 times of water for injection after collecting dry extract powder, mixing, filtering, and sterilizing to obtain injection.
Example 13 preparation method
85g of ginseng, 165g of astragalus, 50g of liquorice, 20g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, water 3020ml of water is added, the decoction is carried out by covering, the decoction is carried out by boiling with strong fire and then decocting with slow fire for keeping micro-boiling, the decoction is carried out for 2h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the total feeding amount of liquid medicine to the decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-15-50 deg.C, freeze-drying temperature-45-75 deg.C, and vacuum degree less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), mixing, and granulating to obtain granule.
Example 14 preparation method
85g of ginseng, 165g of astragalus, 50g of liquorice, 20g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, water 3020ml of water is added, the decoction is carried out by covering, the decoction is carried out by boiling with strong fire and then decocting with slow fire for keeping micro-boiling, the decoction is carried out for 2h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the total feeding amount of liquid medicine to the decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-15-50 deg.C, freeze-drying temperature-45-75 deg.C, and vacuum degree of less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), mixing, and encapsulating to obtain capsule.
Example 15 preparation method
85g of ginseng, 165g of astragalus, 50g of liquorice, 20g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, water 3020ml of water is added, the decoction is carried out by covering, the decoction is carried out by boiling with strong fire and then decocting with slow fire for keeping micro-boiling, the decoction is carried out for 2h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the total feeding amount of liquid medicine to the decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-15-50 deg.C, freeze-drying temperature-45-75 deg.C, and vacuum degree of less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), mixing, and tabletting to obtain tablet.
EXAMPLE 16 preparation method
85g of ginseng, 165g of astragalus, 50g of liquorice, 20g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, water 3020ml of water is added, the decoction is carried out by covering, the decoction is carried out by boiling with strong fire and then decocting with slow fire for keeping micro-boiling, the decoction is carried out for 2h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the total feeding amount of liquid medicine to the decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-15-50 deg.C and-45-75 deg.C under vacuum degree of less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding soluble starch (the ratio of soluble starch to dry extract is 1: 1-1: 4), mixing, drying, and making into pill.
Example 17 preparation method
85g of ginseng, 165g of astragalus, 50g of liquorice, 20g of cinnamon and 20 pieces of ginger are placed in an electronic decoction pot, water 3020ml of water is added, the decoction is carried out by covering, the decoction is carried out by boiling with strong fire and then decocting with slow fire for keeping micro-boiling, the decoction is carried out for 2h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the total feeding amount of liquid medicine to the decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-15-50 deg.C and-45-75 deg.C under vacuum degree of less than 300Pa, and collecting dry extract powder, or adding 15 times of pure water, mixing, filtering, sterilizing, and bottling to obtain oral liquid.
EXAMPLE 18 preparation method
85g of ginseng, 165g of astragalus membranaceus, 50g of liquorice, 20g of cinnamon and 20 ginger slices are placed in an electronic decoction pot, water 3020ml of water is added, the mixture is covered and decocted, the mixture is boiled with strong fire and is decocted with slow fire to keep micro-boiling, the decoction is carried out for 0.5 to 2 hours, filter cloth with 200 meshes is filtered while hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.04 MPa and the temperature of 55 to 60 ℃, the concentration is carried out until the total feeding quantity ratio of the liquid medicine to the decoction pieces is 1: 3, and the temperature of the extract is pre-frozen: freeze-drying at-15-50 deg.C and-45-75 deg.C under vacuum degree of less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding 28 times of water for injection, mixing, filtering, and sterilizing to obtain injection.
Examples 1 to 18 were tested by any of the following test methods
Example 19 detection method
[ PROPERTIES ] the product is brown to tan powder, slightly fragrant, slightly bitter and sweet in taste.
[ IDENTIFICATION ] collecting powder 1g, adding chloroform 40ml, heating under reflux for 1 hr, discarding chloroform solution, volatilizing solvent from residue, adding water 0.5ml, stirring, adding saturated n-butanol 10ml, ultrasonic treating for 30min, collecting supernatant, adding 3 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dryness, and dissolving residue in methanol 1ml to obtain sample solution. Preparing 1g of ginseng reference medicinal material, and preparing a reference medicinal material solution by the same method. Adding methanol into ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 as reference solutions to obtain mixed solutions each containing 2mg of ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 in each 1 ml. Performing thin-layer chromatography (general rule 0502) test, collecting the above test solution, control solution, and control solution 1-2 μ 1 respectively, spotting on the same silica gel G thin-layer plate, spreading with chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) lower layer solution at temperature below 10 deg.C as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the color development of spots is clear, inspecting under sunlight and ultraviolet lamp (365nm), wherein in the chromatogram of the test solution, the same spots are developed at the positions corresponding to those of the control chromatogram, and the negative chromatogram has no spots at the positions corresponding to those of the control chromatogram.
(2) Collecting 1.5g of the product, adding 25mL of water, performing ultrasonic treatment for 30min, filtering, extracting the filtrate with water saturated n-butanol under shaking for 2 times, each time 25mL, mixing n-butanol extractive solutions, washing with ammonia test solution for 2 times, each time 30mL, collecting n-butanol solution, evaporating to dryness, and dissolving the residue with methanol lmL to obtain test solution. And adding water 50ml into 1.5g of astragalus control medicinal material, decocting for 30 minutes, filtering, and preparing the filtrate into a control medicinal material solution by the same method. Adding methanol into astragaloside IV control to obtain solution containing 2mg of astragaloside IV per lmL as control solution. Performing thin layer chromatography (0502 of the four ministerial general rules of the design reside in the Chinese pharmacopoeia 2015), sucking 10 μ l of each of the control solution, and the sample solution, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution at 10 deg.C or below as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; the fluorescent spots with the same color are observed under an ultraviolet lamp (365 nm).
(3) Taking 1g of the product and a negative sample without liquorice respectively, adding 40ml of water, carrying out ultrasonic treatment for 30 minutes, filtering, extracting for 2 times by shaking diethyl ether and 20ml each time, discarding the diethyl ether solution, extracting the water solution by shaking water-saturated n-butyl alcohol for 3 times by shaking and 20ml each time, combining the n-butyl alcohol solutions, extracting for 2 times by shaking and 30ml each time by using an ammonia test solution, combining the ammonia test solutions, and adjusting the pH value to 3-4 by using hydrochloric acid. Extracting with ethyl acetate under shaking for 2 times (30 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1ml methanol to obtain test solution. And (2) adding 50ml of water into 1g of (roasted) liquorice decoction pieces, decocting for 30 minutes, filtering, and preparing a control medicinal solution by the same method. Taking appropriate amount of liquiritin reference substance, adding 70% ethanol to make into solution containing 0.5mg per 1mL as reference substance solution. Performing thin-layer chromatography (general rule 0502) test, collecting the sample solution, Glycyrrhrizae radix-deficient negative sample solution, control solution, and control solution 10 μ l, respectively dropping on the same silica gel G thin-layer plate, spreading with chloroform-methanol-water (13:7:2) subnatant at 5-10 deg.C as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the color development of spots is clear, inspecting under sunlight and ultraviolet lamp (365nm), and displaying spots or fluorescent spots of the same color in the chromatogram of the sample at the positions corresponding to those of the control and control chromatograms.
(4) Taking 1g of the product and each of the arillus-deficient cinnamon negative freeze-dried powder, adding 10ml of water to dissolve the product, shaking and extracting with diethyl ether for 1 time, 25ml each time, combining the diethyl ether solution, volatilizing, adding 2ml of diethyl ether to the residue to dissolve the residue to obtain a test solution and an arillus-deficient cinnamon negative sample solution. Decocting 1g of cortex Cinnamomi decoction pieces in 50ml of water for 30min, filtering, and making into control medicinal solution. Then, a lauric acid control was prepared, and 1ml of a solution containing 0.2mg was prepared with 50% methanol as a control solution. Performing thin layer chromatography (0502 of the four parts of the pharmacopoeia of China 2015), sucking the sample solution, the pulp-deficient Cassia negative sample solution, the control solution and the control solution 3 μ l respectively, and dropping on the same silica gel GF254Developing on the thin layer plate with cyclohexane-diethyl ether-glacial acetic acid (5:5:0.3) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254nm) to show spots or fluorescent spots of the same color in the chromatogram of the sample at the positions corresponding to the chromatogram of the control material and the chromatogram of the control material;
[ EXAMINATION ] the water content should not exceed 12.0% (general method 0832 second method).
[ EXTRACT ] is measured by hot dipping method under the item of alcohol soluble extract measuring method (the general rule 2201 of the four departments in the pharmacopoeia of China 2015 edition), and the extract is 18% -81.0%.
[ fingerprinting ] was measured by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China).
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; a chromatographic column: InertSustain-AQ-C18, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, flow rate of 1mL/min, detection wavelength of 210nm, mobile phase gradient elution program as follows.
Mobile phase gradient elution procedure
Preparation of reference solution A proper amount of calycosin glucoside reference substance is precisely weighed, and 50% methanol is added to prepare 50.4592 μ g/ml solution containing calycosin glucoside per 1 ml.
Preparing a sample solution, precisely weighing about 2.5g of the product, placing the product in a 50mL triangular cone bottle, adding 50% methanol into 50mL of the triangular cone bottle, performing ultrasonic treatment for 45min, filtering, recovering a solvent from a filtrate under reduced pressure till the filtrate is nearly dry, dissolving residues in 20mL of water, extracting for 2 times with water saturated n-butyl alcohol solution, 25mL each time, combining n-butyl alcohol solutions, extracting for 1 time with 30mL of ammonia test solution, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residues in 2mL of water, performing column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating 95% ethanol eluent to dryness, dissolving the residues in 50% methanol, transferring to a 10mL volumetric flask, fixing the volume to the scale, shaking up, filtering, and taking a continuous filtrate to obtain the product.
The determination method comprises precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram for 70 min.
The fingerprint of the sample should respectively present corresponding chromatographic peaks with the same retention time of the chromatographic peak of the reference substance. Calculated according to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprint, the similarity of the sample fingerprint and the comparison fingerprint is not less than 0.85, and the comparison fingerprint is shown in figure 1.
[ CONTENT DETERMINATION ] Ginseng is determined by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China).
The chromatographic condition and system applicability test adopts octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, and water solution as mobile phase B, and the detection wavelength is 203nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1. Elution was carried out according to the mobile phase gradient elution procedure specified below.
Mobile phase gradient elution procedure
Preparation of reference solution A proper amount of ginsenoside Rg1, Rb1 and Re reference is precisely weighed, and methanol is added to obtain 0.2mg mixed solution containing ginsenoside Rg1, Rb1 and Re per 1ml to obtain the final product
Preparing a sample solution, precisely weighing about 1g of the product, dissolving in 12.5ml of water, placing in a separating funnel, extracting with chloroform for 3 times, 15ml each time, discarding a chloroform layer solution, extracting the solution with a water-saturated n-butanol solution for 3 times, 30ml each time, combining n-butanol solutions, washing with an ammonia test solution for 2 times, 30ml each time, discarding the ammonia test solution layer, washing the n-butanol solution with a water-saturated n-butanol solution water layer solution for 20ml, washing for 1 time, discarding a water layer solution, evaporating the n-butanol solution to dryness, dissolving residues with methanol, transferring to a 5ml volumetric flask, adding methanol to fix the volume to the scale, and shaking up to obtain the product.
The determination method comprises precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, determining, and recording chromatogram of 110 min.
The product contains ginsenoside Rg1 (C) per 1g of Ginseng radix42H72O14)、Rb1(C54H92O23)、Re(C48H82O18) The total content of (b) is 0.6 mg/g-2.4 mg/g.
[ CONTENT DETERMINATION ] radix astragali is determined by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China).
Chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; acetonitrile-water (35: 65) is used as a mobile phase; detection by an evaporative light scattering detector. The number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak.
Preparation of control solution A proper amount of astragaloside IV control is precisely weighed, and added with methanol to obtain solution containing 0.5mg per lml.
Preparing a test solution, precisely weighing 1.0g of the product, precisely adding 50ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 45 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, shaking up and extracting with water-saturated n-butanol for 4 times, 25ml each time, combining n-butanol extract, washing with ammonia test solution for 3 times, 30ml each time, discarding ammonia test solution washing liquor, separating n-butanol solution, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product.
The product contains astragaloside IV (C) per 1g of radix astragali41H68O14) The content of the active carbon is 0.10mg/g to 1.40 mg/g.
[ PACKAGE ] vacuum packaging bag is sealed.
[ STORAGE ] the seeds are stored in a cool and dry place.
Example 20 detection method
[ PROPERTIES ] the product is brown to tan powder, slightly fragrant, slightly bitter and sweet in taste;
[ IDENTIFICATION ] collecting 0.5g of the powder, adding 20ml of chloroform, refluxing under heating for 0.5 hr, discarding chloroform solution, volatilizing solvent from the residue, adding 0.5ml of water, stirring to moisten, adding 10ml of saturated n-butanol, ultrasonic treating for 20 min, collecting supernatant, adding 2 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dryness, and dissolving the residue in 1ml of methanol to obtain test solution; preparing 1g of Ginseng radix reference material, and making reference material solution by the same method; adding methanol into ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 as reference solutions to obtain mixed solutions each containing 2mg per 1 ml; performing thin-layer chromatography (0502 of the four ministry of the national pharmacopoeia 2015), sucking the sample solution, the reference medicinal material solution and the reference solution 1-2 mu 1 respectively, and respectively dropping the sample solution, the reference medicinal material solution and the reference solution on the same silica gel G thin-layer plate according to the proportion of 15: 40: 22: 10, taking a lower layer solution of chloroform-ethyl acetate-methanol-water placed at the temperature of below 6-15 ℃ as a developing agent, developing, taking out, airing, spraying a 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clearly developed, respectively placing the solution under sunlight and 365nm ultraviolet lamps for inspection, wherein in the chromatogram of a test sample, the same spots are developed on the positions corresponding to the chromatogram of a reference substance, and a negative chromatogram has no spots on the positions corresponding to the chromatogram of the reference substance;
(2) collecting 1g of the product, adding 15mL of water, performing ultrasonic treatment for 20 min, filtering, extracting the filtrate with water saturated n-butanol under shaking for 2 times (20 mL each time), mixing n-butanol extractive solutions, washing with ammonia test solution for 2 times (20 mL each time), collecting n-butanol solution, evaporating to dry, and dissolving the residue with methanol lmL to obtain test solution; taking 1-2 g of astragalus mongholicus reference medicinal material, adding 40ml of water, decocting for 20 minutes, filtering, and preparing a reference medicinal material solution from the filtrate by the same method; adding methanol into astragaloside IV reference substance to obtain solution containing 2mg of astragaloside IV per lmL as reference substance solution; performing thin layer chromatography (0502 of the four ministerial general rules of the design reside in the Chinese pharmacopoeia 2015), sucking 10 μ l of each of the reference solution, the reference medicinal material solution and the test solution, respectively dropping on the same silica gel G thin layer plate, developing with a lower layer solution of chloroform-methanol-water at a ratio of 13:7:2 and below 5 deg.C as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; observing under 365nm ultraviolet lamp to show fluorescent spots of the same color;
(3) taking 0.5g of each product and a negative sample lacking liquorice, adding 20ml of water, carrying out ultrasonic treatment for 20 minutes, filtering, extracting for 2 times by shaking diethyl ether and 10ml each time, discarding the diethyl ether solution, extracting the water solution by shaking water-saturated n-butyl alcohol for 2 times by shaking and 15ml each time, combining the n-butyl alcohol solutions, extracting for 2 times by shaking and 20ml each time by using an ammonia test solution, combining the ammonia test solutions, and adjusting the pH value to 3-4 by using hydrochloric acid; extracting with ethyl acetate for 2 times (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1ml methanol to obtain test solution and Glycyrrhrizae radix-deficient negative sample solution; taking 0.5g of honey-fried licorice root decoction pieces, adding 40ml of water, decocting for 20 minutes, filtering, and preparing a control medicinal solution by the same method; taking appropriate amount of liquiritin reference substance, adding 70% ethanol to obtain solution containing 0.5mg per 1mL as reference substance solution; performing thin layer chromatography (0502 of the four ministerial rules of the science of Chinese pharmacopoeia 2015), sucking the sample solution, the negative sample solution without liquorice, the reference medicinal material solution and the reference solution by 5 mul respectively, and respectively dropping the two solutions on the same silica gel G thin layer plate according to the proportion of 13:7:2, taking the subnatant of chloroform-methanol-water placed at 5 ℃ as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the color development of the spots is clear, and observing under sunlight and 365nm ultraviolet lamps, wherein spots or fluorescent spots with the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material;
(4) taking 0.5g of the product and each of the lyophilized powder for negative samples of the lean osmanthus, adding 5ml of water to dissolve the product, shaking and extracting with diethyl ether for 2 times, 15ml each time, mixing the diethyl ether solution, volatilizing, and adding 2ml of diethyl ether to the residue to dissolve the residue to obtain a sample solution and a sample solution for negative samples of the lean osmanthus; decocting cortex Cinnamomi decoction pieces 0.5g in water 40ml for 20 min, filtering, and making into control medicinal solution by the same method; taking a cinnamic acid reference substance, and preparing 1ml of 0.2mg solution with 40% methanol as a reference substance solution; performing thin layer chromatography (0502 of the four parts of the pharmacopoeia of China 2015 edition), collecting the above sample solution, cortex Cinnamomi negative sample solution, control solution, and control solution 3 μ l respectively, and dropping on the same silica gel GF254Developing on a thin layer plate with cyclohexane-diethyl ether-glacial acetic acid as developing agent at a ratio of 5:5:0.3, taking out, air drying, inspecting under 254nm ultraviolet lamp, and displaying spots or fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution;
[ EXAMINATION ] the water content can not exceed 12.0% according to the second method (0832, the general rule of the four departments in the 'Chinese pharmacopoeia' 2015 edition);
[ EXTRACT ] is measured by hot dipping method under the item of alcohol soluble EXTRACT determination method (the general rule 2201 of the four departments in the pharmacopoeia of China 2015 edition), the extractive is 18% -81.0%;
[ fingerprinting ] measuring by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China);
octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; a chromatographic column: InertSustain-AQ-C18, acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution is taken as a mobile phase B, the flow rate is 1mL/min, the detection wavelength is 200nm, and the specific gradient elution procedure is as follows:
mobile phase gradient elution procedure
Preparation of reference substance solution A proper amount of calycosin glucoside reference substance is precisely weighed, and 40-60% methanol is added to prepare 50.4592 mug/ml solution of calycosin glucoside per 1ml, so as to obtain the reference substance;
preparing a sample solution, precisely weighing 1.5g of the product, placing the product in a 50mL triangular cone bottle, adding 40mL of 40% methanol, carrying out ultrasonic treatment for 30min, filtering, recovering a solvent from a filtrate under reduced pressure till the solvent is nearly dry, dissolving residues in 20mL of water, extracting for 2 times with a water saturated n-butyl alcohol solution, 15mL each time, combining the n-butyl alcohol solutions, extracting for 1 time with 20mL of an ammonia test solution, removing the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residues in 2mL of water, carrying out column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating the 95% ethanol eluate to dryness, dissolving the residues in 40-60% methanol, transferring to a 10mL volumetric flask, shaking up, filtering, and taking a subsequent filtrate to obtain the product;
the determination method comprises precisely sucking reference solution and test solution 10 μ L respectively, injecting into liquid chromatograph, determining, and recording chromatogram for 70 min;
corresponding chromatographic peaks with same retention time of reference substance chromatographic peaks are respectively presented in the fingerprint of the test sample; calculated according to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprint, the similarity of the sample fingerprint and the comparison fingerprint is not less than 0.85, and the comparison fingerprint is shown in figure 1.
[ content measurement ]: measuring Ginseng radix by high performance liquid chromatography (0512 in the four ministry of communications in 2015 pharmacopoeia);
in chromatographic conditions and system applicability tests, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, an aqueous solution is used as a mobile phase B, the detection wavelength is 190nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1; performing gradient elution according to a specified gradient elution procedure, wherein the gradient elution procedure comprises the following steps:
mobile phase gradient elution procedure
Preparing reference solution by accurately weighing appropriate amount of ginsenoside Rg1, Rb1 and Re reference, and adding methanol to obtain 0.2 mg/1 ml mixed solution containing ginsenoside Rg1, Rb1 and Re;
preparing a test solution, precisely weighing 0.5g of the product, dissolving in 12.5ml of water, placing in a separating funnel, extracting with chloroform for 2 times (10 ml each time), discarding a chloroform layer solution, extracting the solution with a water-saturated n-butanol solution for 2 times (20 ml each time), combining n-butanol solutions, washing with an ammonia test solution for 2 times (20 ml each time), discarding the ammonia test solution layer, washing the n-butanol solution with a water-saturated n-butanol solution for 10ml, washing for 1 time, discarding a water layer solution, evaporating the n-butanol solution to dryness, dissolving residues with methanol, transferring to a 5ml volumetric flask, adding methanol to a constant volume to scale, and shaking up to obtain the product;
the determination method comprises precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram of 110 min;
the product contains 0.6 mg/g-2.4 mg/g of ginseng in each 1g of dry product, calculated by the total content of ginsenoside Rg1, Rb1 and Re;
[ CONTENT DETERMINATION ] measuring radix astragali by high performance liquid chromatography (0512 in the four ministry of communications in 2015 pharmacopoeia of China);
chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; acetonitrile-water (35: 65) is used as a mobile phase; detecting by an evaporative light scattering detector; the number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak;
preparing reference solution by accurately weighing appropriate amount of astragaloside IV reference, and adding methanol to obtain solution containing 0.5mg per lml;
preparing a test solution, precisely weighing 0.5g of the product, precisely adding 40ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 30 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of a subsequent filtrate, shaking up and extracting with water-saturated n-butanol for 2 times (20 ml each time), combining the n-butanol extract, washing with an ammonia test solution for 2 times (20 ml each time), discarding the ammonia test solution washing liquid, separating the n-butanol solution, evaporating to dryness, dissolving the residue with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product.
The product contains astragaloside IV (C) per 1g of radix astragali41H68O14) The content of the active carbon is 0.10mg/g to 1.40 mg/g.
Example 21 detection method
[ PROPERTIES ] the product is brown to tan powder, slightly fragrant in smell, slightly bitter and sweet in taste;
[ IDENTIFICATION ] collecting powder 1.5g, adding chloroform 60ml, heating under reflux for 2 hr, discarding chloroform solution, volatilizing solvent from residue, adding water 0.5ml, stirring, moistening, adding saturated n-butanol 10ml, ultrasonic treating for 40min, collecting supernatant, adding 4 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating, and dissolving residue in methanol 1ml to obtain test solution; preparing 1g of Ginseng radix reference material, and making reference material solution by the same method; adding methanol into ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 as reference solutions to obtain mixed solutions each containing 2mg per 1 ml; performing thin-layer chromatography (0502 of the four ministry of the national pharmacopoeia 2015), sucking the sample solution, the reference medicinal material solution and the reference solution 1-2 mu 1 respectively, and respectively dropping the sample solution, the reference medicinal material solution and the reference solution on the same silica gel G thin-layer plate according to the proportion of 15: 40: 22: 10, taking a lower layer solution of chloroform-ethyl acetate-methanol-water placed below 15 ℃ as a developing agent, developing, taking out, airing, spraying a 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clearly developed, respectively placing the solution under sunlight and 365nm ultraviolet lamps for inspection, wherein in the chromatogram of a test sample, the same spots are developed on the positions corresponding to the chromatogram of a reference substance, and a negative chromatogram has no spots on the positions corresponding to the chromatogram of the reference substance;
(2) taking 2g of the test sample, adding 35mL of water, carrying out ultrasonic treatment for 40 minutes, filtering, extracting the filtrate with water saturated n-butanol for 4 times, 30mL each time, combining n-butanol extract, washing with ammonia test solution for 4 times, 40mL each time, separating n-butanol solution, evaporating to dryness, and adding methanol lmL into residues to dissolve the residues to obtain a test sample solution; taking 1-2 g of astragalus mongholicus reference medicinal material, adding 60ml of water, decocting for 40 minutes, filtering, and preparing a reference medicinal material solution from the filtrate by the same method; adding methanol into astragaloside IV reference substance to obtain solution containing 2mg of astragaloside IV per lmL as reference substance solution; performing thin layer chromatography (0502 of the four ministerial general rules of the design reside in the Chinese pharmacopoeia 2015), sucking 10 μ l of each of the reference solution, the reference medicinal material solution and the test solution, respectively dropping on the same silica gel G thin layer plate, developing with a lower layer solution of chloroform-methanol-water at a ratio of 13:7:2 and below 15 deg.C as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; observing under 365nm ultraviolet lamp to show fluorescent spots of the same color;
(3) taking 1.5g of each product and a negative sample lacking liquorice, adding 60ml of water, carrying out ultrasonic treatment for 40 minutes, filtering, extracting with diethyl ether for 4 times and 30ml each time, discarding the diethyl ether solution, extracting the water solution with water-saturated n-butyl alcohol for 4 times and 25ml each time, combining the n-butyl alcohol solutions, extracting with ammonia test solution for 4 times and 40ml each time, combining the ammonia test solutions, and adjusting the pH value to 3-4 with hydrochloric acid; extracting with ethyl acetate by shaking for 4 times (40 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1ml methanol to obtain test solution and Glycyrrhrizae radix-deficient negative sample solution; adding water 60ml into radix Glycyrrhizae Preparata decoction pieces 1.5g, decocting for 40min, filtering, and making into control medicinal solution by the same method; taking appropriate amount of liquiritin reference substance, adding 70% ethanol to obtain solution containing 0.5mg per 1mL as reference substance solution; performing thin layer chromatography (0502 of the four ministerial rules of the science of Chinese pharmacopoeia 2015), sucking 10 μ l of each of the test solution, the liquorice-lacking negative sample solution, the control solution and the control solution, respectively dropping the solution on the same silica gel G thin layer plate, and performing thin layer chromatography on the solution at the ratio of 13:7:2, taking the subnatant of chloroform-methanol-water placed at 10 ℃ as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the color development of the spots is clear, and observing under sunlight and 365nm ultraviolet lamps, wherein spots or fluorescent spots with the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material;
(4) taking 1.5g of the product and each of the arillus-deficient cinnamon negative freeze-dried powder, adding 15ml of water to dissolve the product, shaking and extracting with diethyl ether for 3 times, 35ml each time, combining the diethyl ether solution, volatilizing, adding 2ml of diethyl ether to the residue to dissolve the residue to obtain a test solution and an arillus-deficient cinnamon negative sample solution; decocting 1.5g cortex Cinnamomi decoction pieces in 60ml water for 40min, filtering, and making into control medicinal solution by the same method; taking a cinnamic acid reference substance, and preparing 1ml of 0.2mg solution with 65% methanol as a reference substance solution; performing thin layer chromatography (0502 of the four parts of the pharmacopoeia of China 2015 edition), collecting the above sample solution, cortex Cinnamomi negative sample solution, control solution, and control solution 3 μ l respectively, and dropping on the same silica gel GF254Developing on a thin layer plate with cyclohexane-diethyl ether-glacial acetic acid as developing agent at a ratio of 5:5:0.3, taking out, air drying, inspecting under 254nm ultraviolet lamp, and displaying spots or fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution;
[ EXAMINATION ] the water content can not exceed 12.0% according to the second method (0832, the general rule of the four departments in the 'Chinese pharmacopoeia' 2015 edition);
[ EXTRACT ] is measured by hot dipping method under the item of alcohol soluble EXTRACT determination method (the general rule 2201 of the four departments in the pharmacopoeia of China 2015 edition), the extractive is 18% -81.0%;
[ fingerprinting ] measuring by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China);
octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; a chromatographic column: InertSustain-AQ-C18, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, flow rate of 1mL/min, detection wavelength of 220nm, specific gradient elution program:
mobile phase gradient elution procedure
Preparing reference solution by precisely weighing appropriate amount of calycosin glucoside reference, and adding 60% methanol to obtain solution containing calycosin glucoside 50.4592 μ g/ml per 1 ml;
preparing a test solution, precisely weighing 3.5g of the product, placing the product in a 50mL triangular cone bottle, adding 60mL of 60% methanol, performing ultrasonic treatment for 60min, filtering, recovering the solvent from the filtrate under reduced pressure till the solution is nearly dry, dissolving the residue in 20mL of water, extracting for 3 times with water-saturated n-butyl alcohol solution, 35mL each time, combining the n-butyl alcohol solutions, extracting for 1 time with 40mL of ammonia test solution, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residue in 2mL of water, performing column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating the 95% ethanol eluate to dryness, dissolving the residue in 60% methanol, transferring to a 10mL volumetric flask, fixing the volume to the scale, shaking up, filtering, and taking a continuous filtrate to obtain the product;
the determination method comprises precisely sucking reference solution and test solution 10 μ L respectively, injecting into liquid chromatograph, determining, and recording chromatogram for 70 min;
corresponding chromatographic peaks with same retention time of reference substance chromatographic peaks are respectively presented in the fingerprint of the test sample; calculating according to the similarity evaluation system of traditional Chinese medicine chromatogram fingerprint, wherein the similarity between the sample fingerprint and the comparison fingerprint is not less than 0.85, and the comparison fingerprint is shown in figure 1;
[ content measurement ]: measuring Ginseng radix by high performance liquid chromatography (0512 in the four ministry of communications in 2015 pharmacopoeia);
in chromatographic conditions and system applicability tests, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, an aqueous solution is used as a mobile phase B, the detection wavelength is 210nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1; performing gradient elution according to a specified gradient elution procedure, wherein the gradient elution procedure comprises the following steps:
mobile phase gradient elution procedure
Preparing reference solution by accurately weighing appropriate amount of ginsenoside Rg1, Rb1 and Re reference, and adding methanol to obtain 0.2 mg/1 ml mixed solution containing ginsenoside Rg1, Rb1 and Re;
preparing a sample solution, precisely weighing 1.5g of the product, dissolving in 12.5ml of water, placing in a separating funnel, extracting with chloroform for 4 times, 20ml each time, discarding a chloroform layer solution, extracting the solution with a water-saturated n-butanol solution for 4 times, 40ml each time, combining n-butanol solutions, washing with an ammonia test solution for 4 times, 40ml each time, discarding the ammonia test solution layer, washing the n-butanol solution with a water-saturated n-butanol solution for 30ml, washing for 1 time, discarding a water layer solution, evaporating the n-butanol solution to dryness, dissolving residues with methanol, transferring to a 5ml volumetric flask, adding methanol to a constant volume to reach a scale, and shaking up to obtain the product;
the determination method comprises precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram of 110 min;
the product contains 0.6 mg/g-2.4 mg/g of ginseng in each 1g of dry product, calculated by the total content of ginsenoside Rg1, Rb1 and Re;
[ CONTENT DETERMINATION ] measuring radix astragali by high performance liquid chromatography (0512 in the four ministry of communications in 2015 pharmacopoeia of China);
chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; acetonitrile-water (35: 65) is used as a mobile phase; detecting by an evaporative light scattering detector; the number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak;
preparing reference solution by accurately weighing appropriate amount of astragaloside IV reference, and adding methanol to obtain solution containing 0.5mg per lml;
preparing a test solution, precisely weighing 1.5g of the product, precisely adding 60ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 60 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of a subsequent filtrate, shaking up and extracting with water-saturated n-butanol for 6 times (40 ml each time), combining the n-butanol extract, washing with an ammonia test solution for 4 times (40 ml each time), discarding the ammonia test solution washing liquid, taking out the n-butanol solution for drying by distillation, dissolving the residue with methanol, transferring the residue into a 5ml measuring flask, adding methanol for diluting to a scale, and shaking up to obtain the product.
The product contains astragaloside IV (C) per 1g of radix astragali41H68O14) The content of the active carbon is 0.10mg/g to 1.40 mg/g.
To further verify the effectiveness of the present invention, the inventors conducted the following tests, specifically as follows:
1 Baoyuan pharmaceutical composition substance benchmark study
1.1 Process description and flow sheet
1.1.1 prescription
3.69g of ginseng, 7.38g of astragalus root, 1.85g of liquorice, 0.74g of cinnamon and one piece of ginger
1.1.2 method of making a material reference
Adding ginger, amplifying the formula amount by 20 times, namely 74g of ginseng, 148g of astragalus, 37g of liquorice, 15g of cinnamon and 20 pieces of ginger, soaking for 40min according to the water addition standard of the granular rhizome medicinal materials in the traditional Chinese medicine formula, wherein the water addition is 7 times of the formula amount, namely 1920ml, filtering by using filter cloth with 1h and 200 meshes, performing low-temperature vacuum concentration on the filtrate to obtain an extract with the feeding amount ratio of 1: 3, performing low-temperature freeze drying to obtain a substance reference corresponding substance, and storing the substance reference substance in a low-temperature drying place.
1.1.4 procedures
1.1.4.1 times of decoction and dosage per decoction
According to the record of the original text, the decocting times are one time, and the amount of the fed decoction is seven times of the daily prescription amount. In order to keep consistency with the ancient book recording process, the decoction times are determined to be one time, and the feeding amount of each decoction is seven times of the daily prescription amount.
1.1.4.2 decoction piece pretreatment
The prescription of the Baoyuan decoction comprises two medicines, namely ginseng and liquorice. In the prescription, the specification of the ginseng decoction pieces is 2mm thin slices, the specification of the astragalus membranaceus decoction pieces is 2-5 mm thick slices, the specification of the liquorice decoction pieces is 2-5 mm thick slices, and the specifications of the cinnamon decoction pieces are different.
1.1.4..3 decoction procedure
(1) Investigation of heating mode
Taking the total amount of ginsenoside Rg1, Rb1 and Re in the extracting solution, the total amount of extracted astragaloside IV and the extraction rate as investigation indexes, and carrying out single-factor comparison test research on two heating modes of open fire and an electronic decoction pot. The two heating modes are different from the comprehensive analysis of the decoction time, the paste yield and the total index components.
(2) Observation of decoction time
Because the decoction end point in the prescription original text is the volume of the extracting solution, the operation is inconvenient in the decoction process by taking the volume of the extracting solution as the decoction end point, and the fixed heating mode is adopted, the research is carried out on the decoction time as the decoction end point instead of the volume of the extracting solution.
1.1.4. 4 investigation of filtration, concentration and drying processes
The extracting solution is inspected by a normal pressure filtration process, and the clarity of the liquid medicine is taken as an index. As a result: the extract is filtered by 200-mesh filter cloth while hot, and the filtrate is clear and free of impurities. Determining the filtering mode as follows: filtering with 200 mesh filter cloth under normal pressure.
Because the volume of the extracting solution is about 1000ml, the extracting solution is concentrated under normal pressure and reduced pressure, and the total amount of the ginsenoside Rg1, Rb1 and Re in the concentrated solution, the content of astragaloside and the transfer rate are used as the investigation indexes. As a result: the content has no obvious difference, and the content and the transfer rate of the vacuum concentration are slightly higher than those of the normal pressure concentration. The concentration mode is determined as follows: concentrating under reduced pressure to obtain extract at ratio of 1: 3.
And (4) freeze-drying the concentrated solution, wherein loose dry paste powder can be collected after the concentrated solution is freeze-dried, and the freeze-drying mode is feasible.
1.1.4..5 packaging
Vacuum sealing bag for inner packing material, and sealing;
1.1.4..6 storage
Storing in a shady and cool warehouse.
1.1.5 basis for determination of Process parameters
According to the methods described in the classical name list: decocting Ginseng radix, radix astragali, Glycyrrhrizae radix, cortex Cinnamomi, rhizoma Zingiberis recens and water. The research adopts an electronic decoction pot for decoction, and researches related parameters of the processes such as pretreatment of material standard decoction pieces, a heating mode, decoction time, filtration, drying and the like by taking the paste yield, the total amount of ginsenoside Rg1, Rb1 and Re, the content of astragaloside and a fingerprint as indexes, thereby determining the material standard process of the Baoyuan decoction.
1.1.5.1 pretreatment
The prescription of the Baoyuan decoction comprises four medicines, namely ginseng, astragalus, liquorice and cinnamon, which are in the specification of decoction pieces and are suitable for decoction of decoction.
1.1.5.2 decocting Process
The process researches and researches two heating modes of open fire and an electronic decoction pot, and the results show that the two heating modes are not different from the comprehensive analysis of the decoction time, the paste yield, the total amount of ginsenoside Rg1, Rb1 and Re and the content of astragaloside. And (3) researching the decoction time instead of the volume of the extracting solution as a decoction endpoint, and determining the standard decoction time of the Baoyuan decoction substance to be 1 h.
1.1.5.3 Process of filtering, concentrating and drying
The solid-liquid separation is determined and filtered by 200-mesh filter cloth when the solid-liquid separation is hot. The volume of the extracting solution is about 1000ml, the extracting solution is concentrated under reduced pressure to an extract with the ratio of the liquid medicine to the total feeding amount of the decoction pieces being 1: 3, the concentrated solution can be collected into loose dry paste powder after being subjected to freeze drying (the pre-freezing temperature is between minus 20 and minus 45 ℃, the freeze drying temperature is between minus 50 and minus 70 ℃, and the vacuum degree is less than 300Pa), and the freeze drying mode is determined to be feasible.
1.1.5.4 Process verification procedure
The method comprises the steps of sampling three batches of medicinal materials, decoction pieces, extracting solution and substance references corresponding to the substance references, determining index component content and fingerprint, analyzing from the total amount of ginsenoside Rg1, Rb1 and Re and the content of astragaloside, verifying that the extracting solution and substance reference data are basically parallel, and the material transfer loss in the process is small, wherein the common peaks in the chromatogram of the medicinal materials, the decoction pieces, the extracting solution and the substance references are analyzed from the fingerprint, and the relative retention time RSD is less than 2.0%.
1.1.5.5 packaging
Vacuum sealing the inner packaging material in a bag, and sealing.
1.1.5.6 storage
Storing in a shade storehouse.
1.1.6 Main Equipment and model
TABLE 1 Equipment model
1.2 study of the Process
1.2.1 pretreatment
According to the ancient book original texts and the examination results, decoction pieces used in the prescription are prepared from medicinal materials according to the corresponding processing standards, wherein the specification of the decoction pieces of ginseng is 2mm thin slices, the specification of the decoction pieces of astragalus membranaceus is 2-5 mm thick slices, the specification of the decoction pieces of liquorice is 2-5 mm thick slices, the decoction pieces of cinnamon are obtained, and the research decoction piece information is shown in table 2.
TABLE 2 decoction piece information for research
1.2.2 decoction
1.2.2.1 examination of Water absorption
The water absorption test of the medicinal materials is carried out because the medicinal part of the ginseng is root, the medicinal part of the astragalus root is root and rhizome, the medicinal part of the liquorice is root and rhizome, and the medicinal part of the cinnamon is dry bark, which are very easy to absorb water.
The experimental method comprises the following steps: taking 74g of ginseng decoction pieces, 148g of astragalus decoction pieces, 37g of liquorice decoction pieces and 15g of cinnamon decoction pieces, uniformly mixing, adding 7 times of water (1920ml), filtering at regular time, weighing and recording the weight of the filtered medicinal materials, and calculating the water absorption weight of the mixed medicinal materials at different times. The results are shown in Table 3:
table 3 water absorption test investigation table
The test result shows that: the soaking time of the mixed medicinal materials is stable after 40min, so the soaking time of the Baoyuan decoction is preferably 40 min.
1.2.2.2 investigation of water addition
The water adding amount of decoction is not specified in the prescription of the Baoyuan decoction, so the water adding amount of the decoction is considered firstly, and three gradients of 7 times, 9 times and 11 times of the water adding amount of the design prescription are considered. The prescription is enlarged twenty times, namely 74g of ginseng (batch number: RS-FS-2018070201Y), 148g of astragalus (batch number: HQ-LX-2017110801Y), 37g of liquorice (batch number: GC-DX-2017110401Y) and 15g of cinnamon (batch number: RG-PN-2018090701Y) are respectively taken and tested according to the water adding amount of 7 times (1920ml), 9 times (2470ml) and 11 times (3020ml), and the test is examined according to the factor table of the water adding amount of decoction, which is shown in Table 4.
TABLE 4 ingredient table for amount of water added for decoction
The values of the factors are ginsenoside Rg1, Rb1, total content of Re, content of astragaloside IV and transfer rate; the solid content is used as the index for investigation, and the detailed results are shown in Table 5
TABLE 5 summary of the factor and content of the amount of water added for decoction
And (4) conclusion: as can be seen from the consideration of water adding amount, the difference between the total content of ginsenoside Rg1, Rb1, Re, astragaloside IV and transfer rate is not obvious, the solid content is 7 times the water adding amount is better, and the optimal water adding amount of 7 times of the standard decoction of Baoyuan decoction, namely 1920ml, is determined by combining the management specification of traditional Chinese medicine decoction rooms in medical institutions.
1.2.2.3 examination of heating methods
The ancient decocting mode is open fire decocting, an electric stove is used as the open fire heating mode, meanwhile, the heating effect of an electronic decocting pot is more stable under the consideration that the heating mode of the electronic decocting pot controls the heating efficiency through power, so that the heating modes of the open fire and the electronic decocting pot are considered by adopting the paste yield, the total amount of ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re and the content of astragaloside as indexes, and if the results of the two heating modes are not different, the electronic decocting pot is selected as the heating mode.
Weighing 4 parts of 74g of ginseng (batch number: RS-FS-2018070201Y), 148g of astragalus (batch number: HQ-LX-2017110801Y), 37g of liquorice (batch number: GC-DX-017110401Y), 15g of cinnamon (batch number: RG-PN-2018090701Y), adding 1920ml of water, placing the mixture into an electronic decoction pot, covering the electronic decoction pot for decoction, decocting the mixture with strong fire until the mixture is boiled, decocting the mixture with slow fire until the mixture is 1000ml, filtering the extracting solution by using 200-mesh filter cloth, and recording the volume of the extracting solution; decocting with open fire and electronic decocting pot (2200W for strong fire and 400W for slow fire) to obtain two extractive solutions, measuring total amount of ginsenoside Rg1, Rb1 and Re, astragaloside A content and extraction rate in the extractive solution according to index component measurement method, and examining influence of heating modes of open fire and electronic decocting pot (2200W for strong fire and 400W for slow fire) on index components. The results are shown in Table 6. The method for measuring the cream yield of the intermediate (extract liquid) comprises the following steps: precisely measuring 20ml of the extractive solution, placing in an evaporating dish W1 dried to constant weight, evaporating to dryness in water bath, drying at 105 deg.C for 3 hr, cooling in a desiccator for 30min, and rapidly precisely weighing W2, as detailed in Table 6.
TABLE 6 examination of heating methods
Test results show that the content determination indexes of the open fire heating mode and the electronic decoction pot have no significant difference in total amount, but the open fire heating mode has shorter decoction time than the electronic decoction pot, the paste yield rate is different, comprehensive analysis shows that the difference of the two heating modes is smaller, and the electronic decoction pot is selected to be preferred for decoction based on convenience in decoction.
1.2.2.4 examination of the decoction time
Baoyuan Tang comes from brief doctor hub (Ming. Sunzhong), for primordial qi deficiency, mental lassitude, soft and slow muscles, short intake of food and unsmooth face
Bai, sleeping and quiet, … … and miscellaneous syndromes are all weak and should be taken. Is prepared from ginseng, astragalus root, liquorice root, cinnamon bark, etc. The decocting method recorded in the original text comprises the following steps: ginseng, astragalus root, licorice, cinnamon, ginger and water. The data unit in the 'preparation method and usage' in the Baoyuan decoction is converted with the modern data unit according to 'historic standard measurement and scale' and 'Chinese historic standard degree, quantity and scale evolution profile'. One piece of money is 3.69g at present, one piece is 0.37 g, namely the 'preparation method' of the Baoyuan decoction decocting process can be described as taking 3.69g of ginseng, 7.38g of astragalus, 1.85g of liquorice, 0.74g of cinnamon and 1 piece of ginger, adding 1920mL of water, soaking for 30min, heating and decocting, and filtering the dregs to obtain the Baoyuan decoction. According to the method recorded in the list of classical famous prescriptions, the ministry of health and the national administration of traditional Chinese medicineThe preparation of the Baoyuan decoction based on the decoction in the management standard (the traditional Chinese medicine issue (2009) 3) is close to the tradition and follows the ancient prescription principle. The heating decoction container is preferably an electronic decoction pot, and the decoction method of the ancient formula is followed, and the water adding amount and the decoction times are specified, so the decoction time is considered.
The original prescription is enlarged by 20 times, 74g of ginseng (batch number: RS-FS-2018070201Y), 148g of astragalus (batch number: HQ-LX-2017110801Y), 37g of liquorice (batch number: GC-DX-2017110401Y), 15g of cinnamon (batch number: RG-PN-2018090701Y) and 20 ginger tablets are respectively taken, and the experiments are carried out according to the decoction time factor table, and the table 7:
TABLE 7 decoction time factor Table
The values of all factors take the total amount of the ginsenosides Rg1, Rb1 and Re and the transfer rate of the astragaloside as investigation indexes, and detailed results are shown in a table 8:
TABLE 8 examination of the transfer Rate of decoction time
And (4) conclusion: in conclusion, the content, transfer rate and solid content of the extract can be known, the content of index components in the decoction is high after 1 hour of decoction, and the transfer rate and solid content are good, so the decoction time of the Baoyuan decoction is preferably 1 hour.
1.2.2.5 summary of the decoction Process
In conclusion, the optimal decocting process of the tentative Baoyuan decoction substance standard comprises the following steps: adding 1920ml of water into 74g of ginseng, 148g of astragalus mongholicus, 37g of liquorice, 15g of cinnamon and 20 pieces of ginger, putting the mixture into an electronic decoction pot, covering the pot for decoction, decocting the mixture with strong fire until the mixture is boiled, decocting the mixture with slow fire for 1 hour, and filtering the mixture through 200-mesh filter cloth to obtain the traditional Chinese medicine.
1.2.3 filtration, concentration and drying
1.2.3.1 examination of filtration methods
In order to avoid the loss of effective components in industrial production, the filtration method was examined.
The test method comprises the following steps: respectively taking 74g of 3 parts of ginseng decoction pieces (batch number: RS-DH-2018070201Y), 148g of astragalus decoction pieces (batch number: HQ-LF-2017110801Y), 37g of liquorice decoction pieces (batch number: GC-HM-2017110601Y), 15g of cinnamon decoction pieces (batch number: RG-RX-2018090701Y) and 20 pieces of ginger, adding 1920ml of water, soaking for 30min, heating and decocting for 1h, respectively filtering without filtering, filtering with 2 layers of gauze and filtering with 200-mesh filter cloth, and respectively calculating the solid content of 3 liquid medicines, wherein the results are shown in Table 9:
TABLE 9 filtration test results
And (4) conclusion: the filtering method has no obvious difference on the clarity of the liquid medicine, but the filtering method of the 'standard of the quality of the Baoyuan decoction' is preferably to use 200-mesh filter cloth for filtering because the original liquid and the solution filtered by 2 layers of gauze are easy to precipitate.
1.2.3.2 concentration method investigation
In order to ensure the minimum loss of active ingredients in the liquid medicine, the method is considered by combining the environmental protection, energy saving and consumption reduction and the actual conditions of enterprises by referring to the current situation and problem analysis of the traditional Chinese medicine extracting solution concentration process and equipment, and adopting two methods of normal-pressure heating concentration and vacuum low-temperature concentration.
The experimental method comprises the following steps: collecting 222g of ginseng decoction pieces (batch number: RS-DH-2018070201Y), 444g of astragalus membranaceus decoction pieces (batch number: HQ-LF-2017110801Y), 111g of liquorice decoction pieces (batch number: GC-HM-2017110601Y), 45g of cinnamon decoction pieces (batch number: RG-RX-2018090701Y) and 60 ginger slices, adding 5760mL of water, soaking for 40min, heating and decocting for 1h, filtering, respectively concentrating 700mL of extracting solution by adopting a normal pressure method and a vacuum method to 100mL, and facilitating the operation of the next working procedure. The solid content of 2 concentrates, the total content of ginsenoside Rg1, Rb1 and Re and the content of astragaloside are respectively calculated, the values of all factors take the total content of ginsenoside Rg1, Rb1 and Re and the transfer rate of astragaloside as investigation indexes, and the results are shown in a table 10:
TABLE 10 condensed method investigation data
The test result shows that: compared with the vacuum concentration, the vacuum concentration is slightly higher than the normal concentration in content and transfer rate. The solid content of the two concentrated solutions is not very different, the time required for normal pressure concentration is 4h, the time required for vacuum concentration is 2h, and the concentration is preferably carried out by adopting a vacuum method to concentrate the two concentrated solutions until the liquid medicine is about 100ml in consideration of the concentration time, environmental protection and energy conservation.
1.2.3.3 investigation of concentration vacuum
After the concentration method is determined to be low-temperature vacuum concentration, whether the vacuum degree influences the effective components or not is determined, so that the vacuum degree is investigated, concentration process parameters are further determined, environmental protection, energy conservation and consumption reduction are combined with the actual conditions of enterprises, and the minimum loss of the effective components in the concentration process is ensured, so that the 'standard of the quality of the Baoyuan soup' is concentrated by adopting a reduced pressure rotary evaporation concentration method, and the vacuum degree of concentration is investigated.
The experimental method comprises the following steps: taking the extracting solution tested by the concentration method, dividing into 4 parts, wherein each part of decoction is 700mL, concentrating by adopting three methods of vacuum degree of-0.04 MPa, -0.06MPa and-0.08 MPa respectively, and concentrating to 100mL so as to facilitate the operation of the next working procedure. The solid content of 3 concentrates, the total content of ginsenoside Rg1, Rb1 and Re and the content of astragaloside are respectively calculated, the values of all factors take the total content of ginsenoside Rg1, Rb1 and Re and the transfer rate of astragaloside as investigation indexes, and the results are shown in Table 11:
TABLE 11 concentration vacuum investigation data
The test result shows that: the vacuum degree has no obvious influence on the solid content, the concentration time of the vacuum degree of-0.04 MPa, -0.06MPa and-0.08 MPa is respectively 4.5h, 3.5h and 2h, and the time is less when the vacuum degree is larger. The total amount and transfer rate of the ginsenoside Rg1, Rb1 and Re are maximum at-0.06 MPa, the content and transfer rate of the astragaloside are maximum when the content and transfer rate of the astragaloside are-0.08 MPa and are higher than-0.04 MPa, and the concentration is carried out by adopting a method with the vacuum degree of-0.08 MPa to-0.06 MPa in consideration of the concentration time, environmental protection, energy conservation and maximum retention of effective components.
1.2.3.4 examination of concentration temperature
In the concentration process of the reduced pressure rotary evaporation concentration method, whether the concentration temperature has influence on effective components or not and how much the energy consumption influence on the concentration process needs to be further examined, so that the concentration temperature in the vacuum concentration process is examined at present, concentration process parameters are further determined, and low-temperature concentration has important significance on the retention of the effective components, so that the test determines that the temperature of 45 ℃, 50 ℃, 55 ℃ and 60 ℃ are the threshold values of the examination temperature.
The experimental method comprises the following steps: respectively taking 222g of ginseng decoction pieces (batch number: RS-DH-2018070201Y), 444g of astragalus membranaceus decoction pieces (batch number: HQ-LF-2017110801Y), 111g of liquorice decoction pieces (batch number: GC-HM-2017110601Y), 45g of cinnamon decoction pieces (batch number: RG-RX-2018090701Y) and 60 ginger slices, adding 5760mL of water, soaking for 40min, heating and decocting for 1h, respectively filtering, respectively dividing obtained filtrate into 4 parts, wherein each part is 800mL, respectively concentrating by adopting four methods of 45 ℃, 50 ℃, 55 ℃ and 60 ℃ to 100mL, so as to facilitate the operation of the next working procedure. The solid contents of the 4 concentrates, the total amounts of the ginsenosides Rg1, Rb1 and Re and the content of the astragaloside are respectively calculated, the values of all the factors take the total amounts of the ginsenosides Rg1, Rb1 and Re and the transfer rate of the astragaloside as investigation indexes, and the results are shown in a table 12:
TABLE 12 concentration temperature survey data
The test result shows that: the temperature has no obvious influence on the solid content, the concentration time at the temperature of 45 ℃, 50 ℃, 55 ℃ and 60 ℃ is 4.5h, 4h, 3h and 2h respectively, and the time is longer when the temperature is lower. The concentration temperature has no significant influence on the total amount of ginsenoside Rg1, Rb1 and Re and the content of astragaloside IV. Therefore, the shorter the time under the low-temperature vacuum concentration condition, the better, so the temperature is preferably 55 ℃ to 60 ℃ for concentration.
1.2.3.5 drying
And (4) freeze-drying the concentrated process investigation solution (the prefreezing temperature is-20 to-50 ℃, the freeze-drying temperature is-45 to-60 ℃, and the vacuum degree is less than 300Pa), and observing the drying condition, wherein the results are shown in a table 13.
TABLE 13 results of reference Process investigation of substances
In conclusion: the preparation process of the tentative baoyuan decoction substance standard comprises the following steps: 74g of ginseng decoction pieces, 148g of astragalus decoction pieces, 37g of liquorice decoction pieces, 15g of cinnamon decoction pieces and 20 pieces of ginger are added with 1920ml of water, soaked for 40min, covered and decocted, boiled with strong fire and boiled with slow fire, decocted for 1h, filtered with 200-mesh filter cloth while hot, concentrated at the vacuum degree of-0.06 MPa to-0.08 MPa and the concentration temperature of 55 ℃ to 60 ℃ preferably, concentrated until the liquid medicine is concentrated to about 100ml, and then freeze-dried and collected to obtain dry paste powder.
1.2.3.6 related Process parameters
The relevant quality control parameters prepared according to the "standard of Baoyuan soup substance" are summarized in the following table 14.
TABLE 14 quality control parameters
The preparation process parameters and quality control indexes of the 'Baoyuan soup material standard' are detailed in the table above, and the process parameters and quality control indexes in the Baoyuan soup production process are adjusted according to the data.
1.2.4 Key Process Steps and intermediates
1.2.4.1 Process validation sample preparation
Weighing 74g of decoction piece ginseng decoction pieces (RS-TH-2018070201Y), 148g of astragalus mongholicus decoction pieces (HQ-LF-2017110801Y), 37g of liquorice decoction pieces (GC-DS-2017110801Y), 15g of cinnamon decoction pieces (RG-CX-2018090701Y) and 20 ginger pieces in parallel, adding 1920ml of water, putting the mixture into an electronic decoction pot, covering the pot, decocting the mixture with strong fire until the mixture is boiled, decocting the mixture with slow fire for 1h, filtering the mixture with 200-mesh filter cloth while the mixture is hot, reserving 50ml of filtrate, decompressing and concentrating the rest of filtrate, reserving 50ml of concentrated solution, and freeze-drying the rest of concentrated solution. (prefreezing temperature is-20 to-45 ℃, cold trap temperature is-50 to-70 ℃, vacuum degree is less than 300Pa), collecting dry paste powder, and calculating the paste yield. Sampling, measuring the water content, extract, total amount of ginsenoside Rg1, Rb1 and Re, and astragaloside content in the dry extract powder, and respectively calculating the content transfer rate in the process. And preparing single-ingredient decoction pieces and medicinal material extracting solution according to the process, and performing fingerprint spectrum determination.
8.2.4.2 Process validation sample determination
The results are shown in tables 15-16, FIG. 2:
TABLE 15 basic process verification and detection results of Baoyuan decoction
TABLE 16 quality control of Baoyuan decoction the results of relative retention time
| |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| Licorice root medicinal material | / | 1.10 | 1.23 | / | / | 2.29 | / | / | / | 3.47 | / | / |
| Astragalus root medicinal material | 1.0 | / | / | 1.49 | 1.77 | 2.35 | / | 2.80 | / | 3.49 | 6.02 | 6.27 |
| |
1.0 | 1.09 | 1.22 | 1.49 | 1.78 | 2.35 | 2.55 | 2.80 | 3.40 | 3.48 | 5.97 | 6.23 |
| |
1.0 | 1.09 | 1.22 | 1.50 | 1.78 | 2.35 | 2.55 | 2.81 | 3.41 | 3.48 | 5.98 | 6.23 |
| |
1.0 | 1.09 | 1.22 | 1.49 | 1.78 | 2.35 | 2.55 | 2.80 | 3.41 | 3.49 | 5.98 | 6.23 |
| |
1.0 | 1.09 | 1.23 | 1.51 | 1.80 | 2.38 | 2.58 | 2.84 | 3.46 | 3.53 | 6.06 | 6.32 |
| |
1.0 | 1.09 | 1.23 | 1.51 | 1.80 | 2.38 | 2.58 | 2.84 | 3.46 | 3.53 | 6.06 | 6.31 |
| |
1.0 | 1.08 | 1.22 | 1.50 | 1.80 | 2.38 | 2.57 | 2.86 | 3.51 | 3.59 | 6.23 | 6.49 |
| |
1.0 | 1.08 | 1.22 | 1.50 | 1.78 | 2.36 | 2.56 | 2.82 | 3.44 | 3.51 | 6.05 | 6.32 |
| |
1.0 | 1.08 | 1.22 | 1.50 | 1.79 | 2.37 | 2.57 | 2.84 | 3.44 | 3.52 | 6.08 | 6.34 |
| |
1.0 | 1.08 | 1.22 | 1.49 | 1.79 | 2.38 | 2.58 | 2.84 | 3.44 | 3.52 | 6.06 | 6.33 |
| Mean value of | 1.0 | 1.09 | 1.22 | 1.50 | 1.79 | 2.36 | 2.56 | 2.83 | 3.44 | 3.51 | 6.05 | 6.31 |
| RSD(％) | 0 | 0.45 | 0.42 | 0.44 | 0.56 | 1.16 | 0.56 | 0.72 | 0.96 | 1.02 | 1.27 | 1.25 |
1.2.4.3 Process determination
Three batches of verification result shows that the data of the extracting solution and the dry paste powder are basically parallel and the material transfer loss is small in the technical process by analyzing the total amount of the ginsenoside Rg1, Rb1 and Re and the total amount of the astragaloside IV; three batches verify that the dry paste powder has no obvious difference by analyzing extracts and water.
When the fingerprint is analyzed, peaks 2, 3, 6 and 10 are licorice chromatographic peaks, and peaks 1, 4, 5, 8, 11 and 12 are astragalus chromatographic peaks. The peak No. 1 is temporarily taken as a reference peak to calculate the relative retention time, and the result shows that the common peak in the chromatogram of the medicinal materials, the decoction pieces, the extracting solution and the dry paste powder has the relative retention time RSD less than 2.0 percent, so that the determined substance standard preparation process is stable and feasible.
In summary, the preferred process for determining the material basis is as follows: 74g of ginseng, 148g of astragalus, 37g of liquorice, 15g of cinnamon and 20 ginger slices are placed in a 9L electronic decoction pot, 1920ml of water is added, the decoction is carried out by covering, the decoction is carried out by boiling with strong fire and then decocting with slow fire for keeping the micro-boiling, the decoction is carried out for 1h, a 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.06 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the total feeding amount of liquid medicine to decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze drying at-20-45 deg.c, freeze drying temperature-50-70 deg.c and vacuum degree lower than 300Pa, and collecting dry extract powder.
1.2.4.4 preparation of Multi-batch Material basis
At present, decoction pieces are collected on the principle of 5 producing areas and not less than 15 batches; in the prescription, ginseng is collected in Xilei city, Ducheng city, Tong city, Jilin province city, Tong city, Fusong county, Jingshan county, 5 production places, Hebei Baocheng city, Nemonguyang county, Gansu Yuyuan county, Likan Gansu county, Gansu Longxi county, 5 production places, licorice is collected in Gansu Dingxi, Gansu Lanzhou, Gansu Zhang, Xinjiang Hami and Dongsheng 5 production places, and cinnamon is collected in Guangxi Nannan county, Guangdong Gangdu city, Guangxi Ganggang city, Guangxi Xixi Ling county, 5 production places, 3 batches of each production place, and 15 batches of substance are prepared. The results of the 15 batches of decoction pieces ordered and combined are shown in Table 17 below.
Note: numbered 1-15, namely 15 combination modes
According to the determined process, the materials are fed by the prescription amount of 20 times a day, extracted, concentrated and freeze-dried. 15 batches of substance-based corresponding real objects are obtained, the determination result of 15 batches of substance-based cream yield is shown in the following table 17, the determination range is 9.85% -17.52%, the average value is 13.97%, the SD value is 2.44, and the range of +/-3 SD value is 6.65-21.28%, so that the substance-based cream yield is determined to be 6.5% -21.5%.
TABLE 17 Multi-batch substance reference batch information
1.3 investigation of Key Mass attributes
(1) Research on key quality attributes of standard material of Baoyuan decoction
Screening quantitative research indexes
And (4) selecting content measurement indexes, and comprehensively determining the content measurement indexes by combining the prescription efficacy and indication and the positions of the prescription medicines in the prescription. As the process research determines that water is a solvent, more water-soluble components are in the quality reference of the Baoyuan decoction, wherein the main components of the monarch drug ginseng and the astragalus root comprise saponins, triterpenes and flavonoids, so the total amount of ginsenoside Rg1, Rb1 and Re of the monarch drug ginsenoside components is determined; the content of astragaloside IV is calculated according to the quality of the comprehensive evaluation substance.
② content determination research
Quantitative study of index component
Detection method
See the text of quality standard of 'Baoyuan decoction reference' for details.
The results of the three verification tests are shown in Table 18:
TABLE 18 Process verification measurements
b summary table of quantitative index detection results
TABLE 19 Process verification measurements
(2) Influencing factors of key quality attributes and control method
Quality of prepared slices
The medicinal materials are collected and fixed in a selected producing area and a selected basic source, and are processed according to determined processing technological parameters strictly, so that the quality of the decoction pieces is ensured.
Material reference process control
a decoction
An electronic medicine decocting pot is adopted as a decocting utensil, an electric heating mode is adopted, boiling is carried out with strong fire, decocting is carried out with slow fire, and technological parameters such as duration of fire, decocting time, filtering material and the like are controlled.
b freeze drying
The drying method is freeze drying, and the water content of the dry extract is controlled to be less than 12.0%.
c packaging and storing
The material standard packaging material is a vacuum packaging bag, sealed and stored in a cool and shady warehouse.
8.3.2 study on correlation between herbs, decoction pieces, intermediates and corresponding substances
The correlation research of medicinal materials, decoction pieces, intermediates and corresponding real objects is carried out by taking key quality attributes (total amount of ginsenoside Rg1, Rb1, Re, total amount of astragaloside IV and finger print) as indexes.
8.3.2.1 method of measurement
(1) Content determination method
The determination method of total amount of corresponding real object (dry extract powder) ginsenoside Rg1, Rb1, and Re and astragaloside IV is described in the standard text of quality standard of BAOYUANTANG.
The preparation method of the intermediate sample comprises the following steps:
total amount of ginsenoside Rg1, Rb1 and Re
Extracting solution: precisely measuring 25ml of extracting solution, placing the extracting solution in a separating funnel, adding chloroform for extraction for 3 times, 15ml each time, removing a chloroform layer, extracting a water layer for 3 times by using water saturated n-butyl alcohol solution, 30ml each time, combining n-butyl alcohol layers, washing for 2 times by using ammonia test solution, 30ml each time, removing a test solution layer, washing the n-butyl alcohol layer for 1 time by using 20ml of n-butyl alcohol saturated water, removing the water layer, drying the n-butyl alcohol layer by distillation, dissolving residues by adding methanol, transferring the residues to a 5ml volumetric flask, adding methanol to a constant volume to scale, shaking up, filtering, and taking a subsequent filtrate to obtain a test solution.
② astragaloside IV
Extracting solution: precisely measuring 25ml of extracting solution, placing the extracting solution into a 50ml triangular conical flask, carrying out ultrasonic treatment (power is 240W and frequency is 45kHz) for 30 minutes, extracting with water saturated n-butyl alcohol for 4 times, each time, obtaining 25ml, combining n-butyl alcohol layers, extracting with ammonia test solution in a shaking way for 3 times, obtaining 30ml each time, removing the ammonia test solution layer, collecting the n-butyl alcohol solution, drying by distillation, dissolving residues with methanol, transferring to a 5ml volumetric flask, adding methanol to a constant volume to a scale, shaking up, filtering, and obtaining a subsequent filtrate to obtain a sample solution.
(2) Fingerprint spectrum measuring method
A method for measuring medicinal materials, decoction pieces, intermediates (extractive solution), and corresponding substances (dry extract powder) comprises marking 10 common peaks according to 15 batches of substance standard fingerprint measurement results, generating reference fingerprint, and calculating similarity.
The preparation method of the medicinal materials, the decoction pieces and the intermediate samples comprises the following steps:
a, liquorice medicinal material solution: weighing 2.5g of Glycyrrhrizae radix powder (sieved with a sieve IV) according to a prescription, precisely weighing, placing in a conical flask with a plug, adding 50% methanol 50ml, performing ultrasonic treatment for 45min, filtering, recovering solvent from the filtrate under reduced pressure to near dryness, dissolving the residue in 20ml of water, extracting with water saturated n-butanol solution for 2 times, each time 25ml, mixing n-butanol solutions, extracting with 30ml of ammonia test solution for 1 time, discarding the ammonia test solution, evaporating the n-butanol layer to dryness, and dissolving the residue in 2ml of water. Passing through a chromatographic column filled with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, collecting 95% ethanol eluate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the product.
b, liquorice decoction piece solution: weighing 2.5g Glycyrrhrizae radix decoction pieces powder (sieved with No. four sieve) according to the prescription, precisely weighing, placing in a conical flask with a plug, adding 50% methanol 50ml, ultrasonic treating for 45min, filtering, recovering solvent from the filtrate under reduced pressure to near dryness, dissolving the residue in 20ml water, extracting with water saturated n-butanol solution for 2 times, each time 25ml, mixing n-butanol solutions, extracting with 30ml ammonia test solution for 1 time, discarding ammonia test solution, evaporating n-butanol layer to dryness, and dissolving the residue in 2ml water. Passing through a chromatographic column filled with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, collecting 95% ethanol eluate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the product.
c, ginseng decoction piece solution: weighing 2.5g of Ginseng radix decoction pieces powder (sieved with a sieve IV) according to a prescription, precisely weighing, placing in a conical flask with a plug, adding 50ml of 50% methanol, performing ultrasonic treatment for 45min, filtering, recovering solvent from the filtrate under reduced pressure until the filtrate is nearly dry, dissolving the residue in 20ml of water, extracting with water saturated n-butanol solution for 2 times, 25ml each time, combining n-butanol solutions, extracting with 30ml of ammonia test solution for 1 time, discarding the ammonia test solution, evaporating the n-butanol layer to dryness, and dissolving the residue in 2ml of water. Passing through a chromatographic column filled with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, collecting 95% ethanol eluate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the product.
d intermediate (extract): precisely measuring 5ml of the extractive solution, placing in a conical flask with a plug, adding 50ml of 50% methanol, performing ultrasonic treatment for 45min, filtering, recovering solvent from the filtrate under reduced pressure, dissolving the residue in 20ml of water, extracting with water saturated n-butanol solution for 2 times (25 ml each time), mixing n-butanol solutions, extracting with 30ml of ammonia solution for 1 time, discarding the ammonia solution, evaporating the n-butanol layer, and dissolving the residue in 2ml of water. Passing through a chromatographic column filled with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, collecting 95% ethanol eluate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the product.
e intermediate (concentrate): precisely measuring 2ml of the concentrated solution, placing in a conical flask with a plug, adding 50ml of 50% methanol, performing ultrasonic treatment for 45min, filtering, recovering solvent from the filtrate under reduced pressure until the filtrate is nearly dry, dissolving the residue in 20ml of water, extracting with water saturated n-butanol solution for 2 times, each time 25ml, mixing n-butanol solutions, extracting with 30ml of ammonia solution for 1 time, discarding the ammonia solution, evaporating the n-butanol layer to dryness, and dissolving the residue in 2ml of water. Passing through a chromatographic column filled with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, collecting 95% ethanol eluate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the product.
1.3.2.2 correlation Studies
(1) Multiple batch correlation study
Measuring contents
The results of the multi-batch content measurement are shown in tables 20 to 21. The total amount and the extracted total amount of multiple batches of astragalosides, ginsenosides Rg1, Rb1 and Re are small in dispersion degree and good in correlation.
TABLE 20 results of astragaloside content determination in different batches
TABLE 21 Total content determination results of ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re of different batches
Fingerprint atlas
Fingerprint spectrum of flavonoid component:
the relative retention time results of multiple batches of fingerprints are shown in a table 22, and the RSD of the common peak of 15 batches of samples is less than 3.0%; the similarity of 15 batches of samples and the comparison fingerprint is calculated by a common peak by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation software system (2012 version), and the similarity calculated according to the fingerprints is more than 0.85, which indicates that the correlation is good.
TABLE 22 relative Retention times
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 1 | 1.00 | 1.08 | 1.21 | 1.47 | 1.75 | 2.35 | 2.53 | 2.80 | 3.35 | 3.45 | 5.88 | 6.13 |
| 2 | 1.00 | 1.08 | 1.21 | 1.47 | 1.75 | 2.35 | 2.54 | 2.81 | 3.38 | 3.47 | 5.98 | 6.23 |
| 3 | 1.00 | 1.08 | 1.21 | 1.48 | 1.77 | 2.39 | 2.58 | 2.86 | 3.44 | 3.53 | 6.05 | 6.30 |
| 4 | 1.00 | 1.08 | 1.21 | 1.48 | 1.76 | 2.36 | 2.54 | 2.81 | 3.39 | 3.49 | 6.02 | 6.28 |
| 5 | 1.00 | 1.08 | 1.21 | 1.48 | 1.77 | 2.36 | 2.55 | 2.83 | 3.41 | 3.51 | 6.05 | 6.31 |
| 6 | 1.00 | 1.08 | 1.21 | 1.48 | 1.76 | 2.36 | 2.55 | 2.83 | 3.42 | 3.51 | 6.09 | 6.35 |
| 7 | 1.00 | 1.08 | 1.21 | 1.49 | 1.78 | 2.37 | 2.55 | 2.83 | 3.43 | 3.52 | 6.11 | 6.38 |
| 8 | 1.00 | 1.08 | 1.21 | 1.49 | 1.78 | 2.37 | 2.56 | 2.84 | 3.43 | 3.53 | 6.13 | 6.40 |
| 9 | 1.00 | 1.09 | 1.22 | 1.49 | 1.78 | 2.37 | 2.56 | 2.84 | 3.44 | 3.52 | 6.10 | 6.36 |
| 10 | 1.00 | 1.09 | 1.22 | 1.49 | 1.77 | 2.36 | 2.55 | 2.82 | 3.41 | 3.50 | 6.01 | 6.27 |
| 11 | 1.00 | 1.08 | 1.22 | 1.49 | 1.77 | 2.36 | 2.56 | 2.82 | 3.41 | 3.50 | 6.03 | 6.29 |
| 12 | 1.00 | 1.09 | 1.22 | 1.49 | 1.77 | 2.37 | 2.56 | 2.83 | 3.43 | 3.52 | 6.06 | 6.32 |
| 13 | 1.00 | 1.09 | 1.22 | 1.49 | 1.77 | 2.37 | 2.56 | 2.83 | 3.43 | 3.51 | 6.07 | 6.33 |
| 14 | 1.00 | 1.08 | 1.22 | 1.49 | 1.77 | 2.37 | 2.56 | 2.83 | 3.43 | 3.52 | 6.08 | 6.35 |
| 15 | 1.00 | 1.08 | 1.22 | 1.49 | 1.78 | 2.38 | 2.57 | 2.85 | 3.45 | 3.53 | 6.11 | 6.37 |
| Mean value of | 1.00 | 1.08 | 1.21 | 1.48 | 1.77 | 2.37 | 2.56 | 2.83 | 3.42 | 3.51 | 6.05 | 6.31 |
| RSD(％) | 0 | 0.24 | 0.32 | 0.45 | 0.50 | 0.45 | 0.53 | 0.55 | 0.75 | 0.66 | 1.05 | 1.09 |
TABLE 23 fifteen lots of material Standard similarity evaluation results
The R contrast maps S2-S16 are sequentially as follows: BYT-2019091001D, BYT-2019091002D, BYT-2019091003D, BYT-2019091604D, BYT-2019091605D, BYT-2019091606D BYT-2019091807D, BYT-2019091808D, BYT-2019091809D, BYT-2019092310D, BYT-2019092311D, BYT-2019092312D, BYT-2019092413D, BYT-2019092414D, BYT-2019092415D
(2) Research on correlation between medicinal materials, decoction pieces, intermediates and corresponding substances in same batch
Preparation of a Material Standard
Preparing 74g of ginseng decoction pieces (RS-TH-2018070201Y), 148g of astragalus membranaceus decoction pieces (HQ-LF-2017110801Y), 37g of liquorice decoction pieces (GC-DS-2017110801Y) and 15g of cinnamon (RG-CX-2018090701Y) according to a determined process to prepare the standard material of the Baoyuan decoction, and reserving 50ml of extracting solution for later use.
Results of correlation study
a measurement result of content
TABLE 24 summary of the results of the measurement of the reference content of ginseng decoction pieces-extract liquid-substance
TABLE 25 summary of the results of the measurement of the content of Astragalus membranaceus decoction pieces-extract liquid-substance basis
b fingerprint spectrum measurement results are shown in FIG. 3
TABLE 26 summary of relative Retention time calculations
Note: decocting the above materials and decoction pieces in water, and making into the same preparation method as the reference materials.
And (4) conclusion: from the results, the chromatograms of the decoction pieces, the extracting solution, the concentrated solution and the substance standard have the same chromatographic peak at corresponding positions, and the relative retention time RSD% < 2.0%, which shows that the consistency of the main chemical components of the decoction pieces to the substance standard meets the requirements of quality standards, and the preparation process is stable.
Results of correlation determination
The results of the determination of the total amount of the ginsenosides Rg1, Rb1 and Re and the content of the astragaloside in the decoction piece-extract-concentrated solution-substance standard show that the transmission of the total amount of the ginsenosides Rg1, Rb1 and Re and the total amount of the astragaloside is consistent with the transmission result in the process verification process.
The result of fingerprint spectrum measurement in the decoction piece-extracting solution-concentrated solution-substance reference process shows that 12 common peaks marked in the substance reference are well transferred, the relative retention time RSD of each peak is less than 2.0 percent, and the correlation is good.
The determination results show that the transmission of fingerprint spectrum, content determination and paste yield in the process of decoction piece-extracting solution-concentrated solution-substance reference research shows certain regularity and good correlation.
1.3.3 analytical methods study
Through literature research, chemical components and key quality attributes of a system in the standard of the Baoyuan soup are summarized, and a method for analyzing and verifying the key quality attributes of the established standard of the material is provided; the test items in the material standard quality standards are summarized in Table 27.
1.3.3.1 method of analysis
1.3.3.2 validation of analytical methods
The analysis methodology verification included in the standard project is executed according to the relevant guiding principle in the current edition of Chinese pharmacopoeia, and the methodology verification data is as follows:
1.3.3.2.1 trait
The actual properties of 15 corresponding batches of the real substance (dry paste powder) are observed and recorded, and the results are shown in a table 28 and fig. 4:
TABLE 28 trait identification results
And (4) conclusion: according to the actual observation condition of 15 batches of corresponding real object (dry paste powder) samples, the product is tentatively brown to tan powder, slightly fragrant, slightly bitter and sweet in taste.
1.3.3.2.2 authentication
The product is a compound preparation prepared by a water decoction process, adopts a thin-layer identification method with strong specificity, rapidness, simplicity and good reproducibility, and carries out identification test research on main characteristic components of ginseng, astragalus, liquorice and cinnamon in the prescription. The results are summarized as follows:
firstly, ginseng
The main components of Ginseng radix are saponins, saccharides, volatile components, organic acids and their esters, sterols and their glycosides, flavonoids, lignin, inorganic elements and vitamins etc. Mainly contains ginsenoside, and the following thin layer groping experiment is carried out on ginseng according to the chemical components.
a method of establishing thin layer chromatography
Refer to the item of Ginseng radix [ identification ] in the chapter of Chinese pharmacopoeia 2015 edition[58]
Collecting powder 1g, adding chloroform 40ml, heating and refluxing for 1 hr, discarding chloroform solution, volatilizing solvent from residue, adding water 0.5ml, stirring and moistening, adding saturated n-butanol 10ml, ultrasonic treating for 30min, collecting supernatant, adding 3 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dryness, and dissolving residue with methanol 1ml to obtain sample solution. Decocting Ginseng radix 1g with appropriate amount of water for 30min, and making into control solution. And adding methanol into ginsenoside Rb1, ginsenoside Re, and ginsenoside Rg1 to obtain mixed solutions each containing 2mg of ginsenoside Rb1, ginsenoside Re, and ginsenoside Rg1 per 1 ml. Performing thin layer chromatography (general rule 0502) test, sucking the above three solutions 5 μ 1, respectively dropping on the same silica gel G thin layer plate, spreading with chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) lower layer solution at below 10 deg.C as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until the color of spots is clear, and respectively inspecting under sunlight and ultraviolet lamp (365 nm):
and (4) analyzing results: in the chromatogram of the test solution, the same spots appear at the corresponding positions of the reference medicinal material and the reference substance, and the negative result is not interfered. The method is feasible by using the ginsenosides Rg1, Rb1 and Re as reference substances and the ginseng reference medicinal material solution as reference substances and inspecting under ultraviolet light, but the spots of the test sample, the reference substance and the reference medicinal material solution are shallow, so the sample amount of the test sample, the reference substance and the reference medicinal material solution is searched in the next step.
The second method comprises the following steps: sample amount searching
Absorbing the three sample solutions prepared by the first method, increasing the sample application amount of the ginsenoside Rg1, Rb1, Re reference substance solution and ginseng decoction piece solution to 10 mu l, changing the sample application amount of the negative sample to 5 mu l, 8 mu l and 10 mu l, respectively applying the sample to the same silica gel G thin layer plate, respectively applying the sample to the lower layer solution which is placed below 10 ℃ of chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) as a developing agent, developing, taking out, airing, spraying with 10% sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clearly developed, and respectively inspecting under sunlight and an ultraviolet lamp (365 nm).
The results show that the sample application amount of the ginsenoside Rg1, Rb1 and Re control solution and the ginseng decoction pieces is 10 mul, the sample application amount of the test solution is 10 mul, the color spots at corresponding positions are all clear and visible, and no tailing phenomenon occurs, so that the concentration of the ginsenoside Rg1, Rb1 and Re control solution is determined to be 10 mul at 1mg/ml sample application amount, the sample application amount of the ginseng control medicinal material solution is determined to be 10 mul at 0.5g/ml sample application amount, and the sample application amount of the test solution is determined to be 10 mul at 10 mul.
Comprehensive analysis, the thin layer identification method is determined as follows: collecting powder 1g, adding chloroform 40ml, heating and refluxing for 1 hr, discarding chloroform solution, volatilizing solvent from residue, adding water 0.5ml, stirring and moistening, adding saturated n-butanol 10ml, ultrasonic treating for 30min, collecting supernatant, adding 3 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dryness, and dissolving residue with methanol 1ml to obtain sample solution. Decocting Ginseng radix 1g with appropriate amount of water for 30min, and making into control solution. Adding methanol into ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 as reference solutions to obtain mixed solutions each containing 2mg of ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 in each 1 ml. Performing thin layer chromatography (general rule 0502) test, sucking the three solutions 1-2 μ 1, respectively dropping on the same silica gel G thin layer plate, spreading with lower layer solution of chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) at temperature below 10 deg.C as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, respectively placing under sunlight and ultraviolet lamp (365nm), observing in the sample chromatogram, at the position corresponding to the control chromatogram, the same spots are developed, and the negative chromatogram has no spots at the position corresponding to the control chromatogram.
b durability examination
Durability experiments were conducted according to the above-described determination method to examine the effects of different development temperatures and relative humidities of self-made boards and machine-made boards (Qingdao ocean chemical plant).
And (4) conclusion: through durability investigation of low-temperature low-humidity, normal-temperature and high-temperature high-humidity environments and silica gel G thin-layer plates of different manufacturers, the durability of the ginseng thin-layer identification condition is good under different influence factors. Therefore, the thin layer identification method is included in the text
c method determination
Collecting powder 1g, adding chloroform 40ml, heating and refluxing for 1 hr, discarding chloroform solution, volatilizing solvent from residue, adding water 0.5ml, stirring and moistening, adding saturated n-butanol 10ml, ultrasonic treating for 30min, collecting supernatant, adding 3 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dryness, and dissolving residue with methanol 1ml to obtain sample solution. Decocting Ginseng radix 1g with appropriate amount of water for 30min, and making into control solution. Adding methanol into ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 as reference solutions to obtain mixed solutions each containing 2mg of ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 in each 1 ml. Performing thin layer chromatography (general rule 0502) test, sucking the three solutions 1-2 μ 1, respectively dropping on the same silica gel G thin layer plate, spreading with lower layer solution of chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) at temperature below 10 deg.C as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, respectively placing under sunlight and ultraviolet lamp (365nm), observing in the sample chromatogram, at the position corresponding to the control chromatogram, displaying the same bluish purple spots, and the negative chromatogram has no spots at the position corresponding to the control chromatogram.
d thin layer identification of corresponding real object (dry paste powder) in different batches
Weighing 1g of corresponding substance (dry extract powder) of different batches, and making into sample solution by the same method. And (5) performing thin-layer identification.
As a result: in the chromatogram of the test sample based on different batches of substances, the same bluish purple spots appear at the positions corresponding to the control chromatogram, and the negative chromatogram has no spots at the positions corresponding to the control chromatogram.
② astragalus root
The radix astragali contains saponins and flavonoids as main chemical components, and also contains polysaccharide. Experiments were performed on Astragalus membranaceus according to the above chemical composition by thin layer chromatography as follows.
a method of establishing thin layer chromatography
The method comprises the following steps: refer to thin layer identification method under item of 'radix astragali granule' in the first part of 'Chinese pharmacopoeia' 2015 edition for groping
Taking 1.5g of the product and negative samples without astragalus root respectively, taking 1.5g of the product powder, adding 25mL of water, carrying out ultrasonic treatment for 30 minutes, filtering, shaking and extracting the filtrate for 2 times by using water saturated n-butyl alcohol, 25mL each time, combining n-butyl alcohol extract, washing for 2 times by using ammonia test solution, 30mL each time, taking n-butyl alcohol solution to evaporate to dryness, and adding methanol lmL into residues to dissolve the residues to obtain test solution. And adding water 50ml into astragalus root decoction pieces 1.5g, decocting for 30min, filtering, and preparing a control medicinal solution by the same method. Adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml, and using as reference substance solution. Performing thin layer chromatography (0502 of the four ministerial rules of the four parts of the book of the Chinese pharmacopoeia 2015), sucking 5 μ l of each of the four solutions, dropping on the same silica gel G thin layer plate, developing with a developing agent of a lower layer solution of chloroform-methanol-water (13:7:2) at a temperature below 10 deg.C, taking out, air drying, spraying with a 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under an ultraviolet lamp (365 nm).
And (4) analyzing results: in the chromatogram of the test sample, the same spots are present at the corresponding positions of the chromatogram of the astragaloside IV and the radix astragali, and the negative is free of interference. The method is feasible by using astragaloside IV as a reference substance and using the radix astragali reference medicinal material solution as a reference substance, and inspecting under ultraviolet light (365nm), wherein the spots are lighter than the test solution, and in order to make the spots more clear, the sample amount of the reference substance and the radix astragali decoction piece solution is further searched.
The second method comprises the following steps: sample amount searching
Sucking the sample solution (BYT-2019091807D) prepared by the 'method one', the astragaloside IV reference substance solution, the astragalus reference medicinal material solution and the negative sample with the sample application amount unchanged by 10 mul, the sample application amount of the sample to be tested is changed to 5 mul, 8 mul and 10 mul, respectively spotting on the same silica gel G thin layer plate, taking a lower layer solution of trichloromethane-methanol-water (13:7:2) as a developing agent, developing, taking out, airing, spraying with a 10% sulfuric acid ethanol solution, and heating at 105 ℃ until the spots are clearly developed. Spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; the film was observed under an ultraviolet lamp (365 nm).
The result shows that the sample application amount of the astragaloside control solution and the astragalus mongholicus decoction piece is 10 mul, the sample application amount of the test solution is 5 mul, 8 mul and 10 mul respectively, the color spots at corresponding positions are clear and visible, and no tailing phenomenon occurs, so that the concentration of the astragaloside control solution is determined to be 10 mul of sample application amount of 2mg/ml, the sample application amount of the astragalus mongholicus decoction piece solution is 10 mul of 1.5g/ml, and the sample application amount of the test solution is 10 mul. Comprehensive analysis, confirmation of thin layer identification method: dissolving 1.5g of the product in 25mL of water, performing ultrasonic treatment for 30 minutes, filtering, extracting the filtrate with 25mL of water-saturated n-butanol under shaking for 2 times, mixing the n-butanol extractive solutions, washing with ammonia test solution for 2 times, 30mL each time, collecting the n-butanol extractive solution, evaporating, and dissolving the residue in lmL methanol to obtain a test solution. And adding water 50ml into 1.5g of astragalus control medicinal material, decocting for 30 minutes, filtering, and preparing a control medicinal material solution by the same method. Adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml, and using as reference substance solution. Performing thin layer chromatography (0502 of the four ministerial rules of the republic of China pharmacopoeia 2015), sucking 5 μ l of each of the four solutions, dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution as developing agent, taking out, air drying, spraying with 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under ultraviolet lamp (365 nm).
b durability examination
And (3) carrying out a durability test according to the determined method, and inspecting influence factors such as thin-layer plates (Qingdao ocean chemical plants) of the self-made silica gel G plate and the machine-made silica gel G plate, different development high temperature and high humidity (temperature 42 ℃ and humidity 75%), relative low temperature and low humidity (temperature 4 ℃ and humidity 45%) and the like.
And (4) conclusion: through durability investigation of low-temperature, high-temperature, normal-temperature, low-humidity and high-humidity environments and different silica gel G thin-layer plates, the durability of the identification condition of the astragalus membranaceus thin layer is good under different influence factors. The thin layer method is hereby incorporated into the text.
c method determination
Collecting 1.5g of the product, adding 25mL of water, performing ultrasonic treatment for 30min, filtering, extracting the filtrate with water saturated n-butanol under shaking for 2 times, each time 25mL, mixing n-butanol extractive solutions, washing with ammonia test solution for 2 times, each time 30mL, collecting n-butanol solution, evaporating to dryness, and dissolving the residue with methanol lmL to obtain test solution. And adding water 50ml into 1.5g of astragalus control medicinal material, decocting for 30 minutes, filtering, and preparing the filtrate into a control medicinal material solution by the same method. Adding methanol into astragaloside IV control to obtain solution containing 2mg of astragaloside IV per lmL as control solution. Performing thin layer chromatography (0502 of the four ministerial general rules of the design reside in the Chinese pharmacopoeia 2015), sucking 10 μ l of each of the control solution, and the sample solution, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution at 10 deg.C or below as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; the fluorescent spots with the same color are observed under an ultraviolet lamp (365 nm).
d thin layer identification of corresponding real object (dry paste powder) in different batches
Weighing 1.5g of corresponding substance (dry extract powder) of different batches, and making into sample solution by the same method. And (5) performing thin-layer identification.
And (4) conclusion: in the chromatogram of the test sample based on different batches of substances, spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance.
③ Liquorice
The main chemical components of Glycyrrhrizae radix are triterpenes and flavonoids, and in addition, it also contains polysaccharide, organic acid and coumarin. Performing thin layer chromatography experiment on Glycyrrhrizae radix according to the above chemical components
a method of establishing thin layer chromatography
The method comprises the following steps: thin-layer identification method under item of 'liquorice' in first edition of 'Chinese pharmacopoeia' 2015 edition
Taking 1g of a corresponding substance (freeze-dried powder) and the liquorice-deficient negative freeze-dried powder, adding 40ml of water, carrying out ultrasonic treatment for 30 minutes, filtering, extracting with diethyl ether for 2 times and 10ml each time, discarding the diethyl ether solution, extracting the water solution with water-saturated n-butyl alcohol for 3 times and 20ml each time, combining the n-butyl alcohol solutions, extracting with ammonia test solution for 2 times and 30ml each time, combining the ammonia test solutions, and adjusting the pH value to 3-4 with hydrochloric acid. Extracting with ethyl acetate under shaking for 2 times (30 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1ml methanol to obtain test solution. Taking 1g of honey-fried licorice root decoction pieces, adding 50ml of water, decocting for 30 minutes, filtering, and preparing a control medicinal solution by the same method. Taking appropriate amount of liquiritin reference substance, adding 70% ethanol to make into solution containing 0.5mg per 1mL as reference substance solution. According to thin layer chromatography 2015 version four (general rule 0502) test, sucking each 5 μ l of the above four solutions, respectively dropping on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution, developing with chloroform-methanol-water (13:7:2) subnatant at 10 deg.C as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight and ultraviolet lamp (365 nm).
And (4) conclusion: in the chromatogram of the test sample, spots with the same color are displayed at the corresponding positions of the chromatogram of the liquorice control medicinal material and the control sample, but the spots are not clear in color development and poor in separation effect, and the sample application amount of 5 mu l is small, so that the quantification is difficult and inaccurate, and the sample application amount is groped.
The second method comprises the following steps: sample amount searching
Sucking sample solution (BYT-2019091001D) prepared by the first method, the sample application amount of astragaloside IV control solution, astragalus mongholicus decoction piece solution and negative samples is not changed to 10 mu l, the sample application amount of the samples is changed to 5 mu l, 8 mu l and 10 mu l, respectively dropping the samples on a silica gel G thin layer plate prepared by 1% sodium hydroxide solution, taking a subnatant placed at 5-10 ℃ of trichloromethane-methanol-water (13:7:2) as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clearly developed, and inspecting under sunlight and an ultraviolet lamp (365 nm).
And (4) analyzing results: in the chromatogram of the test solution, spots with the same color appear at the positions corresponding to the chromatograms of the liquorice control drug and the reference drug, and no spots are trailing seriously, so that the spot application amount of the test solution, the reference drug and the reference drug solution is determined to be 10 mu l.
The thin layer identification method is determined by comprehensive analysis: taking 1g of a corresponding substance (freeze-dried powder) and the liquorice-deficient negative freeze-dried powder, adding 40ml of water, carrying out ultrasonic treatment for 30 minutes, filtering, extracting with diethyl ether for 2 times and 10ml each time, discarding the diethyl ether solution, extracting the water solution with water-saturated n-butyl alcohol for 3 times and 20ml each time, combining the n-butyl alcohol solutions, extracting with ammonia test solution for 2 times and 30ml each time, combining the ammonia test solutions, and adjusting the pH value to 3-4 with hydrochloric acid. Extracting with ethyl acetate under shaking for 2 times (30 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1ml methanol to obtain test solution. Taking 1g of honey-fried licorice root decoction pieces, adding 50ml of water, decocting for 30 minutes, filtering, and preparing a control medicinal solution by the same method. Taking appropriate amount of liquiritin reference substance, adding 70% ethanol to make into solution containing 0.5mg per 1mL as reference substance solution. According to thin layer chromatography 2015 version four (general rule 0502) test, 10 μ l of each of the three solutions is sucked and respectively spotted on the same silica gel G thin layer plate prepared by 1% sodium hydroxide solution, the subnatant placed at 5-10 ℃ with trichloromethane-methanol-water (13:7:2) is used as a developing agent, the developing solution is taken out, the developing solution is dried in the air, 10% sulphuric acid ethanol solution is sprayed, the solution is heated at 105 ℃ until spots are developed and cleared, and the solution is inspected under sunlight and an ultraviolet lamp (365 nm).
b durability examination
And (3) carrying out a durability test according to the determination method, and inspecting influence factors such as self-made boards, machine-made boards (Qingdao ocean chemical plants), different spreading temperatures and humidity.
And (4) conclusion: through durability investigation of low-temperature low-humidity, normal-temperature and high-temperature environments, the thin layer identification condition has good durability under different influence factors.
c method determination
Taking 1g of a corresponding substance (freeze-dried powder) and a negative sample lacking liquorice, adding 40ml of water, carrying out ultrasonic treatment for 30 minutes, filtering, extracting with diethyl ether for 2 times and 20ml each time, discarding the diethyl ether solution, extracting the water solution with water-saturated n-butyl alcohol for 3 times and 20ml each time, combining the n-butyl alcohol solutions, extracting with ammonia test solution for 2 times and 30ml each time, combining the ammonia test solutions, and adjusting the pH value to 3-4 with hydrochloric acid. Extracting with ethyl acetate under shaking for 2 times (30 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1ml methanol to obtain test solution. And (2) adding 50ml of water into 1g of (roasted) liquorice decoction pieces, decocting for 30 minutes, filtering, and preparing a control medicinal solution by the same method. Taking appropriate amount of liquiritin reference substance, adding 70% ethanol to make into solution containing 0.5mg per 1mL as reference substance solution. Performing thin layer chromatography (general rule 0502) test, sucking 10 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) subnatant at 5-10 deg.C as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution, heating at 105 deg.C until the color of spots is clear, inspecting under sunlight and ultraviolet lamp (365nm), and displaying spots or fluorescent spots of the same color in the chromatogram of the test sample at the positions corresponding to those of the control and control chromatogram.
d thin layer identification of corresponding substance (lyophilized powder) in different batches
1g of corresponding substances (lyophilized powder) of different batches are respectively weighed and prepared into a sample solution by the same method for preparing the sample. And (5) performing thin-layer identification.
And (4) conclusion: in the chromatogram of the test sample based on different batches of substances, spots or fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the reference medicinal material and the chromatogram of the reference substance.
Fourthly, cinnamon
Cinnamon mainly contains cinnamaldehyde, cinnamic acid, diterpene and its glycoside, flavanol and its polymer, and in addition, flavonoids, polyphenols, etc. The following experiments of thin layer chromatography were performed on cinnamon according to the above chemical composition.
a method of establishing thin layer chromatography
The method comprises the following steps: reference document "thin layer chromatography identification of cassia twig and tuckahoe in wuling tablets" for groping
Taking 1g of the corresponding substance (lyophilized powder) and each of the cortex Cinnamomi negative lyophilized powder, adding 10ml of water to dissolve, extracting with diethyl ether under shaking for 1 time, 25ml each time, mixing the diethyl ether solution, volatilizing, and dissolving the residue with 2ml of diethyl ether to obtain a sample solution and a cortex Cinnamomi negative sample solution. Decocting 1g of cortex Cinnamomi decoction pieces in 50ml of water for 30min, filtering, and making into control medicinal solution. Then, a lauric acid control was prepared, and 1ml of a solution containing 0.2mg was prepared with 50% methanol as a control solution. Performing thin layer chromatography (0502 of the four Provisions of the national pharmacopoeia 2015 edition), sucking 3 μ l of each of the four solutions, and dropping on the same silica gel GF254Spreading on thin layer plate with cyclohexane-diethyl ether-glacial acetic acid (5:5:0.3) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm).
And (4) conclusion: in the chromatogram of the test sample, spots with the same color are displayed at the corresponding positions of the chromatogram of the cinnamon reference medicinal material and the chromatogram of the reference product, but the spots are not clear in color development and poor in separation effect, and the sample application amount of 5 mul is small, so that the quantification is difficult and inaccurate, and the sample application amount is groped.
The second method comprises the following steps: sample amount searching
Sucking 10 μ l of each of the four solutions, and dropping on the same silica gel GF254Spreading on thin layer plate with cyclohexane-diethyl ether-glacial acetic acid (5:5:0.3) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm).
And (4) analyzing results: in the chromatogram of the test solution, spots with the same color appear at the positions corresponding to the chromatograms of the cinnamon control medicinal material and the reference medicinal material, and no spots are trailing seriously, so that the spot application amount of the test solution, the reference medicinal material and the reference medicinal material solution is determined to be 10 mu l.
The thin layer identification method is determined by comprehensive analysis: taking 1g of corresponding substance (lyophilized powder) and each of the lyophilized powder for negative samples of the lean osmanthus, adding 10ml of water to dissolve, extracting with diethyl ether under shaking for 1 time, 25ml each time, mixing diethyl ether solution, volatilizing, and adding 2ml of diethyl ether to the residue to dissolve to obtain test solution and negative sample solution of the lean osmanthus. Decocting 1g of cortex Cinnamomi decoction pieces in 50ml of water for 30min, filtering, and making into control medicinal solution. Then, a lauric acid control was prepared, and 1ml of a solution containing 0.2mg was prepared with 50% methanol as a control solution. Performing thin layer chromatography (0502 of the four Provisions of the national pharmacopoeia 2015 edition), sucking 3 μ l of each of the four solutions, and dropping on the same silica gel GF254Spreading on thin layer plate with cyclohexane-diethyl ether-glacial acetic acid (5:5:0.3) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm).
b durability examination
And (3) carrying out a durability test according to the determination method, and inspecting influence factors such as self-made boards, machine-made boards (Qingdao ocean chemical plants), different spreading temperatures and humidity.
And (4) conclusion: through durability investigation of low-temperature low-humidity, normal-temperature and high-temperature environments, the thin layer identification condition has good durability under different influence factors.
c method determination
Taking 1g of the corresponding substance (lyophilized powder) and each of the cortex Cinnamomi negative lyophilized powder, adding 10ml of water to dissolve, extracting with diethyl ether under shaking for 1 time, 25ml each time, mixing the diethyl ether solution, volatilizing, and dissolving the residue with 2ml of diethyl ether to obtain a sample solution and a cortex Cinnamomi negative sample solution. Decocting 1g of cortex Cinnamomi decoction pieces in 50ml of water for 30min, filtering, and making into control medicinal solution. Then, a lauric acid control was prepared, and 1ml of a solution containing 0.2mg was prepared with 50% methanol as a control solution. Performing thin layer chromatography (0502 in the four parts of the pharmacopoeia of China 2015 edition)Dropping 3 μ l of each solution on the same silica gel GF254Spreading on thin layer plate with cyclohexane-diethyl ether-glacial acetic acid (5:5:0.3) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254nm)
d thin layer identification of corresponding substance (lyophilized powder) in different batches
1g of corresponding real object (lyophilized powder) of different batches is respectively weighed, and prepared into a test solution by the same method for preparing the test. And (5) performing thin-layer identification.
And (4) conclusion: in the chromatogram of the test sample based on different batches of substances, spots or fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the reference medicinal material and the chromatogram of the reference substance.
8.3.3.2.3 examination
(ii) water content
The reference moisture content of the material was measured in 15 lots according to a moisture measurement method (the second method 0832, general rule of four parts of the edition of "Chinese pharmacopoeia" 2015), and the results are shown in Table 29.
TABLE 29 moisture test results
The results show that: the minimum value of the moisture of 15 batches of material is 6.3%, the maximum value is 11.8%, and the average value is 8.7%, so that the moisture of the temporary dry paste is not more than 12.0%.
② extract
Most of chemical components or major components in the Chinese medicinal materials can be dissolved out in water or alcohol, and then extract measurement is carried out. Because the quality standard of the Baoyuan decoction is water extraction, the significance of selecting water as a solvent to establish extract measurement is not great, and the internal quality is difficult to reflect, when the solvent is selected, the solubility of the major components is considered, ethanol is selected to perform extract measurement to control the quality of the material standard
According to the extract measuring method (the alcohol-soluble hot dipping method 2201 in the four departments of the version 2015 in the Chinese pharmacopoeia): weighing about 2g of the product, precisely weighing, placing in a 100ml conical flask, precisely adding 50ml of ethanol, sealing, weighing, standing for 1 hr, connecting with a reflux condenser tube, heating to boil, and keeping slightly boiling for 1 hr. After cooling, the flask was taken down, the stopper was sealed, the weight was weighed again, the lost weight was made up with ethanol, shaken well, filtered through a drying filter, 25ml of the filtrate was measured precisely, placed in an evaporation dish dried to constant weight, dried on a water bath, dried at 105 ℃ for 3 hours, placed in a desiccator for cooling for 30 minutes, and the weight was weighed precisely and quickly. The content (%) of the alcohol-soluble extract in the test sample was calculated. The extract content of 15 batches of classical name material standard was determined and the results are shown in table 30.
TABLE 30 measurement results of reference extract of different batches of material
The results show that: the minimum value of 15 batches of substance reference extracts prepared from different batches of decoction pieces is 27.8 percent, the maximum value is 70.1 percent, the average value is 49.5 percent, and the plus or minus 3SD value of the average value is 18.4 to 80.5 percent. Considering various influencing factors and the like in the actual production process, the content of the alcohol-soluble extract of the product is tentatively set to be 18.0-81.0%.
1.3.3.2.4 fingerprint map
Due to the simple qualitative identification and quantitative analysis of index components, the quality of the reference quality of the substance is difficult to reflect. As a standard reference for measuring whether a substance reference corresponding real object is basically consistent with a substance reference, the quality of the standard reference should be enhanced by specificity identification and multi-component and overall quality control. Therefore, the quality control based on material quality control emphasizes the overall quality control mainly based on fingerprint.
Instrument and reagent
Liquid chromatograph: saimei fly-U3000
A chromatographic column: Inertsustatin-AQ-C18 (250 mm. times.4.6, 5 μm)
Reagent: acetonitrile is chromatographically pure; the methanol is chromatographic pure, and the water is ultrapure water; other reagents were analytically pure.
Sample preparation: 15 batches of 'Baoyuan decoction quality standard'
Comparison products: ammonium glycyrrhizinate (batch No. 110731-201720) and calycosin glucoside (batch No. 111920-201606) were purchased from China institute for testing food and drug
② investigation of chromatographic conditions
The method comprises the following steps: reference document "HPLC-UV fingerprint spectrum research of radix astragali drug"
Chromatographic conditions and System suitability test InertSustain-AQ-C18 was a chromatographic column; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the following table; the flow rate was 1mL/min, the column temperature was 25 ℃, the detection wavelength was 210nm, and the mobile phase gradient elution is shown in Table 31.
TABLE 31 mobile phase gradient elution table
Preparation of reference solution A proper amount of calycosin glucoside and ammonium glycyrrhizinate reference substance is precisely weighed, 50% methanol is added to prepare 1ml solution containing calycosin glucoside and glycyrrhizic acid,
preparing a sample solution, precisely weighing about 2.5g of the product, placing the product in a 50mL triangular cone bottle, adding 50% methanol into 50mL of the triangular cone bottle, performing ultrasonic treatment for 45min, filtering, recovering a solvent from a filtrate under reduced pressure till the filtrate is nearly dry, dissolving residues in 20mL of water, extracting for 2 times with water saturated n-butyl alcohol solution, 25mL each time, combining n-butyl alcohol solutions, extracting for 1 time with 30mL of ammonia test solution, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residues in 2mL of water, performing column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating 95% ethanol eluent to dryness, dissolving the residues in 50% methanol, transferring to a 10mL volumetric flask, fixing the volume to the scale, shaking up, filtering, and taking a continuous filtrate to obtain the product.
The measurement method comprises precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, measuring, and recording 60min chromatogram, as shown in FIG. 5.
The second method comprises the following steps: the reference document "HPLC-UV fingerprint study of astragalus membranaceus medicinal material" changes the gradient of the mobile phase, and other conditions are unchanged, and the specific gradient is shown in table 32 below, and is shown in fig. 6.
TABLE 32 mobile phase gradient elution Table
The third method comprises the following steps: the reference document "HPLC-UV fingerprint study of astragalus root crude drug" changes the mobile phase gradient, the other conditions are not changed, and the specific gradient is as shown in table 33 below.
TABLE 33 mobile phase gradient elution Table
The method four comprises the following steps: the reference document "HPLC-UV fingerprint study of astragalus root crude drug" changes the mobile phase gradient, the other conditions are unchanged, and the specific gradient is as shown in table 34 below.
TABLE 34 mobile phase gradient elution Table
The method five comprises the following steps: the reference document "HPLC-UV fingerprint study of astragalus root crude drug" changes the mobile phase gradient, the other conditions are unchanged, and the specific gradient is as shown in table 35 below.
TABLE 35 mobile phase gradient elution Table
And (4) analyzing results: the first, second, third and fourth methods are gradient elution, the chromatogram effect separation effect is not ideal, and under the condition of the fifth method, the separation degree between chromatographic peaks is good, so that the mobile phase system under the gradient is selected.
Selection of preparation method of test solution
Reference document "HPLC-UV fingerprint spectrum research of radix astragali drug"
a. 50% methanol solution: precisely weighing about 2.5g of BAOYUANG decoction, placing in a conical flask, precisely adding 50% methanol 50ml, performing ultrasonic treatment (100W, 40kHz) for 45min, filtering, collecting filtrate, and filtering with 0.45 μm microporous membrane.
b. Extraction with water-saturated n-butanol: taking about 2.5g of the product, precisely weighing, placing in a 50mL triangular cone bottle, adding 50mL of 50% methanol, carrying out ultrasonic treatment for 45min, filtering, recovering the solvent from the filtrate under reduced pressure until the solvent is nearly dry, dissolving the residue in 20mL of water, extracting for 2 times with water saturated n-butyl alcohol solution, 25mL each time, mixing the n-butyl alcohol solution, extracting for 1 time with 30mL of ammonia test solution, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residue in 2mL of water, carrying out column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, eluting with 100mL of 95% ethanol, evaporating the 95% ethanol eluate to dryness, dissolving the residue in 50% methanol, transferring to a 10mL volumetric flask, fixing the volume to the scale, shaking up, filtering, and taking the subsequent filtrate to obtain the product.
c. And (3) ethyl acetate extraction: taking about 2.5g of the product, precisely weighing, placing in a 50mL triangular conical flask, adding 50mL of 50% methanol, carrying out ultrasonic treatment for 45min, filtering, recovering the solvent from the filtrate under reduced pressure until the solvent is nearly dry, dissolving the residue in 20mL of water, extracting for 2 times with 25mL of ethyl acetate solution each time, combining the ethyl acetate solutions, extracting for 1 time with 30mL of ammonia test solution, discarding the ammonia test solution, volatilizing the ethyl acetate layer solution, dissolving the residue in 2mL of water, passing through a column chromatography filled with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating the 95% ethanol eluate to dryness, dissolving the residue in 50% methanol, transferring to a 10mL volumetric flask, fixing the volume to scale, shaking up, filtering, and taking a subsequent filtrate to obtain the product.
According to the above chromatographic conditions, chromatograms of different extraction methods under the same chromatographic conditions were recorded, and the results are shown in FIG. 10.
The results show that: from the chromatogram, it is known that the first and third methods have unsatisfactory separation effect of chromatographic peak detection, and the second method has good separation effect of peaks, so the second method is selected as the preparation method of the test sample.
Selection of peak attribution and reference substance
a attribution of herb flavor
According to the substance standard preparation method, freeze-dried powder of single medicinal material and freeze-dried powder of negative sample lacking single medicinal material are prepared, the sample solution is prepared according to the determined preparation method of the sample solution of fingerprint spectrum, the determination is carried out according to the determined chromatographic conditions, and the chromatographic peak in the fingerprint spectrum of the freeze-dried powder belongs to which medicinal slices, as shown in figure 11 and table 36.
TABLE 36 attribution list of herbs and flavors
And (4) experimental conclusion: the substance standard chromatographic peak is mainly the characteristic peak of astragalus and liquorice, and the blank of the mobile phase is free from interference.
b selection of reference
The calycosin glucoside chromatographic peak has moderate retention time and high response value, and can achieve baseline separation, so that calycosin glucoside is selected as a reference peak of a reference substance, calycosin glucoside is marked as a peak S, and calycosin glucoside is used as a reference substance.
Preliminary determination of method
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; a chromatographic column: Inertsustatin-AQ-C18, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, flow rate of 1mL/min, detection wavelength of 210nm, specific gradient as shown in Table 37
TABLE 37 mobile phase gradient elution Table
Preparation of reference solution A proper amount of calycosin glucoside reference substance is precisely weighed, and 50% methanol is added to prepare 50.4592 μ g/ml solution containing calycosin glucoside per 1 ml.
Preparing a sample solution, precisely weighing about 2.5g of the product, placing the product in a 50mL triangular cone bottle, adding 50% methanol into 50mL of the triangular cone bottle, performing ultrasonic treatment for 45min, filtering, recovering a solvent from a filtrate under reduced pressure till the filtrate is nearly dry, dissolving residues in 20mL of water, extracting for 2 times with water saturated n-butyl alcohol solution, 25mL each time, combining n-butyl alcohol solutions, extracting for 1 time with 30mL of ammonia test solution, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residues in 2mL of water, performing column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating 95% ethanol eluent to dryness, dissolving the residues in 50% methanol, transferring to a 10mL volumetric flask, fixing the volume to the scale, shaking up, filtering, and taking a continuous filtrate to obtain the product.
The determination method comprises precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram for 70 min.
(analysis of methodology)
a specificity and integrity test
In order to investigate whether the blank solvent has interference, the blank solvent has no interference. Meanwhile, the integrity of the sample is also examined, and the result basically meets the principle of maximum information content (see table 38, figure 12)
TABLE 38 mobile phase gradient elution Table
And (4) analyzing results: and (3) the blank is found to be free of interference through comparison of a blank solvent spectrum and a chromatographic peak of the test solution.
b precision test
2.5g of this product (lot: BYT-2019091604D) is precisely weighed, and is continuously injected for 6 times according to the preparation and determination items of the test solution, the relative retention time and the relative peak area are calculated, and the specific determination results of the relative retention time and the relative peak area are shown in tables 39-40.
TABLE 39 calculation of fingerprint precision relative to retention time
TABLE 40 summary of relative peak areas of fingerprint precision
c stability test
2.5g of this product (lot number: BYT-2019091604D) is precisely weighed, and is subjected to sample injection measurement for 0, 3, 6, 9, 12 and 24 hours after preparation according to the text of preparation and measurement items of the test solution, and the relative retention time and the relative peak area are calculated, wherein the specific measurement results of the relative retention time and the relative peak area are shown in the following tables 41 to 42, and fig. 14.
TABLE 41 calculation of stability versus Retention time for finger prints
TABLE 42 summary of stability of finger prints versus peak area
The result shows that the relative retention time difference is not large, and the RSD is less than 2.0 percent; relative peak area RSD is less than 12.0%. Indicating that the stability in the test solution is good within 24 hours.
d repeatability test
About 2.5g of this product (lot: BYT-2019091604D) (6 parts in total) was weighed out precisely and subjected to the following procedures in the text of test solution preparation and measurement. The relative retention time and the relative peak area were calculated, and the specific measurement results of the relative retention time and the relative peak area are shown in tables 43 to 44 below, and fig. 15.
TABLE 43 calculation of fingerprint repeatability versus retention time
TABLE 44 summary of relative peak areas of repeatability of finger prints
The result shows that the relative retention time difference is not large, and the RSD is less than 2.0 percent; the relative peak area RSD is less than 10.0 percent, which shows that the repeatability of the method is good.
Seventhly, establishing the fingerprint map
a calibration of common peaks
Measuring chromatographic peaks of 15 batches of samples by adopting a method under the text (fingerprint), analyzing the obtained fingerprint, selecting chromatographic peaks with better stability and proper response value in the 15 batches of sample fingerprints as common peaks, and calibrating 12 common peaks in total. The results are shown in FIG. 16.
b establishing a control map
The following comparison fingerprint R is generated by correcting 12 marked common peaks according to the measurement result of 15 batches of substance standard fingerprints by adopting 'Chinese medicine chromatogram fingerprint similarity evaluation software system 2012 edition' issued by the State pharmacopoeia Committee. The results are shown in FIG. 1.
c calculation of similarity
The similarity between 15 batches of samples and the reference fingerprint is calculated by common peaks by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation software system, and the result is shown in a table 45.
TABLE 45 fifteen lots of material Standard similarity evaluation results
The R contrast maps S2-S16 are sequentially as follows: BYT-2019091001D, BYT-2019091002D, BYT-2019091003D, BYT-2019091604D, BYT-2019091605D, BYT-2019091606D BYT-2019091807D, BYT-2019091808D, BYT-2019091809D, BYT-2019092310D, BYT-2019092311D, BYT-2019092312D, BYT-2019092413D, BYT-2019092414D, BYT-20180515D
The fingerprint of the corresponding real object (lyophilized powder) of the above 15 batches of Baoyuan decoction is generated into a comparison fingerprint R, so that the similarity of the corresponding real object (lyophilized powder) of the 15 batches is more than 0.85 by comparison, and the similarity of the corresponding real object (lyophilized powder) of different batches is good. The similarity between the temporary sample fingerprint and the reference fingerprint should not be less than 0.85.
1.3.3.2.5 content determination
And comprehensively determining content measurement indexes by combining the prescription efficacy indication and the positions of the prescription medicines in the prescription. As the process research determines that water is a solvent, water-soluble components in the quality standard of the Baoyuan decoction are more, wherein the main component of the monarch drug ginseng is saponin components, so the content of the total amount of the ginsenoside Rg1, Rb1 and Re can be measured. Although the ministerial drug astragalus mainly contains flavonoids, saponins and the like, the pharmacological action of the ministerial drug astragalus is related to the effect of the standard substance of the decoction for protecting the vitality, so the content of the astragaloside in the astragalus is measured to comprehensively evaluate the standard substance quality.
Firstly, ginseng
Measuring according to high performance liquid chromatography (Chinese pharmacopoeia 2015 edition four parts (general rules 0512)
a instruments and reagents
Liquid chromatograph: saimei Fei-U3000;
a chromatographic column: InertSustain-AQ-C18(250 mm. times.4.6, 5 μm);
reagent: acetonitrile is chromatographic pure, and water is ultrapure water; other reagents were analytically pure.
Comparison products: the reference substances of the ginsenosides Rg1, Rb1 and Re (the batch numbers: 110703-201832, 110704-201726 and 110754-201827) are all purchased from China institute of food and drug testing).
b selection of chromatographic conditions
a) Selection of detection wavelength
A ginsenoside Rg1 reference substance, a ginsenoside Re reference substance and a ginsenoside Rb1 reference substance are precisely weighed, methanol is added to prepare a proper reference substance solution (the concentration of the ginsenoside Rg1 is 33.6024 mu g/ml, the batch number is 110703-201731, the concentration of the ginsenoside Re is 99.533 mu g/ml, the batch number is 110704-201726, the concentration of the ginsenoside Rb1 is 58.506 mu g/ml, the batch number is 1107544-201324), and scanning is carried out at the wavelength of 190-500 nm, so that the ginsenoside Rg1 has a maximum absorption peak at 202, the ginsenoside Rb1 has a maximum absorption peak at 204, and the ginsenoside Re has a maximum absorption peak at 203.
Summarizing and explaining: ginsenoside Rg1 has the maximum absorption peak at 202, ginsenoside Rb1 has the maximum absorption peak at 203, ginsenoside Re has the maximum absorption peak at 204, the detection wavelength of the ginsenoside Rg1, Rb1 and Re in pharmacopeia is 203nm, the maximum absorption peaks of the ginsenosides Rg1, Rb1 and Re comprise the detection wavelengths of the ginsenoside Rg1, Rb1 and Re in pharmacopeia, and 203nm is selected as the preferable detection wavelength for taking the measurement of the ginsenosides Rg1, Rb1 and Re into consideration and the chromatographic conditions of related references.
b) Selection of mobile phase
The method comprises the following steps: reference document "determination of ginsenoside content in Sijunzi decoction and Lizhong pill formula"
Chromatographic conditions and system applicability test the octadecylsilane chemically bonded silica was used as filler, acetonitrile was used as mobile phase a, aqueous solution was used as mobile phase B, detection wavelength was 203nm, flow rate was 1mL/min, and mobile phase gradient elution is shown in table 46.
TABLE 46 mobile phase gradient elution Table
Preparing a sample solution, precisely weighing about 1g of the product, dissolving in 12.5ml of water, placing in a separating funnel, extracting with chloroform for 3 times, 15ml each time, discarding a chloroform layer solution, extracting the solution with a water-saturated n-butanol solution for 3 times, 30ml each time, combining n-butanol solutions, washing with an ammonia test solution for 2 times, 30ml each time, discarding the ammonia test solution layer, washing the n-butanol solution with a water-saturated n-butanol solution water layer solution for 20ml, washing for 1 time, discarding a water layer solution, evaporating the n-butanol solution to dryness, dissolving residues with methanol, transferring to a 5ml volumetric flask, adding methanol to fix the volume to the scale, and shaking up to obtain the product.
As a result: the separation effect of the method is poor.
The second method comprises the following steps: reference for content determination of ginsenoside Rb1, Rg1 and Re in 'Shuangshen tablet'
The chromatographic conditions and the system applicability test take octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A, an aqueous solution as a mobile phase B, the detection wavelength of 203nm and the flow rate of 1mL/min, and the specific gradient is as follows:
TABLE 47 mobile phase gradient elution Table
Preparing a sample solution, precisely weighing about 1g of the product, adding 40 chloroform, refluxing for 1 hour, cooling, volatilizing the residue, adding 30ml of methanol, heating and refluxing for 2 hours, cooling, filtering, evaporating the filtrate to dryness, dissolving the residue in water (washing and dissolving the residue for 3 times, respectively 15ml, 10ml and 10ml, combining washing solutions), adding 1ml of 3% ammonia water solution, extracting with water-saturated n-butanol for 4 times, 35ml each time, combining the n-butanol solution, evaporating to dryness, dissolving the residue in methanol, transferring to a 5ml volumetric flask, adding methanol to a constant volume to a scale, and shaking uniformly to obtain the product.
The experimental results are as follows: the separation effect of the method is poor.
The third method comprises the following steps: according to the chromatographic conditions under the content measurement item of 'ginseng' in the first part of 'Chinese pharmacopoeia' 2015, samples are injected to the test samples treated by the first and second methods, wherein the chromatographic conditions are as follows:
chromatographic conditions and systematic usability tests take octadecylsilane chemically bonded silica as a filler; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, the detection wavelength is 203nm, and the specific gradient is shown in the following table 48.
TABLE 48 mobile phase gradient elution Table
Experimental analysis: under the gradient condition, a method I for sample treatment is adopted, and the separation degree between chromatographic peaks is good, so that the method I for sample treatment is selected, and the chromatographic condition adopts the content determination chromatographic condition under the ginseng item of Chinese pharmacopoeia for further investigation.
c examination of preparation method of test solution
a) Examination of extraction methods
Taking about 1g (total 3 groups) of the product (lot number: BYT-2019091001D), precisely weighing, adding 12.5ml of water for dissolving, respectively carrying out ultrasonic treatment for 30min, carrying out reflux treatment for 30min, placing the mixture without treatment in a separating funnel, adding chloroform for extracting for 3 times, 15ml each time, discarding chloroform layer liquid, extracting the solution for 3 times with water-saturated n-butanol solution for 30ml each time, combining n-butanol solution, washing for 2 times with ammonia test solution for 30ml each time, discarding the ammonia test solution layer, washing the n-butanol solution for 1 time with water-saturated n-butanol solution for 20ml, discarding the water layer solution, evaporating the n-butanol solution to dryness, dissolving the residue with methanol, transferring to a 5ml volumetric flask, adding methanol for volume fixing to scale, and shaking up to obtain the product. Precisely sucking 10 μ l of the subsequent filtrate, injecting into a liquid chromatograph, measuring, and calculating to obtain the final product. The results are shown in Table 49.
Table 49 results of examination of extraction modes
The results show that the content of samples with different extraction modes is not very different, and the direct treatment is selected in consideration of the convenience of extraction.
b) Examination of extraction vehicle
Taking about 1g (total 3 groups) of the product (lot number: BYT-2019091001D), precisely weighing, adding 12.5ml of water to dissolve, placing in a separating funnel, adding chloroform to extract for 3 times, 15ml each time, discarding chloroform layer liquid, extracting solution with petroleum ether, ethyl acetate and water saturated n-butyl alcohol solution for 3 times, 30ml each time, combining n-butyl alcohol solutions, washing with ammonia test solution for 2 times, 30ml each time, discarding ammonia test solution layer, washing petroleum ether layer, ethyl acetate layer and n-butyl alcohol layer with water saturated n-butyl alcohol solution water layer solution 20ml, washing for 1 time, discarding water layer solution, evaporating n-butyl alcohol solution, dissolving residue with methanol, transferring to a 5ml volumetric flask, adding methanol to constant volume to scale, and shaking up to obtain the product. Precisely sucking 10 μ l of the subsequent filtrate, injecting into a liquid chromatograph, measuring, and calculating to obtain the final product. The results are given in Table 50 below.
TABLE 50 results of the extraction vehicle study
The results show that: extracting with water saturated n-butanol solution to obtain ginsenoside Rg1, Rb1 and Re with high total content, so selecting water saturated n-butanol as extraction solvent.
Preliminary determination of the method of measuring the content
The chromatographic condition and system applicability test adopts octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, and water solution as mobile phase B, and the detection wavelength is 203nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1. Gradient elution was performed as specified in table 51 below.
TABLE 51 gradient elution Table
Preparation of reference solution A proper amount of ginsenoside Rg1, Rb1 and Re reference is precisely weighed, and methanol is added to obtain 0.2mg mixed solution containing ginsenoside Rg1, Rb1 and Re per 1ml to obtain the final product
Preparing a sample solution, precisely weighing about 1g of the product, dissolving in 12.5ml of water, placing in a separating funnel, extracting with chloroform for 3 times, 15ml each time, discarding a chloroform layer solution, extracting the solution with a water-saturated n-butanol solution for 3 times, 30ml each time, combining n-butanol solutions, washing with an ammonia test solution for 2 times, 30ml each time, discarding the ammonia test solution layer, washing the n-butanol solution with a water-saturated n-butanol solution water layer solution for 20ml, washing for 1 time, discarding a water layer solution, evaporating the n-butanol solution to dryness, dissolving residues with methanol, transferring to a 5ml volumetric flask, adding methanol to fix the volume to the scale, and shaking up to obtain the product.
The determination method comprises precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, determining, and recording chromatogram of 110 min.
d methodological investigation
a) Specificity test
The total content of ginsenoside Rg1, Rb1 and Re is used as index components of Baoyuan decoction, in order to examine whether the medicinal materials interfere with each other, the medicinal materials are taken according to the prescription ratio to prepare a negative sample (batch number: BYT-RS-20190917-D01) by the same method, and a negative sample solution without ginseng is prepared according to the treatment method of a test sample, and a sample injection chromatogram is recorded. The result shows that no chromatographic peak exists in the negative chromatogram in the retention time corresponding to the ginsenosides Rg1, Rb1 and Re, the determination between the two medicines is free of interference, and the determination of the contents of the ginsenosides Rg1, Rb1 and Re in the product by the method has specificity.
The result shows that no chromatographic peak exists in the negative chromatogram in the retention time corresponding to the ginsenosides Rg1, Rb1 and Re, the determination between the two medicines is free of interference, and the determination of the total content of the ginsenosides Rg1, Rb1 and Re in the product by the method has specificity.
b) Investigation of linear relationships
Precisely sucking a reference substance mixed solution (the concentration of ginsenoside Rg1 is 0.2016mg/ml, the batch number is 110703-201731, the concentration of ginsenoside Rg1 is 0.223mg/ml, the batch number is 110704-201726, the concentration of ginsenoside Rg1 is 0.2106mg/ml, the batch number is 110754-201324), injecting samples of 2 muL, 4 muL, 8 muL, 12 muL, 16 muL and 20 muL, recording a chromatogram, measuring a peak area, and regressing the sample injection amount (C) by using the peak area (A) to obtain a standard curve. The results are given in Table 52 below.
TABLE 52 Linear relationship of Total amounts of ginsenoside Rg1, Rb1, Re
Regression equation of ginsenoside Rg 1: y is 0.0041x +0.0447, and the correlation coefficient of the total amount of the ginsenoside Rg1 is as follows: r is 0.9997, and the ginsenoside Rg1 has good linearity in the range of 403.2-4032 μ g.
Regression equation of ginsenoside Re: y is 0.0017x +0.1657, and the correlation coefficient of the ginsenoside Re: when r is 0.9994, ginsenoside Re has good linearity in the range of 0.446-4460 mug.
Regression equation for ginsenoside Rb 1: y is 0.0031x +0.0597, correlation coefficient of ginsenoside Rb 1: when r is 0.9999, ginsenoside Rb1 is linear in the range of 421.2 μ g-4212 μ g.
c) Precision of the instrument
Taking mixed reference solution of ginsenoside Rg1, Rb1 and Re, carrying out continuous sample injection for 6 times according to text determination method, and determining results are shown in Table 53.
TABLE 53 Instrument precision test results (n ═ 6)
d) Stability test
About 1g of this product (lot: BYT-2019091807D) was weighed out precisely, and was subjected to sample injection measurement for 0, 2, 4, 8, 12, and 24 hours after preparation according to the procedure for determining the preparation and measurement method of the test solution, and the measurement results are shown in Table 54 below.
TABLE 54 stability test results
e) Repeatability test
About 1g (total 6 parts) of this product (lot No. BYT-2019091807D) was weighed out precisely and subjected to the following procedures in the text of test solution preparation and measurement. The results are shown in Table 55 below.
Table 55 repeatability test results (n ═ 6)
f) Sample application recovery test
Taking the product (batch: BYT-2019091807D), precisely weighing about 0.5g and 6 parts in total, precisely adding 1.1ml of mixed reference substance solution of ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re with the concentration of 0.2016 (batch: 110703-.
TABLE 56 sample Loading recovery test results (n ═ 6)
f determination method confirmation
The above research result tests show that the method for measuring the total content of the ginsenosides Rg1, Rb1 and Re in the material object (freeze-dried powder) corresponding to the Baoyuan decoction has strong specificity, and the precision, the stability, the repeatability, the sample adding and recycling and the like all accord with the regulations, so the liquid chromatography condition can be used for measuring the total content of the ginsenosides Rg1, Rb1 and Re in the material object (freeze-dried powder) corresponding to the Baoyuan decoction, and the specific measurement conditions are as follows:
the chromatographic condition and system applicability test adopts octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, and water solution as mobile phase B, and the detection wavelength is 203nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1. The gradient elution was performed as specified in table 57 below.
TABLE 57 gradient elution Table
Preparation of reference solution A proper amount of ginsenoside Rg1, Rb1 and Re reference is precisely weighed, and methanol is added to obtain 0.2mg mixed solution containing ginsenoside Rg1, Rb1 and Re per 1ml to obtain the final product
Preparing a sample solution, precisely weighing about 1g of the product, dissolving in 12.5ml of water, placing in a separating funnel, extracting with chloroform for 3 times, 15ml each time, discarding a chloroform layer solution, extracting the solution with a water-saturated n-butanol solution for 3 times, 30ml each time, combining n-butanol solutions, washing with an ammonia test solution for 2 times, 30ml each time, discarding the ammonia test solution layer, washing the n-butanol solution with a water-saturated n-butanol solution water layer solution for 20ml, washing for 1 time, discarding a water layer solution, evaporating the n-butanol solution to dryness, dissolving residues with methanol, transferring to a 5ml volumetric flask, adding methanol to fix the volume to the scale, and shaking up to obtain the product.
The determination method comprises precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, determining, and recording chromatogram of 110 min.
g multiple batches of corresponding substance (freeze-dried powder) content determination
The total content of ginsenoside Rg1, Rb1 and Re in 15 batches of corresponding real objects (lyophilized powder) was determined according to the preparation and determination method of test solution, and the results are shown in Table 58.
TABLE 58 Total content determination results of ginsenoside Rg1, Rb1, and Re in corresponding real object (lyophilized powder) of different batches
From the above table, it can be seen that: the minimum value of the total content of the ginsenosides Rg1, Rb1 and Re is 0.929mg/g, the maximum value is 1.973mg/g, the average value is 1.480mg/g, the SD value is 0.281, the average value minus 3 times of the SD value is 0.638mg/g, and the average value plus 3 times of the SD value is 2.322 mg/g.
The total content of the provisional ginsenosides Rg1, Rb1 and Re is in the range of 0.6mg/g to 2.4mg/g by combining with the actual requirements of production.
② astragalus root
Liquid chromatograph: a Saimer fly Ultimate 3000;
and (3) chromatographic column: GL Sciences Lnc
AQ-C18(5μm，250×4.6mm)；Welch 
AQ-C18 (5 μm, 250X 4.6mm), etc.
Shanghai Yueping scientific instruments Co., Ltd FA1104 (one in ten thousand)
Japan Shimadzu science and technology Co., Ltd AUW1220D (one hundred thousand)
Acetonitrile is chromatographically pure: TEDIA production; methanol is chromatographically pure: TEDIA production; phosphoric acid is chromatographically pure, and Aladdin is produced; methanol was analytically pure: hongsheng fine chemical company, Heizhou city; water is ultrapure water, Onhancon technologies, Inc.
Astragaloside IV reference substance (batch number: 110781-.
Preliminary determination of content determination method
According to the content determination of the astragalus root granules in the Chinese pharmacopoeia
Precisely weighing 1.0g of the product, precisely adding 50ml of water, weighing, carrying out ultrasonic treatment (with the power of 720W and the frequency of 40kHz) for 45 minutes, weighing again, supplementing the lost weight with water, shaking up, filtering, precisely taking 25ml of subsequent filtrate, shaking up and extracting with water-saturated n-butyl alcohol for 4 times, 25ml each time, combining n-butyl alcohol extracts, washing with ammonia test solution for 3 times, 30ml each time, discarding the ammonia test solution washing solution, separating n-butyl alcohol solution, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product. Octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-water (35: 65) is used as a mobile phase; detection by an evaporative light scattering detector. The number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak.
Chromatographic conditions and system applicability test with octadecylsilane chemically bonded silica as filler and acetonitrile-water (35: 65) as mobile phase; detection by an evaporative light scattering detector. The number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak.
Preparation of control solution A proper amount of astragaloside IV control is precisely weighed, and added with methanol to obtain solution containing 0.4mg per lml.
Preparing a test solution, precisely weighing 1.0g of the product, precisely adding 50ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 30 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, shaking up and extracting with water-saturated n-butanol for 4 times, 25ml each time, combining n-butanol extract, washing with ammonia test solution for 3 times, 30ml each time, discarding ammonia test solution washing liquor, separating n-butanol solution, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product.
The determination method comprises precisely sucking 10 μ L and 20 μ L of reference solution and 20 μ L of test solution, subjecting to liquid chromatograph, determining, and calculating with external standard two-point method logarithmic equation.
a methodology examination
a) Specificity test
The content of astragaloside is used as one of index components of the Baoyuan decoction, in order to examine whether the medicinal materials are mutually interfered, the medicinal materials are taken according to the prescription proportion to prepare a negative sample (batch number: BYT-HQ-20190917-D01) by the same method, and a negative sample solution without astragaloside is prepared according to the treatment method of a test sample and is injected to record a chromatogram. The result shows that no chromatographic peak exists in the negative chromatogram in the retention time corresponding to the astragaloside IV, the determination of medicines is free of interference, and the method for determining the astragaloside IV content in the product has specificity.
a) Investigation of linear relationships
Precisely sucking 2 μ l, 4 μ l, 8 μ l, 12 μ l, 16 μ l and 20 μ l of astragaloside IV reference substance (batch No. 110781-. The sample amount (μ g) was taken as the abscissa and the peak area as the ordinate, and a standard curve was drawn. The results are given in Table 59 below.
The regression equation: 4.7063x-4.5398
Correlation coefficient: r-0.9972
The result shows that the astragaloside IV has good linearity in the range of 1.0036-10.036 mu g
TABLE 59 astragaloside IV Linear relationship
b) Precision of the instrument
Taking astragaloside IV reference substance solution, and continuously injecting sample for 6 times according to text determination method, wherein the determination result is shown in Table 60.
TABLE 60 Instrument precision test results (n ═ 6)
The results show that: RSD is less than 2%, and the precision of the instrument is good.
c) Stability test
1g of the product (lot number: BYT-2019091807D) is weighed, and is subjected to operation according to the items of preparation and determination of a test sample solution, and is respectively placed for 0, 2, 4, 8, 12 and 24 hours after preparation for sample injection determination, and the determination results are shown in the following table 61.
TABLE 61 stability test results
The results show that: RSD is less than 2 percent, and the stability of the sample is good.
d) Repeatability test
The experimenter (A) takes about 1g (5 parts in total) of this product (batch number: BYT-2019091807D), precisely weighs it, and operates according to the text of the preparation and measurement of the test solution. The results are shown in Table 62 below.
Table 62 repeatability test results (n ═ 6)
The result shows that RSD is less than 3.0%, which indicates that the repeatability of the method is good.
e) Sample application recovery test
0.5g of an extract quality standard sample (the content of astragaloside is 0.661mg/g) and 6 parts in total are precisely weighed, 0.28ml of astragaloside control solution (the concentration is 0.5018mg/ml, the batch number is 110781-.
TABLE 63 astragaloside IV accuracy test results (n ═ 6)
As a result, the average recovery rate is 98.32%, and the RSD is less than 2.26%, which shows that the method has good accuracy.
f determination method confirmation
The research result tests show that the method for measuring the content of astragaloside in the material object (freeze-dried powder) corresponding to the Baoyuan soup has strong specificity, the stability, the repeatability, the accuracy and the like all accord with the regulations, and the durability of the chromatographic condition is good, so that the liquid chromatographic condition can be used for measuring the content of astragaloside in the material object (freeze-dried powder) corresponding to the Baoyuan soup, and the specific measuring conditions are as follows:
chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; acetonitrile-water (35: 65) is used as a mobile phase; detection by an evaporative light scattering detector. The number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak.
Preparation of control solution A proper amount of astragaloside IV control is precisely weighed, and added with methanol to obtain solution containing 0.5mg per lml.
Preparing a test solution, precisely weighing 1.0g of the product, precisely adding 50ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 45 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, shaking up and extracting with water-saturated n-butanol for 4 times, 25ml each time, combining n-butanol extract, washing with ammonia test solution for 3 times, 30ml each time, discarding ammonia test solution washing liquor, separating n-butanol solution, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product.
The determination method comprises precisely sucking 10 μ L and 20 μ L of reference solution and 20 μ L of test solution, subjecting to liquid chromatograph, and calculating with external standard two-point method.
g multiple batches of corresponding substance (freeze-dried powder) content determination
The astragaloside content in 15 batches of the corresponding real object (lyophilized powder) was determined according to the procedures of preparation and determination of test solution, and the results are shown in Table 64.
TABLE 6415 content of astragaloside IV in corresponding real object (lyophilized powder)
The results show that: 15 batches of corresponding real object (lyophilized powder) astragaloside IV with the range of 0.479-1.037 mg/g, the average content of 0.772mg/g, the subtraction 3SD value of 0.19mg/g, the addition 3SD value of 1.35mg/g, and considering various influence factors in the actual production process, etc., the product is tentatively calculated according to the dry product, and each 1g astragaloside IV (C) is contained41H68O14) The amount of the substance is 0.10-1.40 mg (. + -. 3 times SD).
1.3.4 Mass analysis of corresponding entities
1.3.4.1 preparation of corresponding entities
The traditional Chinese medicine ingredients in the prescription are prepared according to a determined process, which is detailed in 'process research', 15 batches of qualified decoction pieces in the prescription are prepared according to a material standard, the dosage of each batch is 20 times daily prescription amount, about 30g, 15 batches of material standard are prepared in parallel, and the specific preparation results are shown in the following table 65.
TABLE 65 fifteen lot Material benchmarks
1.3.4.2 corresponding substance determination method
(1) And (3) measuring the cream yield: the determination method is detailed in 'process research'.
(2) And (3) moisture determination: according to a second method for determining water content of 0832 according to general regulations of the four departments of the edition of 'Chinese pharmacopoeia' 2015, 2g of a test sample is taken to be spread in a flat weighing bottle which is dried to constant weight, the thickness is not more than 5mm, the loose test sample is not more than 10mm, the test sample is precisely weighed, the bottle cap is opened to be dried for 5 hours at 105 ℃, the bottle cap is covered and moved into a drier to be cooled for 30 minutes, the test sample is precisely weighed, and the test sample is dried for 1 hour at the temperature, cooled and weighed until the difference between two successive weighing times is not more than 5 mg. The test article is calculated to have a water content of less than 12 (%) based on the weight loss.
(3) And (3) extract determination: the alcohol-soluble hot-dipping method is determined according to a rule 2201 of China pharmacopoeia (general regulations of China pharmacopoeia) 2015: taking a proper amount of the product, grinding, precisely weighing about 2g, placing in a 100ml conical flask, precisely adding 50ml of ethanol, sealing, weighing, standing for 1 hour, performing extract measurement by adopting a hot dipping method, precisely weighing 25ml of filtrate, placing in an evaporation dish dried to constant weight, drying by distillation on a water bath, drying at 105 ℃ for 3 hours, cooling in a dryer for 30 minutes, and rapidly precisely weighing. The content of alcohol-soluble extract in the sample is calculated to be 18.0-81.0% (%).
(4) Fingerprint spectrum determination: the method for measuring the medicinal materials, the decoction pieces, the intermediates and the corresponding material objects is detailed in 8.4 quality standard texts, 12 common peaks are marked according to the measurement results of 15 batches of substance reference fingerprints, a reference fingerprint is generated, and the similarity is calculated. The preparation method of the medicinal materials, the decoction pieces and the intermediate samples comprises the following steps:
a, liquorice medicinal material solution: weighing 2.5g of Glycyrrhrizae radix powder (sieved with a sieve IV) according to a prescription, precisely weighing, placing in a conical flask with a plug, adding 50% methanol 50ml, performing ultrasonic treatment for 45min, filtering, recovering solvent from the filtrate under reduced pressure to near dryness, dissolving the residue in 20ml of water, extracting with water saturated n-butanol solution for 2 times, each time 25ml, mixing n-butanol solutions, extracting with 30ml of ammonia test solution for 1 time, discarding the ammonia test solution, evaporating the n-butanol layer to dryness, and dissolving the residue in 2ml of water. Passing through a chromatographic column filled with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, collecting 95% ethanol eluate, evaporating to dryness, dissolving the residue in 50% methanol, transferring to a 10ml volumetric flask, metering to a certain volume, shaking up, and filtering to obtain the product.
b, liquorice decoction piece solution: weighing 2.5g Glycyrrhrizae radix decoction pieces powder (sieved with No. four sieve) according to the prescription, precisely weighing, placing in a conical flask with a plug, adding 50% methanol 50ml, ultrasonic treating for 45min, filtering, recovering solvent from the filtrate under reduced pressure to near dryness, dissolving the residue in 20ml water, extracting with water saturated n-butanol solution for 2 times, each time 25ml, mixing n-butanol solutions, extracting with 30ml ammonia test solution for 1 time, discarding ammonia test solution, evaporating n-butanol layer to dryness, and dissolving the residue in 2ml water. Passing through a chromatographic column filled with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, collecting 95% ethanol eluate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the product.
c, ginseng decoction piece solution: weighing 2.5g of Ginseng radix decoction pieces powder (sieved with a sieve IV) according to a prescription, precisely weighing, placing in a conical flask with a plug, adding 50ml of 50% methanol, performing ultrasonic treatment for 45min, filtering, recovering solvent from the filtrate under reduced pressure until the filtrate is nearly dry, dissolving the residue in 20ml of water, extracting with water saturated n-butanol solution for 2 times, 25ml each time, combining n-butanol solutions, extracting with 30ml of ammonia test solution for 1 time, discarding the ammonia test solution, evaporating the n-butanol layer to dryness, and dissolving the residue in 2ml of water. Passing through a chromatographic column filled with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, collecting 95% ethanol eluate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the product.
d intermediate (extract): precisely measuring 5ml of the extractive solution, placing in a conical flask with a plug, adding 50ml of 50% methanol, performing ultrasonic treatment for 45min, filtering, recovering solvent from the filtrate under reduced pressure, dissolving the residue in 20ml of water, extracting with water saturated n-butanol solution for 2 times (25 ml each time), mixing n-butanol solutions, extracting with 30ml of ammonia solution for 1 time, discarding the ammonia solution, evaporating the n-butanol layer, and dissolving the residue in 2ml of water. Passing through a chromatographic column filled with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, collecting 95% ethanol eluate, evaporating to dryness, dissolving the residue in 50% methanol, transferring to a 10ml volumetric flask, metering to a certain volume, shaking up, and filtering to obtain the product.
e intermediate (concentrate): precisely measuring 2ml of the concentrated solution, placing in a conical flask with a plug, adding 50ml of 50% methanol, performing ultrasonic treatment for 45min, filtering, recovering solvent from the filtrate under reduced pressure until the filtrate is nearly dry, dissolving the residue in 20ml of water, extracting with water saturated n-butanol solution for 2 times, each time 25ml, mixing n-butanol solutions, extracting with 30ml of ammonia solution for 1 time, discarding the ammonia solution, evaporating the n-butanol layer to dryness, and dissolving the residue in 2ml of water. Passing through a chromatographic column filled with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, collecting 95% ethanol eluate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the product.
(5) Content determination: the total content of corresponding ginsenoside Rg1, Rb1 and Re, and astragaloside IV determination method are described in 8.4 quality standard text. The preparation method of the intermediate sample comprises the following steps:
firstly, extracting solution
The total content of the ginsenosides Rg1, Rb1 and Re: precisely absorbing 25ml of extracting solution, placing in a separating funnel, adding chloroform for extraction for 3 times, 15ml each time, removing a chloroform layer, extracting a water layer with water-saturated n-butanol for 3 times, 30ml each time, combining n-butanol solutions, washing with an ammonia test solution for 2 times, 30ml each time, removing the ammonia test solution layer, washing the n-butanol layer with 20ml of n-butanol saturated water for 1 time, removing the water layer, evaporating the n-butanol solution to dryness, adding a proper amount of methanol into residues for dissolving, transferring to a 5ml volumetric flask, and adding methanol for constant volume until the volume reaches the scale.
Content of astragaloside: precisely measuring 25ml of the extractive solution, performing ultrasonic treatment for 30min, extracting with water saturated n-butanol solution for 4 times (25 ml each time), mixing n-butanol solutions, extracting with ammonia solution under shaking for 3 times (30 min each time), discarding ammonia solution layer, collecting n-butanol solution, evaporating, dissolving residue with methanol, transferring to 5ml volumetric flask, and adding methanol to desired volume.
② concentrated solution
The total content of the ginsenosides Rg1, Rb1 and Re: precisely absorbing 10ml of the extracting solution, placing the extracting solution in a separating funnel, adding chloroform for extraction for 3 times, 15ml each time, removing a chloroform layer, extracting a water layer with water-saturated n-butyl alcohol for 3 times, 30ml each time, combining n-butyl alcohol solutions, washing with an ammonia test solution for 2 times, 30ml each time, removing the ammonia test solution layer, washing the n-butyl alcohol layer with 20ml of n-butyl alcohol-saturated water for 1 time, removing the water layer, evaporating the n-butyl alcohol solution, dissolving residues with a proper amount of methanol, transferring the residues into a 5ml volumetric flask, and adding methanol to fix the volume to a scale.
Content of astragaloside: precisely measuring 10ml of the extract in a 25ml volumetric flask, adding water to a constant volume to a scale, carrying out ultrasonic treatment for 30min, extracting with water saturated n-butyl alcohol solution for 4 times, each time of 25ml, combining n-butyl alcohol solutions, extracting with ammonia test solution by shaking for 3 times and each time of 30min, discarding the ammonia test solution layer, collecting the n-butyl alcohol solution, evaporating to dryness, adding methanol to the residue to dissolve the residue, transferring to a 5ml volumetric flask, and adding methanol to a constant volume to a scale to obtain the product.
1.3.4.3 measurement results
The results are summarized in Table 66 below.
1.3.4.4 determination of object key quality attribute range
(1) Rate of paste discharge
The standard cream yield range of 15 batches of materials is 9.85-17.52%, the average cream yield is 13.97%, and the standard cream yield ranges from 6.65-21.28% in a 3SD floating range; the average value +/-70% of the floating range is 9.78% -18.16%. The range of the temporary cream yield is 6.00-22.00%.
(2) Moisture content
The 15 batches of the material have the reference moisture range of 6.3-11.8 percent, the average moisture of 8.7 percent and the average value +/-3 SD value range of 4.05-13.38 percent, so the tentative moisture is not more than 12.0 percent.
(3) Extract of plant
The 15 batches of material standard extract range is 27.8-70.1%, the average extract is 49.47%, the SD value is 0.1, the average value +/-3 SD value range is 18.43-80.52%, and the tentative extract range is 18.0-81.0%.
(4) Finger print
15 batches of the standard finger prints of the material of the Baoyuan decoction generate comparison finger prints, the similarity of the 15 batches of the material standard is more than 0.85 by comparing the 15 batches of the material standard in sequence, the similarity of the material standard among different batches is good, and the limit of the similarity of the tentative finger prints is more than or equal to 0.85.
(5) Determination of content
15 batches of substance standard ginsenoside Rg1, Rb1 and Re have total content range of 0.929 mg/g-1.973 mg/g and average content of 1.480mg/g, which are all within the range of +/-30% and 3 SD; referring to + -30% and 3SD floating range, considering various influence factors in actual production process, etc., the product is tentatively calculated according to dry product, and every 1g contains ginsenoside Rg1 (C)42H72O14)、Rb1 (C54H92O23)、Re(C48H82O18) The total content of (b) is 0.6 mg/g-2.4 mg/g.
15 batches of materials have a standard astragaloside IV range of 0.479 mg/g-1.037 mg/g and an average content of 0.772mg/g, all of which are within 3SD, refer to a fluctuation range of +/-30 percent and 3SD, and simultaneously consider various influence factors and the like in the actual production process, the product is tentatively calculated according to dry products, and each 1g of the product contains astragaloside IV (C) for every 1g of astragalus41H68O14) The content of the active carbon is 0.10mg/g to 1.40 mg/g.
1.4 prescription of method for detecting Baoyuan composition
1.4.1 prescription
This recipe comes from the brief doctor hub (Ming & Sun Shi hong). For a detailed study, see "data 5 prescription examination and historical leather data".
1.4.2 preparation of
Decocting according to the method described in the list of classical name prescriptions. The method is characterized in that an electronic decoction pot (with the capacity of 9L) is adopted for decoction, and relevant parameters such as pretreatment, a heating mode, decoction time, filtration and drying processes of material standard decoction pieces are researched by taking the extraction rate, the total content of ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re, the extraction content of astragaloside IV and a fingerprint spectrum as indexes, so that the material standard process of the Baoyuan decoction is determined. According to the determined process, the prescription amount is amplified by 20 times, 15 batches of decoction liquid are extracted, filtered, concentrated and dried. 15 batches of substance-based corresponding real objects are obtained, and the result shows that the substance-based preparation process is stable and feasible.
The standard preparation method of the Baoyuan decoction substance comprises the following steps: 3.69g of ginseng, 7.38g of astragalus, 1.85g of liquorice, 0.74g of cinnamon and one piece of ginger are added, the formula amount is enlarged by 20 times, namely 74g of ginseng, 148g of astragalus, 37g of liquorice, 25g of cinnamon and 20 pieces of ginger are added, according to the water adding amount standard of the rhizome medicinal materials of the traditional Chinese medicine formula, the water adding amount is 7 times of the formula amount, namely 1920ml of the medicinal materials, the medicinal materials are soaked for 40min, decocted for 1h, filtered by 200-mesh filter cloth, filtrate is subjected to low-temperature vacuum concentration to obtain extract with the feeding amount ratio of 1: 3, and the extract is subjected to low-temperature freeze drying to obtain a substance standard corresponding object which is stored in a low-temperature drying place. (see "technical study" for details).
1.4.3 Properties
According to the actual observation condition of multiple batches of samples, the product is tentatively brown to tan powder, slightly fragrant, slightly bitter and sweet in taste.
1.4.4 identification
The product is prepared from four traditional Chinese medicines, is a compound preparation prepared by a water decoction process, adopts a thin-layer identification method with strong specificity, rapidness, simplicity and good reproducibility, carries out identification test research on main characteristic components of ginseng, astragalus, liquorice and cinnamon in the prescription, and determines that the ginseng, the astragalus, the liquorice and the cinnamon are brought into quality standards.
(1) Ginseng radix
The main components of Ginseng radix are saponins, saccharides, volatile components, organic acids and their esters, sterols and their glycosides, flavonoids, lignin, inorganic elements and vitamins etc.
Determining a thin layer identification method: collecting powder 1g, adding chloroform 40ml, heating and refluxing for 1 hr, discarding chloroform solution, volatilizing solvent from residue, adding water 0.5ml, stirring and moistening, adding saturated n-butanol 10ml, ultrasonic treating for 30min, collecting supernatant, adding 3 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dryness, and dissolving residue with methanol 1ml to obtain sample solution. Preparing 1g of ginseng reference medicinal material, and preparing a reference medicinal material solution by the same method. Adding methanol into ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 as reference solutions to obtain mixed solutions each containing 2mg of ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 in each 1 ml. Performing thin layer chromatography (general rule 0502) test, sucking the three solutions 1-2 μ 1, respectively dropping on the same silica gel G thin layer plate, spreading with lower layer solution of chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) at temperature below 10 deg.C as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, respectively placing under sunlight and ultraviolet lamp (365nm), observing in the sample chromatogram, at the position corresponding to the control chromatogram, displaying the same bluish purple spots, and the negative chromatogram has no spots at the position corresponding to the control chromatogram.
Through durability investigation of silica gel G thin-layer plates in low-temperature, normal-temperature and high-humidity environments and different manufacturers, the ginseng thin-layer identification condition is good in durability under different influence factors. In the chromatogram of the test sample based on different batches of substances, spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance. The thin layer method is hereby incorporated into the text.
(2) Radix astragali
The radix astragali contains saponins and flavonoids as main chemical components, and also contains polysaccharide. Performing thin layer chromatography experiments on radix astragali according to the above chemical components, and determining the method.
Determining a thin layer identification method: collecting 1.5g of the product, adding 25mL of water, performing ultrasonic treatment for 30min, filtering, extracting the filtrate with water saturated n-butanol under shaking for 2 times, each time 25mL, mixing n-butanol extractive solutions, washing with ammonia test solution for 2 times, each time 30mL, collecting n-butanol solution, evaporating to dryness, and dissolving the residue with methanol lmL to obtain test solution. And adding water 50ml into 1.5g of astragalus control medicinal material, decocting for 30 minutes, filtering, and preparing the filtrate into a control medicinal material solution by the same method. Adding methanol into astragaloside IV control to obtain solution containing 2mg of astragaloside IV per lmL as control solution. Performing thin layer chromatography (0502 of the four ministerial general rules of the design reside in the Chinese pharmacopoeia 2015), sucking 10 μ l of each of the control solution, and the sample solution, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution at 10 deg.C or below as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; the fluorescent spots with the same color are observed under an ultraviolet lamp (365 nm).
Through durability investigation of silica gel G thin-layer plates in low-temperature, normal-temperature and high-humidity environments and different manufacturers, the ginseng thin-layer identification condition is good in durability under different influence factors. In the chromatogram of the test sample based on different batches of substances, spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance. The thin layer method is hereby incorporated into the text.
(3) Licorice root, radix Glycyrrhizae
The main chemical components of Glycyrrhrizae radix are triterpenes and flavonoids, and in addition, it also contains polysaccharide, organic acid and coumarin. Performing thin layer chromatography experiments on Glycyrrhrizae radix according to the above chemical components, and determining the method.
Determining a thin layer identification method: taking a corresponding substance (freeze-dried powder) and 1g of liquorice-lacking negative freeze-dried powder, adding 40ml of water, carrying out ultrasonic treatment for 30 minutes, filtering, extracting for 2 times by shaking with ether, 10ml each time, discarding ether liquid, extracting water liquid by shaking with water-saturated n-butyl alcohol for 3 times by shaking, 20ml each time, combining n-butyl alcohol liquids, extracting for 2 times by shaking with ammonia test solution, 30ml each time, combining the ammonia test solutions, and adjusting the pH value to 3-4 by using hydrochloric acid. Extracting with ethyl acetate for 2 times (30 ml each time), mixing ethyl acetate solutions, evaporating to dry, and dissolving the residue with 1ml methanol to obtain test solution. Taking 1g of honey-fried licorice root decoction pieces, adding 50ml of water, decocting for 30 minutes, filtering, and preparing a control medicinal solution by the same method. Taking appropriate amount of liquiritin reference substance, adding 70% ethanol to make into solution containing 0.5mg per 1mL as reference substance solution. According to thin layer chromatography 2015 version four (general rule 0502) test, sucking 10 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution, developing with chloroform-methanol-water (13:7:2) subnatant placed at 5-10 deg.C as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until spot color development is clear, and inspecting under sunlight and ultraviolet lamp (365 nm).
Through durability examination of silica gel G thin layer plates in low-temperature, normal-temperature and high-humidity environments and different manufacturers, the identification condition of the honey-fried licorice root thin layer has good durability under different influence factors. In the chromatogram of the test sample based on different batches of substances, spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance. The thin layer method is hereby incorporated into the text.
(4) Cortex Cinnamomi
Cinnamon mainly contains cinnamaldehyde, cinnamic acid, diterpene and its glycoside, flavanol and its polymer, and in addition, flavonoids, polyphenols, etc. Experiments were performed on cinnamon by thin layer chromatography based on the above chemistry as follows.
Determining a thin layer identification method: taking 1g of corresponding substance (lyophilized powder) and each of the lyophilized powder for negative samples of the lean osmanthus, adding 10ml of water to dissolve, extracting with diethyl ether under shaking for 1 time, 25ml each time, mixing diethyl ether solution, volatilizing, and adding 2ml of diethyl ether to the residue to dissolve to obtain test solution and negative sample solution of the lean osmanthus. Decocting 1g of cortex Cinnamomi decoction pieces in 50ml of water for 30min, filtering, and making into control medicinal solution. Then, a lauric acid control was prepared, and 1ml of a solution containing 0.2mg was prepared with 50% methanol as a control solution. Performing thin layer chromatography (0502 of the four Provisions of the national pharmacopoeia 2015 edition), sucking 3 μ l of each of the four solutions, and dropping on the same silica gel GF254Developing on the thin layer plate with cyclohexane-diethyl ether-glacial acetic acid (5:5:0.3) as developing agent, taking out, air drying, inspecting under ultraviolet lamp (254nm), and displaying spots or fluorescent spots of the same color in the sample chromatogram at the positions corresponding to the reference medicinal material chromatogram and the reference substance chromatogram. The thin layer method is hereby incorporated into the text.
1.4.5 examination
(1) Moisture content
According to a water determination method (the second method of 0832 in the general rule of four parts of the edition of Chinese pharmacopoeia 2015), the minimum value of the standard water of 15 batches of materials is 6.3 percent, the maximum value is 11.8 percent, the average value is 8.7 percent, and the plus or minus 3SD value of the average value is 4.05 to 13.38 percent, so that the temporary dry paste water content is not more than 12.0 percent.
1.4.6 extracts
Most of chemical components or major components in the Chinese medicinal materials can be dissolved out in water or alcohol, and then extract measurement is carried out. Because the quality standard of the Baoyuan decoction is water extraction, the significance of establishing extract measurement by selecting water as a solvent is not large, and the internal quality is difficult to reflect, when the solvent is selected, the solubility of the major components is considered, and ethanol is selected for extract measurement to control the quality of the material standard.
The minimum value of 15 batches of the reference extract was 27.79%, the maximum value was 70.07%, the average value was 49.47%, and the ± 3SD value of the average value was 18.43% to 80.52%, measured by extract measurement method (2201 alcohol-soluble hot dipping method in the four departments of the pharmacopoeia of China 2015). Considering various influencing factors and the like in the actual production process, the content of the alcohol-soluble extract of the product is tentatively set to be 18.0-81.0%.
1.4.7 finger print
Due to the simple qualitative identification and quantitative analysis of index components, the quality of the reference quality of the substance is difficult to reflect. As a standard reference for measuring whether a substance reference corresponding real object is basically consistent with a substance reference, the quality of the standard reference should be enhanced by specificity identification and multi-component and overall quality control. Therefore, the quality control based on material quality control emphasizes the overall quality control mainly based on fingerprint.
Searching the mobile phase composition and gradient of the method and the preparation method of a sample, performing medicinal flavor attribution, peak identification and reference substance selection on each spectrum peak in the substance standard fingerprint (selecting calycosin glucoside with moderate retention time and high response value which achieves baseline separation as a reference substance), and primarily determining the fingerprint determination method.
The methodological verification is carried out on the preliminarily determined fingerprint spectrum method, the method has good specificity, the blank solvent has no interference, and simultaneously the principle of the maximum information content is basically satisfied, and the method has the advantages that the precision, the repeatability and the stability (within 24 hours) all accord with the regulations (the relative retention time RSD of each spectrum peak is less than 2 percent), all the common peak peaks in the chromatogram are sharp in shape, symmetrical and good in separation degree, so the method is determined as the fingerprint spectrum determination method and is listed as the standard text:
octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; a chromatographic column: InertSustain-AQ-C18, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, flow rate of 1mL/min, detection wavelength of 210nm, specific gradient see Table 67 below.
TABLE 67 mobile phase gradient elution Table
Preparation of reference solution A proper amount of calycosin glucoside reference substance is precisely weighed, and 50% methanol is added to prepare 0.2068mg/ml solution containing calycosin glucoside per 1ml to obtain the product.
Preparing a sample solution, precisely weighing about 2.5g of the product, placing the product in a 50mL triangular cone bottle, adding 50% methanol into 50mL of the triangular cone bottle, performing ultrasonic treatment for 45min, filtering, recovering a solvent from a filtrate under reduced pressure till the filtrate is nearly dry, dissolving residues in 20mL of water, extracting for 2 times with water saturated n-butyl alcohol solution, 25mL each time, combining n-butyl alcohol solutions, extracting for 1 time with 30mL of ammonia test solution, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residues in 2mL of water, performing column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating 95% ethanol eluent to dryness, dissolving the residues in 50% methanol, transferring to a 10mL volumetric flask, fixing the volume to the scale, shaking up, filtering, and taking a continuous filtrate to obtain the product.
The determination method comprises precisely sucking reference solution and test solution 10 μ L respectively, injecting into liquid chromatograph, determining, and recording chromatogram for 70 min.
And (3) measuring 15 batches of substance references according to the determination method, analyzing the obtained fingerprints, selecting chromatographic peaks with better stability and proper response value in the 15 batches of substance reference fingerprints as common peaks, and calibrating 12 common peaks in total. The HPLC chromatogram peak of the physical object (dry paste powder) is automatically matched according to 15 batches of Baoyuan decoction substance standards by adopting' traditional Chinese medicine chromatogram fingerprint similarity evaluation software system 2012 edition issued by the State pharmacopoeia Committee to form a common pattern diagram, and a comparison spectrum is established. The similarity of 15 batches of samples and the comparison fingerprint is calculated by the common peak and is more than 0.85, so the similarity of the product and the comparison fingerprint of the standard of the Baoyuan decoction substance is tentatively determined and is not less than 0.85 by selecting the fingerprint as the evaluation standard of the Baoyuan decoction substance.
1.4.8 content determination
And comprehensively determining content measurement indexes by combining the prescription efficacy indication and the positions of the prescription medicines in the prescription. As the process research determines that water is used as solvent, the quality standard of the Baoyuan decoction has more water-soluble components, wherein, the main components of the monarch drug ginseng are monoterpene and glycosides thereof, thus being capable of measuring the content of the total amount of the glycosides thereof, namely ginsenoside Rg1, Rb1 and Re. Although the ministerial drug astragalus mainly contains flavonoids, saponins and the like, the pharmacological action of the ministerial drug astragalus is related to the effect of the standard substance of the decoction for protecting the vitality, so the content of the astragaloside in the astragalus is measured to comprehensively evaluate the standard substance quality.
The method for measuring the total content of ginsenoside Rg1, Rb1 and Re comprises the steps of measuring the total content of ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re, preparing a test sample, and measuring the detection wavelength to preliminarily determine the content.
The methodology of the preliminary determination method is verified, the specificity of the method is good, the ginseng-deficient negative sample has no interference, the purity of the target peak is qualified, and the linearity of the ginsenoside Rg1 is good within the range of the sample injection amount of 403.2-4032 mug. The ginsenoside Re has good linearity in the range of 466-4660 mug. Ginsenoside Rb1 was linear well in the range of 421.2. mu.g to 4212. mu.g. The method has the advantages that the precision (RSD is less than 2.0 percent), the repeatability (RSD is less than 3.0 percent), the stability (RSD is less than 3.0 percent in 24 hours) and the accuracy (average recovery rate is 96.656 percent and RSD is 1.98 percent) all meet the requirements, and the astragaloside IV has good linearity in the range of the sample injection amount of 1.0036-10.036 mug. And the method has the advantages that the precision (RSD is less than 2.0 percent), the repeatability (RSD is less than 3.0 percent) and the stability (RSD is less than 3.0 percent in 24 hours) have accuracy (the average recovery rate is 98.32 percent and the RSD is 2.26 percent) which meet the requirements. The durability of the chromatographic conditions is good. Therefore, the liquid chromatography condition can be used for measuring the total content of the ginsenosides Rg1, Rb1 and Re in the corresponding real object (freeze-dried powder) of the Baoyuan decoction and the astragaloside IV content, and the specific measurement conditions are as follows:
in the ginseng chromatographic condition and system applicability test, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, an aqueous solution is used as a mobile phase B, the detection wavelength is 203nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1. The gradient elution was performed as specified in table 68 below.
TABLE 68 gradient elution Table
Preparation of reference solution A proper amount of ginsenoside Rg1, Rb1 and Re reference is precisely weighed, and methanol is added to obtain 0.2mg mixed solution containing ginsenoside Rg1, Rb1 and Re per 1ml to obtain the final product
Preparing a sample solution, precisely weighing about 1g of the product, dissolving in 12.5ml of water, placing in a separating funnel, extracting with chloroform for 3 times, 15ml each time, discarding a chloroform layer solution, extracting the solution with a water-saturated n-butanol solution for 3 times, 30ml each time, combining n-butanol solutions, washing with an ammonia test solution for 2 times, 30ml each time, discarding the ammonia test solution layer, washing the n-butanol solution with a water-saturated n-butanol solution water layer solution for 20ml, washing for 1 time, discarding a water layer solution, evaporating the n-butanol solution to dryness, dissolving residues with methanol, transferring to a 5ml volumetric flask, adding methanol to fix the volume to the scale, and shaking up to obtain the product.
The determination method comprises precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, determining, and recording chromatogram of 110 min.
The product contains ginsenoside Rg1 (C) per 1g of Ginseng radix42H72O14)、Rb1(C54H92O23)、Re(C48H82O18) The total content of (b) is 0.6 mg/g-2.4 mg/g.
The chromatographic condition and system applicability test adopts octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, and water solution as mobile phase B, and the detection wavelength is 203nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1. The gradient elution was performed as specified in table 69 below.
TABLE 69 gradient elution Table
Preparation of reference solution A proper amount of ginsenoside Rg1, Rb1 and Re reference is precisely weighed, and methanol is added to obtain 0.2mg mixed solution containing ginsenoside Rg1, Rb1 and Re per 1ml to obtain the final product
Preparing a sample solution, precisely weighing about 1g of the product, dissolving in 12.5ml of water, placing in a separating funnel, extracting with chloroform for 3 times, 15ml each time, discarding a chloroform layer solution, extracting the solution with a water-saturated n-butanol solution for 3 times, 30ml each time, combining n-butanol solutions, washing with an ammonia test solution for 2 times, 30ml each time, discarding the ammonia test solution layer, washing the n-butanol solution with a water-saturated n-butanol solution water layer solution for 20ml, washing for 1 time, discarding a water layer solution, evaporating the n-butanol solution to dryness, dissolving residues with methanol, transferring to a 5ml volumetric flask, adding methanol to fix the volume to the scale, and shaking up to obtain the product.
The determination method comprises precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, determining, and recording chromatogram of 110 min.
The product contains ginsenoside Rg1 (C) per 1g of Ginseng radix42H72O14)、Rb1(C54H92O23)、Re(C48H82O18) The total content of (b) is 0.6 mg/g-2.4 mg/g.
15 batches of substance standard ginsenoside Rg1, Rb1 and Re have the total content of 0.929 mg/g-1.973 mg/g and the average content of 1.480mg/g which are all within +/-30 percent3SD range; referring to + -30% and 3SD floating range, considering various influence factors in actual production process, etc., the product is tentatively calculated according to dry product, and every 1g contains ginsenoside Rg1 (C)42H72O14)、Rb1 (C54H92O23)、Re(C48H82O18) The total content of (b) is 0.6 mg/g-2.4 mg/g.
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests of astragalus; acetonitrile-water (35: 65) is used as a mobile phase; detection by an evaporative light scattering detector. The number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak.
Preparation of control solution A proper amount of astragaloside IV control is precisely weighed, and added with methanol to obtain solution containing 0.5mg per lml.
Preparing a test solution, precisely weighing 1.0g of the product, precisely adding 50ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 45 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, shaking up and extracting with water-saturated n-butanol for 4 times, 25ml each time, combining n-butanol extract, washing with ammonia test solution for 3 times, 30ml each time, discarding ammonia test solution washing liquor, separating n-butanol solution, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product.
The product contains astragaloside IV (C) per 1g of radix astragali41H68O14) The content of the active carbon is 0.10mg/g to 1.40 mg/g.
15 batches of materials have a standard astragaloside IV range of 0.479 mg/g-1.037 mg/g and an average content of 0.772mg/g, all of which are within 3SD, refer to a fluctuation range of +/-30 percent and 3SD, and simultaneously consider various influence factors and the like in the actual production process, the product is tentatively calculated according to dry products, and each 1g of the product contains astragaloside IV (C) for every 1g of astragalus41H68O14) The content of the active carbon is 0.10mg/g to 1.40 mg/g.
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A detection method of a Baoyuan medicinal composition is characterized by detecting a product prepared by the following preparation method, wherein the preparation method comprises the following steps: 74 parts of ginseng, 148 parts of astragalus, 37 parts of liquorice, 15 parts of cinnamon and 20 parts of ginger are placed in an electronic decoction pot, 1920ml of water is added, the decoction is carried out by covering, the strong fire boiling is changed into the slow fire boiling for keeping the micro boiling, the decoction is carried out for 1h, 200-mesh filter cloth is filtered while the decoction is hot, the filtrate is subjected to vacuum concentration at the vacuum degree of-0.08 MPa to-0.06 MPa and the temperature of 55 ℃ to 60 ℃, the concentration is carried out until the ratio of the liquid medicine to the total feeding amount of decoction pieces is 1: 3, and the pre-freezing temperature of the extract is as follows: freeze-drying at-20-45 deg.C, freeze-drying temperature-50-70 deg.C, vacuum degree less than 300Pa, and collecting dry extract powder, or collecting dry extract powder, adding pharmaceutically acceptable adjuvants, and making into pharmaceutically acceptable dosage form;
the specific detection method comprises the following steps:
the characteristics are as follows: the product is brown to tan powder, slightly fragrant, slightly bitter and sweet;
and (3) identification: 1) taking 0.5-1.5 g of the product powder, adding 20-60 ml of chloroform, heating and refluxing for 0.5-2 hours, removing chloroform liquid, volatilizing solvent from decoction dregs, adding 0.5ml of water, stirring and wetting, adding 10ml of saturated n-butyl alcohol, carrying out ultrasonic treatment for 20-40 minutes, absorbing supernate, adding 2-4 times of ammonia test solution, shaking uniformly, standing and layering, taking supernatant liquid, evaporating to dryness, and adding 1ml of methanol to residues for dissolving to obtain a test solution; preparing 1g of Ginseng radix reference material, and making reference material solution by the same method; adding methanol into ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 as reference solutions to obtain mixed solutions each containing 2mg per 1 ml; according to the test of thin-layer chromatography of 0502 of the four ministry of the pharmacopoeia 2015 year edition, the test solution, the reference medicinal material solution and the reference solution are respectively absorbed by 1-2 mu 1 and respectively spotted on the same silica gel G thin-layer plate according to the proportion of 15: 40: 22: 10, taking a lower layer solution of chloroform-ethyl acetate-methanol-water which is placed below 10 ℃ as a developing agent, developing, taking out, airing, spraying a 10% sulfuric acid ethanol solution, heating at 105 ℃ until the color development of spots is clear, respectively placing the spots under sunlight and 365nm ultraviolet lamps for inspection, and developing the same spots in the chromatogram of the test sample at the positions corresponding to the chromatogram of the reference sample;
2) taking 1-2 g of the product, adding 15-35 mL of water, carrying out ultrasonic treatment for 20-40 minutes, filtering, shaking and extracting filtrate with water saturated n-butyl alcohol for 2-4 times, 20-30 mL each time, combining n-butyl alcohol extract, washing with ammonia test solution for 2-4 times, 20-40 mL each time, taking n-butyl alcohol solution to evaporate, and adding methanol lmL into residues to dissolve the residues to obtain a test solution; taking 1-2 g of astragalus mongholicus reference medicinal material, adding 40-60 ml of water, decocting for 20-40 minutes, filtering, and preparing a reference medicinal material solution from the filtrate by the same method; adding methanol into astragaloside IV reference substance to obtain solution containing 2mg per lmL as reference substance solution; performing thin-layer chromatography test according to 0502 of general rules of the four parts of the national pharmacopoeia 2015 edition, sucking 10 μ l of the reference solution, the reference medicinal material solution and the test sample solution respectively, respectively dropping the reference solution, the reference medicinal material solution and the test sample solution on the same silica gel G thin-layer plate, taking out a lower layer solution which is placed at the temperature of below 10 ℃ of trichloromethane-methanol-water according to the ratio of 13:7:2 as a developing agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, and heating at 105 ℃ until spots are clearly developed; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; observing under 365nm ultraviolet lamp to show fluorescent spots of the same color;
3) taking 0.5-1.5 g of the product and a negative sample without liquorice respectively, adding 20-60 ml of water, performing ultrasonic treatment for 20-40 minutes, filtering, performing shake extraction for 2-4 times by using ether, 10-30 ml each time, discarding ether liquid, performing shake extraction for 2-4 times by using water-saturated n-butyl alcohol for 15-25 ml each time, combining n-butyl alcohol liquid, performing shake extraction for 2-4 times by using ammonia test solution, 20-40 ml each time, combining ammonia test solutions, and adjusting the pH value to 3-4 by using hydrochloric acid; extracting with ethyl acetate for 2-4 times by shaking, wherein 20-40 ml of ethyl acetate is extracted each time, combining ethyl acetate solutions, evaporating to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a test sample solution and a liquorice-lacking negative sample solution; taking 0.5-1.5 g of honey-fried licorice root decoction pieces, adding 40-60 ml of water, decocting for 20-40 minutes, filtering, and preparing a control medicinal solution by the same method; taking appropriate amount of liquiritin reference substance, adding 70% ethanol to obtain solution containing 0.5mg per 1mL as reference substance solution; according to the test of thin-layer chromatography of 0502 of the four ministry of the pharmacopoeia 2015 year edition, 5-10 mul of the test solution, the liquorice-lacking negative sample solution, the control medicinal material solution and the control solution are respectively absorbed and respectively spotted on the same silica gel G thin-layer plate according to the proportion of 13:7:2, taking the subnatant of chloroform-methanol-water placed at 5-10 ℃ as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the color of the spots is clear, and observing under sunlight and 365nm ultraviolet lamps, wherein spots or fluorescent spots with the same color appear in the chromatogram of the test solution and at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material;
4) taking 0.5-1.5 g of the product and the cinnamomum japonicum negative freeze-dried powder respectively, adding 5-15 ml of water to dissolve the product, shaking and extracting the product by using ether for 1-3 times, 15-35 ml each time, combining ether solutions, volatilizing the ether solutions, adding 2ml of ether to the residues to dissolve the residues to obtain a test solution and a cinnamomum japonicum negative sample solution; taking 0.5-1.5 g of cinnamon decoction pieces, adding 40-60 ml of water, decocting for 20-40 minutes, filtering, and preparing a control medicinal solution by the same method; taking a cinnamic acid reference substance, and preparing 1ml of 0.2mg solution by using 40-65% methanol to serve as a reference substance solution; according to the test of thin layer chromatography 0502 of the four general rules of the Chinese pharmacopoeia 2015 year edition, 10 mul of the test solution, the negative sample solution of the cinnamomum japonicum, the solution of the control medicinal material and the solution of the control are respectively sucked and spotted on the same silica gel GF254Developing on a thin layer plate with cyclohexane-diethyl ether-glacial acetic acid as developing agent at ratio of 5:5:0.3, taking out, air drying, and inspecting under 254nm ultraviolet lamp to show spots or fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution;
and (4) checking: the water content is measured according to the second method 0832 of the general rule of four parts of the Chinese pharmacopoeia 2015 edition to be not more than 12.0 percent;
extract: according to the alcohol-soluble extract determination method-Chinese pharmacopoeia 2015 edition four parts general rule 2201 item under the hot dipping method determination, the extract is 18% -81.0%;
fingerprint spectrum: measuring by high performance liquid chromatography according to 0512 general rules of four parts of the national pharmacopoeia 2015 edition;
octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; a chromatographic column: InertSustain-AQ-C18, acetonitrile is used as a mobile phase A, a 0.1% phosphoric acid solution is used as a mobile phase B, the flow rate is 1mL/min, the detection wavelength is 200-220 nm, and the specific gradient elution procedure is as follows:
mobile phase gradient elution procedure
Preparation of reference substance solution A proper amount of calycosin glucoside reference substance is precisely weighed, and 40-60% methanol is added to prepare 50.4592 mug/ml solution of calycosin glucoside per 1ml, so as to obtain the reference substance;
preparing a sample solution, taking 1.5-3.5 g of the product, precisely weighing, placing the product in a 50mL triangular conical flask, adding 40-60 mL of 40-60% methanol, carrying out ultrasonic treatment for 30-60min, filtering, recovering the solvent from the filtrate under reduced pressure until the solvent is nearly dry, dissolving the residue in 20mL of water, extracting with a water-saturated n-butyl alcohol solution for 2-3 times, 15-35 mL each time, combining the n-butyl alcohol solution, extracting with 20-40 mL of an ammonia test solution for 1 time, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residue in 2mL of water, carrying out column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating the 95% ethanol eluate, dissolving the residue in 40-60% methanol, transferring to a volumetric flask, fixing the volume to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the product;
the determination method comprises precisely sucking reference solution and sample solution 10 μ L respectively, injecting into liquid chromatograph, determining, and recording chromatogram for 70 min;
corresponding chromatographic peaks with same retention time of reference substance chromatographic peaks are respectively presented in the fingerprint of the test sample; calculating according to the similarity evaluation system of traditional Chinese medicine chromatogram fingerprint, wherein the similarity between the sample fingerprint and the comparison fingerprint is not less than 0.85, and the comparison fingerprint is shown in figure 1;
content determination: measuring Ginseng radix with high performance liquid chromatography according to 0512 in the four-part general rules of the Chinese pharmacopoeia 2015 edition;
in chromatographic conditions and system applicability tests, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, an aqueous solution is used as a mobile phase B, the detection wavelength is 190-210 nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1; performing gradient elution according to a specified gradient elution procedure, wherein the gradient elution procedure comprises the following steps:
mobile phase gradient elution procedure
Preparing reference solution by accurately weighing appropriate amount of ginsenoside Rg1, Rb1 and Re reference, and adding methanol to obtain 0.2 mg/1 ml mixed solution containing ginsenoside Rg1, Rb1 and Re;
preparing a test sample solution, taking 0.5-1.5 g of the product, precisely weighing, adding 12.5ml of water for dissolving, placing the product in a separating funnel, adding chloroform for extracting for 2-4 times, 10-20 ml each time, discarding a chloroform layer solution, extracting the solution for 2-4 times with a water saturated n-butanol solution for 20-40 ml each time, combining n-butanol solutions, washing for 2-4 times with an ammonia test solution for 20-40 ml each time, discarding an ammonia test solution layer, washing n-butanol solution with a water saturated n-butanol solution water layer solution for 10-30 ml, washing for 1 time, discarding a water layer solution, evaporating the n-butanol solution to dryness, adding methanol for dissolving residues, transferring the residues into a 5ml volumetric flask, adding methanol for metering to a scale, and shaking up to obtain the product;
the determination method comprises precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram of 110 min;
the product contains 0.6 mg/g-2.4 mg/g of ginseng in each 1g of dry product, calculated by the total content of ginsenoside Rg1, Rb1 and Re;
measuring content of radix astragali by high performance liquid chromatography according to 0512 of the general rules of four parts in 2015 pharmacopoeia;
chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; in a ratio of 35: 65 taking acetonitrile-water as a mobile phase; detecting by an evaporative light scattering detector; the number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak;
preparing reference solution by accurately weighing appropriate amount of astragaloside IV reference, and adding methanol to obtain solution containing 0.5mg per lml;
preparing a test solution, namely taking 0.5-1.5 g of the product, precisely weighing, precisely adding 40-60 ml of water, weighing, ultrasonically treating for 30-60 minutes, weighing again, complementing the weight loss with water, shaking up, filtering, precisely taking 25ml of subsequent filtrate, shaking and extracting for 2-6 times with water-saturated n-butyl alcohol, each time 20-40 ml, combining n-butyl alcohol extract, washing for 2-4 times with ammonia test solution, each time 20-40 ml, discarding ammonia test solution washing liquid, separately taking n-butyl alcohol solution, evaporating to dryness, dissolving residues with methanol, transferring the residues into a 5ml measuring flask, adding methanol to dilute to a scale, and shaking up to obtain the test solution;
the content of astragalus root in 1g of the product is 0.10 mg/g-1.40 mg/g calculated as astragaloside IV according to the dry product.
2. The assay of claim 1, wherein the pharmaceutically acceptable formulation is a solid formulation or a liquid formulation.
3. The detection method according to claim 2, wherein the solid preparation is a granule, a capsule, a tablet, a pill, a powder, or a lyophilized powder injection.
4. The detection method according to claim 2, wherein the liquid preparation is an injectable preparation or an oral liquid.
5. The detection method according to claim 1, wherein the discrimination method is:
1) taking 1g of the product powder, adding 40ml of trichloromethane, heating and refluxing for 1 hour, removing trichloromethane liquid, volatilizing solvent from medicine residue, adding 0.5ml of water, stirring and moistening, adding 10ml of saturated n-butyl alcohol, performing ultrasonic treatment for 30 minutes, sucking supernatant, adding 3 times of ammonia test solution, shaking uniformly, standing for layering, taking supernatant, evaporating to dryness, and adding 1ml of methanol to dissolve residues to obtain a test solution; preparing 1g of Ginseng radix reference material, and making reference material solution by the same method; adding methanol into ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 as reference solutions to obtain mixed solutions each containing 2mg per 1 ml; according to the thin-layer chromatography test of 0502 of the four ministry of the national pharmacopoeia 2015 year edition, the test solution, the reference medicine solution and the reference solution are respectively sucked to be 1-2 mu 1 and respectively spotted on the same silica gel G thin-layer plate according to the proportion of 15: 40: 22: 10, taking a lower layer solution of chloroform-ethyl acetate-methanol-water which is placed below 10 ℃ as a developing agent, developing, taking out, airing, spraying a 10% sulfuric acid ethanol solution, heating at 105 ℃ until the color development of spots is clear, respectively placing the spots under sunlight and 365nm ultraviolet lamps for inspection, and developing the same spots in the chromatogram of the test sample at the positions corresponding to the chromatogram of the reference sample;
2) collecting 1.5g of the product, adding 25mL of water, performing ultrasonic treatment for 30min, filtering, extracting the filtrate with water saturated n-butanol under shaking for 2 times, each time 25mL, mixing n-butanol extractive solutions, washing with ammonia test solution for 2 times, each time 30mL, collecting n-butanol solution, evaporating to dry, and dissolving the residue with methanol lmL to obtain test solution; adding water 50ml into radix astragali control 1.5g, decocting for 30min, filtering, and making into control solution; adding methanol into astragaloside IV reference substance to obtain solution containing 2mg per lmL as reference substance solution; performing thin-layer chromatography test according to 0502 of general rules of the four parts of the national pharmacopoeia 2015 edition, sucking 10 μ l of each of a reference solution, a reference medicinal material solution and a test sample solution, respectively dropping the reference solution, the reference medicinal material solution and the test sample solution on the same silica gel G thin-layer plate, taking out a lower layer solution which is placed at the temperature of below 10 ℃ of trichloromethane-methanol-water according to the ratio of 13:7:2 as a developing agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, and heating at 105 ℃ until spots are clearly developed; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; observing under 365nm ultraviolet lamp to show fluorescent spots of the same color;
3) taking 1g of each product and a negative sample without liquorice, adding 40ml of water, carrying out ultrasonic treatment for 30 minutes, filtering, shaking and extracting with diethyl ether for 2 times, 20ml each time, discarding the diethyl ether solution, shaking and extracting the water solution with water-saturated n-butyl alcohol for 3 times, 20ml each time, combining the n-butyl alcohol solutions, shaking and extracting with ammonia test solution for 2 times, 30ml each time, combining the ammonia test solutions, and adjusting the pH value to 3-4 with hydrochloric acid; extracting with ethyl acetate for 2 times (30 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1ml methanol to obtain test solution and Glycyrrhrizae radix-deficient negative sample solution; adding water 50ml into radix Glycyrrhizae Preparata decoction pieces 1g, decocting for 30min, filtering, and making into control medicinal solution by the same method; taking appropriate amount of liquiritin reference substance, adding 70% ethanol to obtain solution containing 0.5mg per 1mL as reference substance solution; according to the test of thin-layer chromatography of 0502 of the four ministry of the pharmacopoeia 2015 year edition, 10 mul of each of the test solution, the liquorice-lacking negative sample solution, the reference medicinal material solution and the reference solution are respectively spotted on the same silica gel G thin-layer plate according to the proportion of 13:7:2, taking the subnatant of chloroform-methanol-water placed at 5-10 ℃ as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the color of the spots is clear, and observing under sunlight and 365nm ultraviolet lamps, wherein spots or fluorescent spots with the same color appear in the chromatogram of the test solution and at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material;
4) dissolving the product and the lyophilized powder with water 10ml each 1g, extracting with diethyl ether under shaking for 1 time, 25ml each time, volatilizing the diethyl ether solution, dissolving the residue with diethyl ether 2ml to obtain test solution and pulp-deficient cortex Cinnamomi negative sample solution; decocting 1g of cortex Cinnamomi decoction pieces in 50ml of water for 30min, filtering, and making into control medicinal solution; taking a cinnamic acid reference substance, and preparing 1ml of 0.2mg solution with 50% methanol as a reference substance solution; according to the test of thin layer chromatography 0502 of the four general rules of the Chinese pharmacopoeia 2015 year edition, 10 mul of each of the test solution, the negative sample solution of the cinnamomum zeylanicum, the solution of the control medicinal material and the solution of the control is absorbed and spotted on the same silica gel GF254Developing on the thin layer plate with cyclohexane-diethyl ether-glacial acetic acid as developing agent at ratio of 5:5:0.3, taking out, air drying, and inspecting under 254nm ultraviolet lamp to show spots or fluorescent spots of the same color in the chromatogram of the sample at the positions corresponding to the chromatogram of the control material and the chromatogram of the control material.
6. The detection method according to claim 1, wherein the fingerprint detection method comprises:
fingerprint spectrum: measuring by high performance liquid chromatography according to 0512 general rules of four parts of the national pharmacopoeia 2015 edition;
octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; a chromatographic column: InertSustain-AQ-C18, acetonitrile is used as a mobile phase A, 0.1% phosphoric acid solution is used as a mobile phase B, the flow rate is 1mL/min, the detection wavelength is 210nm, and the specific mobile phase gradient elution program is as follows:
mobile phase gradient elution procedure
Preparing reference solution by precisely weighing appropriate amount of calycosin glucoside reference, and adding 50% methanol to obtain solution containing calycosin glucoside 50.4592 μ g/ml per 1 ml;
preparing a sample solution, precisely weighing 2.5g of the product, placing the product in a 50mL triangular cone bottle, adding 50% methanol into 50mL of the triangular cone bottle, performing ultrasonic treatment for 45min, filtering, recovering a solvent from a filtrate under reduced pressure till the solvent is nearly dry, dissolving residues in 20mL of water, extracting for 2 times with water-saturated n-butyl alcohol solution, 25mL each time, combining the n-butyl alcohol solution, extracting for 1 time with 30mL of ammonia test solution, removing the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residues in 2mL of water, performing column chromatography with 10mL of D101 macroporous adsorption resin, eluting with 50mL of water, then eluting with 100mL of 95% ethanol, evaporating the 95% ethanol eluate to dryness, dissolving the residues in 50% methanol, transferring to a 10mL volumetric flask, fixing the volume to the scale, shaking up, filtering, and taking a continuous filtrate to obtain the product;
the determination method comprises precisely sucking reference solution and sample solution 10 μ L respectively, injecting into liquid chromatograph, determining, and recording chromatogram for 70 min;
corresponding chromatographic peaks with same retention time of reference substance chromatographic peaks are respectively presented in the fingerprint of the test sample; calculated according to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprint, the similarity of the sample fingerprint and the comparison fingerprint is not less than 0.85, and the comparison fingerprint is shown in figure 1.
7. The detection method according to claim 1, wherein the method for measuring the content of ginseng comprises:
measuring Ginseng radix with high performance liquid chromatography according to 0512 in the four-part general rules of the Chinese pharmacopoeia 2015 edition;
in chromatographic conditions and system applicability tests, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, an aqueous solution is used as a mobile phase B, the detection wavelength is 203nm, and the number of theoretical plates is not less than 5000 according to the total peak of ginsenoside Rg 1; performing gradient elution according to a specified gradient elution procedure, wherein the mobile phase gradient elution procedure comprises the following steps:
mobile phase gradient elution procedure
Preparing reference solution by accurately weighing appropriate amount of ginsenoside Rg1, Rb1 and Re reference, and adding methanol to obtain 0.2 mg/1 ml mixed solution containing ginsenoside Rg1, Rb1 and Re;
preparing a sample solution, precisely weighing 1g of the product, dissolving the product in 12.5ml of water, placing the solution in a separating funnel, extracting the solution for 3 times by adding chloroform, extracting 15ml of the solution each time, removing a chloroform layer solution, extracting the solution for 3 times by using a water-saturated n-butanol solution, extracting 30ml of the solution each time, combining n-butanol solutions, washing the solution for 2 times by using an ammonia test solution, removing the ammonia test solution layer each time by 30ml, removing the ammonia test solution layer by using a water-saturated n-butanol solution water layer solution 20ml of the n-butanol solution, washing for 1 time, removing the water layer solution, evaporating the n-butanol solution to dryness, dissolving residues by adding methanol, transferring the residues into a 5ml volumetric flask, adding methanol to fix the volume to the scale, and shaking up to obtain the product;
the determination method comprises precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram of 110 min;
the content of the product is 0.6 mg/g-2.4 mg/g calculated by the total content of ginsenoside Rg1, Rb1 and Re in every 1g of ginseng.
8. The detection method according to claim 1, wherein the method for determining the content of astragalus comprises the following steps:
measuring content of radix astragali by high performance liquid chromatography according to 0512 of the general rules of four parts in 2015 pharmacopoeia;
chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; in a ratio of 35: 65 taking acetonitrile-water as a mobile phase; detecting by an evaporative light scattering detector; the number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak;
preparing reference solution by accurately weighing appropriate amount of astragaloside IV reference, and adding methanol to obtain solution containing 0.5mg per lml;
preparing a test solution, namely taking 1.0g of the product, precisely weighing, precisely adding 50ml of water, weighing, ultrasonically treating for 45 minutes, weighing again, supplementing the lost weight with water, shaking up, filtering, precisely weighing 25ml of a subsequent filtrate, shaking and extracting for 4 times with water-saturated n-butyl alcohol, 25ml each time, combining n-butyl alcohol extract, washing for 3 times with ammonia test solution, 30ml each time, discarding ammonia test solution washing liquor, separating n-butyl alcohol solution, evaporating to dryness, dissolving residues with methanol, transferring the residues into a 5ml measuring flask, adding methanol to dilute to a scale, and shaking up to obtain the product;
the content of astragalus root in 1g of the product is 0.10 mg/g-1.40 mg/g calculated as astragaloside IV according to the dry product.
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