Mass balancing method for CDD-2101 intermediate

Abstract

The present invention provides a mass balancing method for a CDD-2101 intermediate. The mass balancing method comprises: measuring the content of one or more of the following components of a CDD-2101 intermediate: total flavone, total phenols, total anthraquinone, total triterpenes, total alkaloids, total sugar, total protein, lignan and total fat. The method of the present invention provides a systematic measurement means for various main components in the CDD-2101 intermediate, measures the content of nine components therein, exhibits high sensitivity and high specificity, and can efficiently, rapidly, comprehensively and accurately control the mass of the traditional Chinese medicine composition.

Description

A quality balance method of CDD-2101 intermediate TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine component analysis, and particularly relates to a quality balance method of CDD-2101 intermediate. BACKGROUND

[0002] In the preparation and research process of drugs, the research and quality control of intermediates are indispensable parts. Intermediate testing is a key quality control procedure before drugs enter the market, which is related to the safety of consumers. Flavonoids, phenolic compounds, anthraquinones, lignans, terpenoids and alkaloids are widely present in traditional Chinese medicines and are effective components of many traditional Chinese medicine compounds. At the same time, a large number of pharmacological studies have confirmed that macromolecular components such as sugars, proteins and fats in traditional Chinese medicines all exhibit different degrees of pharmacological activity. Therefore, the determination of the contents of main components in intermediates is an important content in the quality evaluation, quality control, extraction process purification, pharmacokinetics and metabolomics of traditional Chinese medicines.

[0003] CDD-2101 granules are derived from the ancient prescription of Mazi Ren Pills in Treatise on Febrile Diseases and Miscellaneous Diseases, and are composed of hemp seeds, rhubarb, bitter almonds, magnolia bark, white peony root and fried bitter orange fruit. It has been reported that thin layer chromatography and high performance liquid chromatography can be used to identify and determine one or more active components in hemp seeds, rhubarb, bitter almonds, magnolia bark, white peony root and fried bitter orange fruit, but there is a lack of analysis method for the determination of the contents of multiple main components contained in the traditional Chinese medicine compound.

[0004] SUMMARY

[0005] To solve the above technical problems, the purpose of the present application is to provide a quality balance method of CDD-2101 intermediate, which can systematically detect multiple main components in CDD-2101 intermediate.

[0006] To achieve the above purpose, the present application provides a quality balance method of CDD-2101 intermediate, the raw materials of the CDD-2101 intermediate include the following medicinal materials: hemp seeds, rhubarb, magnolia bark, white peony root, and fried bitter orange fruit; the quality balance method includes determining the contents of one or more of the following components in the CDD-2101 intermediate: total flavonoids, total phenols, total anthraquinones, total triterpenes, total alkaloids, total sugars, total proteins, lignans and total fats.

[0007] According to the specific embodiment of the present application, preferably, the quality balance method includes one or more of the following methods:

[0008] (1) The contents of total flavonoids in the CDD-2101 intermediate are determined by sodium nitrite colorimetry;

[0009] (2) Total phenol in CDD-2101 intermediate is determined by ferric chloride-potassium ferricyanide colorimetry;

[0010] (3) Total anthraquinone in CDD-2101 intermediate is determined by magnesium acetate ethanol colorimetry;

[0011] (4) Total triterpenoids in CDD-2101 intermediate is determined by vanillin glacial acetic acid colorimetry after purification by macroporous resin;

[0012] (5) Total alkaloids in CDD-2101 intermediate is determined by Reinecke ammonium salt colorimetry;

[0013] (6) Total sugar in CDD-2101 intermediate is determined by 3,5-dinitrosalicylic acid colorimetry;

[0014] (7) Total protein in CDD-2101 intermediate is determined by Coomassie brilliant blue method;

[0015] (8) Lignans in CDD-2101 intermediate is determined by high performance liquid chromatography;

[0016] (9) Total fat in CDD-2101 intermediate is determined by Soxhlet extraction with diethyl ether and evaporation.

[0017] According to the specific embodiment of the present application, preferably, the content determination method of total flavonoids comprises the following steps:

[0018] The extraction solvent is mixed with the CDD-2101 intermediate, ultrasonic treatment and / or heating reflux are performed, and the extraction solvent is used to make up the lost weight; centrifugation is performed, the supernatant is taken, 5wt% (mass fraction) sodium nitrite solution and 4wt% sodium hydroxide solution are sequentially added, a blank control reagent is set, and the absorbance is determined at 250-700nm wavelength according to the ultraviolet-visible spectrophotometry;

[0019] The extraction solvent is selected from methanol, 70vol% (volume fraction) methanol aqueous solution, 80vol% ethanol aqueous solution or 60vol% ethanol aqueous solution.

[0020] According to the specific embodiment of the present application, more preferably, the content determination method of total flavonoids comprises the following steps:

[0021] (1) Preparation of reference solution:

[0022] An appropriate amount of rutin reference substance is accurately weighed, 60vol% ethanol aqueous solution (denoted as 60% ethanol, and the other reagents are the same) is added to prepare a solution containing 0.2mg per 1ml, and the solution is obtained.

[0023] (2) Preparation of standard curve:

[0024] Precisely pipette 1 ml, 2 ml, 3 ml, 4 ml, 5 ml and 6 ml of the control solution into 25 ml volumetric flasks, respectively, and add water to 6.0 ml, add 1 ml of 5% sodium nitrite solution, mix well, stand for 6 minutes, add 1 ml of 10% aluminum nitrate solution, mix well, stand for 6 minutes, add 10 ml of sodium hydroxide test solution, then add water to the calibration mark, shake well, stand for 15 minutes, set up a blank control reagent, and immediately determine the absorbance at a wavelength of 250-700 nm by ultraviolet-visible spectrophotometry; plot a standard curve with the absorbance as the vertical coordinate and the concentration as the horizontal coordinate.

[0025] (3) Determination method:

[0026] Take 0.2 g of CDD-2101 intermediate powder, accurately weigh, and place it in a conical flask with a plug. Precisely add 25 ml of methanol, 70% methanol, 80% ethanol or 60% ethanol, tightly plug, weigh, ultrasonic treatment or heating reflux for 30 minutes, take out, cool, re-weigh, make up the weight loss with 60% ethanol, shake well, centrifuge (4000 r / min) for 10 minutes, and take the supernatant as the test sample solution.

[0027] Precisely pipette 1 ml of the test sample solution into a 25 ml volumetric flask, and determine the absorbance according to the method under item "(2) Preparation of standard curve" from "add water to 6.0 ml"; read the weight of rutin (mg) in the test sample solution from the standard curve, and calculate the percentage content of total flavonoids in the test sample as dry product.

[0028] According to the specific embodiment of the present application, preferably, the total phenol content determination method comprises the following steps:

[0029] Mix the extraction solvent with the CDD-2101 intermediate, ultrasonic treatment, and make up the weight loss with the extraction solvent; centrifuge the solution, take the supernatant, centrifuge, dilute with the extraction solvent, and then sequentially add 0.3 wt% sodium dodecyl sulfate solution, a solution mixed from 0.6 wt% ferric chloride solution and 0.9 wt% potassium ferricyanide solution at a volume ratio of 1:0.9, and 0.1 mol / L hydrochloric acid solution; set up a blank control reagent, and determine the absorbance at a wavelength of 500-850 nm by ultraviolet-visible spectrophotometry;

[0030] The extraction solvent is selected from methanol, ethanol, 70 vol% ethanol aqueous solution (denoted as 70% ethanol, and the same applies to other reagents) or 50 vol% ethanol aqueous solution.

[0031] According to the specific embodiment of the present application, more preferably, the total phenol content determination method comprises the following steps:

[0032] (1) Preparation of the control solution:

[0033] An appropriate amount of gallic acid control sample was precisely weighed and dissolved in 70% ethanol to obtain a solution containing 25 μg per 1 ml.

[0034] (2) Preparation of the standard curve:

[0035] 0.2 ml, 0.5 ml, 0.8 ml, 1.0 ml, 1.2 ml, and 1.4 ml of the control sample solution were precisely measured and placed in 25 ml volumetric flasks, 2.0 ml of a 0.3 wt% sodium dodecyl sulfate solution and 1.0 ml of a 0.6 wt% ferric chloride-0.9 wt% potassium ferricyanide (volume ratio 1:0.9) mixed solution were added, and the mixture was uniformly mixed and placed in the dark for 5 minutes, 0.1 mol / L hydrochloric acid solution was added to the calibration mark, and the mixture was uniformly shaken and placed in the dark for 20 minutes; a blank control reagent was set, and the absorbance was measured at a wavelength of 500-850 nm by ultraviolet-visible spectrophotometry, the absorbance was taken as the vertical coordinate, and the concentration was taken as the horizontal coordinate to draw a standard curve.

[0036] (3) Determination method:

[0037] 0.1 g of CDD-2101 intermediate powder was precisely weighed, 25 ml of methanol, ethanol, 70% ethanol, or 50% ethanol was precisely added, weighed, ultrasonically treated for 30 minutes, removed, cooled, re-weighed, the weight loss was made up with 70% ethanol, uniformly shaken, centrifuged (4000 r / min) for 10 minutes, 1.0 ml of the supernatant was precisely measured and placed in a 10 ml volumetric flask, diluted to the calibration mark with 70% ethanol, and uniformly shaken to obtain a test sample solution.

[0038] 1.0 ml of the test sample solution was precisely measured and placed in a 25 ml volumetric flask, and the absorbance was determined according to the method in the preparation of the standard curve from “add anhydrous ethanol to 5.0 ml” to read the weight of gallic acid in the test sample solution (mg) from the standard curve, and the percentage content of total phenol in the test sample was calculated as dry product.

[0039] According to the specific embodiment of the present application, preferably, the total anthraquinone content determination method comprises the following steps:

[0040] The extraction solvent is mixed with the CDD-2101 intermediate, heated to reflux, and the lost weight is made up with the extraction solvent; after filtration, the filtrate is taken and the solvent is evaporated, the residue is first treated with 8wt% hydrochloric acid solution under ultrasonic, then heated to reflux in a water bath with chloroform, and separated into chloroform layer and acid layer by standing; the chloroform layer is taken, the acid layer is extracted with chloroform again, and the chloroform liquids are combined and evaporated; the residue is dissolved in methanol, colored with 1wt% magnesium acetate ethanol solution, and set a blank control reagent; the absorbance is measured at 300-700nm by ultraviolet-visible spectrophotometry;

[0041] The extraction solvent is selected from methanol, 80vol% ethanol aqueous solution or ethanol.

[0042] According to the specific embodiment of the present application, more preferably, the total anthraquinone content determination method comprises the following steps:

[0043] (1) Preparation of reference solution:

[0044] Take emodin reference substance, accurately weigh, add methanol to prepare a solution containing 0.2mg per 1ml, and obtain it.

[0045] (2) Preparation of standard curve:

[0046] Accurately take 0.2ml, 0.5ml, 1.0ml, 1.4ml, 1.8ml and 2.0ml of the reference solution, respectively, and place them in 10ml volumetric flasks, dilute to the mark with 1% magnesium acetate ethanol solution, shake well, stand for color development for 30 minutes, set a blank control reagent, and measure the absorbance at 300-700nm by ultraviolet-visible spectrophotometry; take the absorbance as the vertical coordinate and the concentration as the horizontal coordinate to draw the standard curve.

[0047] (3) Determination method:

[0048] Take 0.4g of CDD-2101 intermediate powder, accurately weigh, accurately add 25-50ml of methanol, 80% ethanol or ethanol, weigh, heat to reflux for 1 hour, take out, cool, make up the lost weight with methanol, shake well, filter, accurately take 10ml of the filtrate, place it in a flask, evaporate the solvent, add 15ml of 8% hydrochloric acid solution to the residue, ultrasonic treat for 2 minutes, then add 15ml of chloroform, heat to reflux in a water bath for 1 hour, take out, cool, place in a separatory funnel, wash the container with a small amount of chloroform and add it to the separatory funnel, separate the chloroform layer, shake the acid liquid with chloroform for 2-4 times, each time 20ml, combine the chloroform liquids, evaporate, add methanol to the residue to dissolve and transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, and use it as the test solution;

[0049] Accurately take 1 ml of the test solution, and determine the absorbance from the beginning of "putting into a 10 ml flask" according to the method under the preparation of the standard curve, read the weight (mg) of emodin in the test solution from the standard curve, and calculate the percentage content of total anthraquinones in the test solution as the dried product, to obtain the total anthraquinones.

[0050] According to the specific embodiment of the present application, preferably, the content determination method of total triterpenes comprises the following steps:

[0051] The extraction solvent is mixed with the CDD-2101 intermediate, heated to reflux and / or ultrasonic treatment, and the lost weight is supplemented with the extraction solvent; centrifugation is performed, the supernatant is evaporated to remove the solvent, and the residue is dissolved with water and / or ethanol and then added to an AB-8 macroporous resin column, eluted with water and 50-80 vol% ethanol, respectively, the eluate is discarded, and then eluted with ethanol, the eluate is collected, evaporated to dryness, and the residue is dissolved with ethanol to obtain the test solution;

[0052] Vanillin glacial acetic acid solution with a newly prepared concentration of 0.05 g / ml is sequentially added to the test solution, mixed with perchloric acid, heated in a 70℃ water bath, then placed in an ice bath for cooling, taken out, and then glacial acetic acid is added and shaken to mix; the corresponding reagent is used as a blank, the absorbance is determined at a wavelength of 400-700 nm according to the ultraviolet-visible spectrophotometry method;

[0053] The extraction solvent is selected from 80 vol% ethanol aqueous solution, ethanol or methanol.

[0054] According to the specific embodiment of the present application, more preferably, the content determination method of total triterpenes comprises the following steps:

[0055] (1) Preparation of the reference solution:

[0056] An appropriate amount of oleanolic acid reference substance is accurately weighed and dissolved in ethanol to prepare a solution containing 0.2 mg per 1 ml, to obtain the reference solution.

[0057] (2) Preparation of the standard curve:

[0058] Accurately take 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml and 0.5 ml of the reference solution into 15 ml stoppered test tubes, evaporate to dryness in a water bath, and cool, accurately add 0.2 ml of newly prepared vanillin glacial acetic acid solution (accurately weigh 0.5 g of vanillin, add glacial acetic acid to dissolve to 10 ml, to obtain the solution) and 0.8 ml of perchloric acid, mix, heat in a 70℃ water bath for 15 minutes, immediately place in an ice bath for cooling for 5 minutes, take out, accurately add 5 ml of glacial acetic acid, shake to mix, use the corresponding reagent as a blank, immediately determine the absorbance at a wavelength of 400-700 nm according to the ultraviolet-visible spectrophotometry method, take the absorbance as the vertical coordinate, and the concentration as the horizontal coordinate, to draw the standard curve;

[0059] (3) Assay:

[0060] Take 0.2 g of CDD-2101 intermediate powder, accurately weigh, accurately add 80% ethanol, ethanol or methanol 25 ml, weigh, heat reflux or ultrasonic treatment for 30 minutes, take out, cool, make up the weight loss with 80% ethanol, shake well, centrifuge (4000 r / min) for 10 minutes, accurately suck 15 ml of supernatant, place in an evaporating dish, evaporate ethanol in a water bath, dissolve the residue with 5 ml of water in portions, add to the AB-8 macroporous resin column (column height 12 cm, inner diameter 1.5 cm), add 30% ethanol 5 ml to the residue in the evaporating dish in portions to dissolve, add the solution to the same macroporous resin column, add water 30 ml, 50-80% ethanol 80 ml, elute, discard the eluent, and then elute with ethanol 150 ml, collect the eluent, evaporate to dryness, dissolve the residue in ethanol and dilute to 5 ml in a volumetric flask, shake well, and obtain;

[0061] Accurately take 1.0 ml, place it in a 15 ml stoppered test tube, and according to the method under the preparation of the standard curve, from "water bath evaporation", measure the absorbance according to the method, read the weight of oleanolic acid in the test solution (mg) from the standard curve, and calculate the percentage content of total triterpenes in the dry product, and obtain.

[0062] According to the specific embodiment of the present application, preferably, the total alkaloid content determination method comprises the following steps:

[0063] Mix the extraction solvent with the CDD-2101 intermediate, heat reflux, make up the weight loss with the extraction solvent, evaporate the filtrate to dryness, dissolve the residue in 0.1 mol / L hydrochloric acid solution, centrifuge to obtain the supernatant, add activated carbon to a boiling water bath to decolorize, filter, wash the residue with 0.1 mol / L hydrochloric acid, filter together, add 2 wt% Reichardt ammonium salt solution, dilute with 0.1 mol / L hydrochloric acid solution, and place it in 2°C for 60-120 minutes, take the filtrate as the test solution; use 0.1 mol / L hydrochloric acid solution as the blank reagent, and measure the absorbance at 400-700 nm according to the ultraviolet-visible spectrophotometric method;

[0064] Among them, the extraction solvent is selected from 1 vol% methanol hydrochloride solution, 1 vol% hydrochloric acid 70 vol% methanol aqueous solution, 1 vol% ethanol hydrochloride solution or 1 vol% hydrochloric acid 70 vol% ethanol aqueous solution.

[0065] According to the specific embodiment of the present application, preferably, the total alkaloid content determination method comprises the following steps:

[0066] (1) Preparation of the reference solution:

[0067] Take the betaine control sample, accurately weigh, add 0.1 mol / L hydrochloric acid to prepare a solution containing 1.5 mg per 1 ml, and you get it;

[0068] (2) Preparation of standard curve:

[0069] Accurately take 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml of the control solution, respectively, into 10 ml volumetric flasks, accurately add 2 wt% of the Riesammonium salt solution 2 ml (newly prepared before use, filtered before use), and then add 0.1 mol / L hydrochloric acid solution to dilute to the mark, shake well, and place in a 2°C refrigerator for 2 hours. Take it out and filter it with a 0.22 μm microporous filter. Take the filtrate as the control sample solution;

[0070] Accurately take 4 ml of the newly prepared 2 wt% Riesammonium salt solution into a 20 ml volumetric flask, dilute to the mark with 0.1 mol / L hydrochloric acid solution, shake well, and place in a 2°C refrigerator for 2 hours. Take it out and filter it with a 0.22 μm microporous filter. Take the filtrate as the blank control solution;

[0071] With 0.1 mol / L hydrochloric acid solution as the reagent blank, the absorbance is determined at 400-700 nm wavelength by ultraviolet-visible spectrophotometry (Chinese Pharmacopoeia 2020 edition Part 0401). The absorbance difference (the difference between the absorbance of the blank control and the control sample) is used as the vertical coordinate, and the concentration is used as the horizontal coordinate. Draw the standard curve;

[0072] (3) Determination method:

[0073] Take about 5 g of CDD-2101 intermediate powder, accurately weigh, accurately add 1% hydrochloric acid methanol, 1% hydrochloric acid 70% methanol, 1% hydrochloric acid ethanol or 1% hydrochloric acid 70% ethanol solution 100 ml, weigh, heat reflux for 1 hour, cool, weigh again, make up the weight loss with methanol, shake well, filter, accurately take 50 ml of the filtrate, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution and dilute to 20 ml in a volumetric flask, shake well, centrifuge (4000 r / min) for 10 minutes, accurately take 10 ml of the supernatant, add 0.8-1.2 g of activated carbon, heat in a boiling water bath for 2 minutes, take it out, filter into a 20 ml volumetric flask, add 0.1 mol / L hydrochloric acid 6 ml for washing, filter into the volumetric flask together, accurately add 2 wt% of the Riesammonium salt solution 4 ml (newly prepared before use, filtered before use), then add 0.1 mol / L hydrochloric acid solution to dilute to the mark, shake well, place in a 2°C refrigerator for 60-120 minutes, take it out, filter with a 0.22 μm microporous filter, take the filtrate as the test sample solution;

[0074] With 0.1 mol / L hydrochloric acid solution as reagent blank, the absorbance was determined at 522 nm wavelength according to the ultraviolet-visible spectrophotometry (General Rule 0401 in Chinese Pharmacopoeia 2020 Edition), the absorbance difference (the absorbance difference between the blank control and the test sample) was calculated, the weight (mg) of betaine in the test sample solution was read from the standard curve, the percentage content of total alkaloids in the test sample was calculated based on the dried product, and the percentage content of total alkaloids in the test sample was obtained.

[0075] According to the specific embodiment of the present application, preferably, the total sugar content determination method comprises the following steps:

[0076] Water is used as the extraction solvent to mix with the CDD-2101 intermediate, ultrasonic treatment and / or heating reflux are performed, the supernatant is taken after centrifugation and 6 mol / L hydrochloric acid solution is added, after being heated in a boiling water bath for 30 minutes, 1 drop of phenolphthalein indicator solution is added after cooling, neutralized to light red with 5 mol / L sodium hydroxide solution, and then diluted with water and shaken well; the diluted solution is taken and added to 3,5-dinitrosalicylic acid solution in a boiling water bath at 100°C for 5 minutes, then quickly cooled with cold water, quantitatively added with water, and a test sample solution is obtained; a blank solution is prepared in the same way with water; and the absorbance is determined at 470-700 nm wavelength according to the ultraviolet-visible spectrophotometry.

[0077] According to the specific embodiment of the present application, more preferably, the total sugar content determination method comprises the following steps:

[0078] (1) Preparation of the reference solution:

[0079] An appropriate amount of D-anhydrous glucose reference substance is accurately weighed and added to water to prepare a solution containing 1 mg per 1 ml, and the reference solution is obtained;

[0080] (2) Preparation of the standard curve:

[0081] 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml and 0.7 ml of the reference solution are accurately taken and placed in 25 ml volumetric flasks, respectively, 1.5 ml of 3,5-dinitrosalicylic acid solution is added to each, shaken well, heated in a boiling water bath for 5 minutes, quickly cooled with cold water, added with water to the calibration mark, shaken well, and a blank solution is prepared in the same way with 1.0 ml of water; the absorbance is determined at 470-700 nm wavelength according to the ultraviolet-visible spectrophotometry (General Rule 0401), and the standard curve is drawn with the absorbance as the vertical coordinate and the concentration as the horizontal coordinate;

[0082] (3) Determination method:

[0083] Take CDD-2101 intermediate powder 0.1 g, accurately weigh, accurately add water 10-50 ml, ultrasonic treatment or heating reflux 30 minutes, cool, centrifugal (4000 r / min) 10 minutes, accurately take supernatant 5.0 ml, add 6 mol / L hydrochloric acid solution 5 ml, place in boiling water bath and heat for 30 minutes, then take out, cool, add phenolphthalein indicator 1 drop, neutralize to light red with 5 mol / L sodium hydroxide solution, dilute to 25 ml with water, shake well, accurately take 1.0 ml, place in 25 ml volumetric flask, according to the method under the preparation of standard curve, from "add 3,5-dinitrosalicylic acid 1.5 ml", measure the absorbance according to the method, measure the absorbance at 470-700 nm wavelength), read the weight of D-anhydrous glucose in the test solution (mg) from the standard curve, calculate the percentage of total sugar in the test sample as dry product, and the percentage of total sugar in the test sample is obtained.

[0084] According to the specific embodiment of the present application, preferably, the total protein content determination method comprises the following steps:

[0085] Water is used as the extraction solvent to mix with CDD-2101 intermediate, ultrasonic treatment is used to dissolve the intermediate, water is added to make up the lost weight after cooling; centrifugal is used to take the supernatant, and coomassie brilliant blue solution is added to mix and react; a blank control reagent is set, and the absorbance is measured at 250-700 nm wavelength according to the ultraviolet-visible spectrophotometry.

[0086] According to the specific embodiment of the present application, more preferably, the total protein content determination method comprises the following steps:

[0087] (1) Preparation of reference solution:

[0088] Take serum albumin (bovine) reference substance in an appropriate amount, accurately weigh, and add water to prepare a solution containing 0.1 mg per 1 ml, and the reference solution is obtained;

[0089] (2) Preparation of standard curve:

[0090] Accurately take 0.1 ml, 0.25 ml, 0.5 ml, 0.8 ml, 1.0 ml, and 1.2 ml of the reference solution, and accurately add 5.0 ml of coomassie brilliant blue solution to each, mix immediately, stand for 2 minutes, set a blank control reagent, and measure according to the ultraviolet-visible spectrophotometry (general rule 0401); measure the absorbance at 250-700 nm wavelength, take the absorbance as the vertical coordinate, and take the concentration as the horizontal coordinate, and draw a standard curve;

[0091] (3) Determination method:

[0092] Take 0.2 g of CDD-2101 intermediate powder, accurately weigh, accurately add 50 ml of water, weigh, ultrasonic treatment for 30 minutes to dissolve, take out, cool, add water to make up for the loss of weight, shake well, centrifuge (4000 r / min) for 10 minutes, accurately take 1.0 ml of supernatant, according to the method under the preparation of the standard curve, from "accurately add 5.0 ml of coomassie brilliant blue solution", the absorbance is determined according to the method, and the weight (mg) of serum albumin (bovine) in the test solution is read from the standard curve. The percentage of total protein in the test sample is calculated as dry product, and the percentage of total protein in the test sample is obtained.

[0093] According to the specific embodiment of the present application, preferably, the content determination method of lignans comprises the following steps:

[0094] The extraction solvent is mixed with the CDD-2101 intermediate, ultrasonic treatment is performed, and after cooling, methanol is added to make up for the loss of weight; the filtrate is taken as the test solution, injected into the liquid chromatograph to determine the total amount of magnolol and honokiol as the content of lignans;

[0095] The extraction solvent is selected from methanol, 70 vol% methanol aqueous solution, ethanol or 70 vol% ethanol aqueous solution;

[0096] The chromatographic conditions include:

[0097] The chromatographic column is a Waters XBridge C18 chromatographic column with a specification of 4.6 mm x 250 mm x 5 μm (4.6 mm x 250 mm, 5 μm);

[0098] The column temperature is 25℃;

[0099] The mobile phase is a mixed solution of methanol-acetonitrile-water with a volume ratio of 50:20:30;

[0100] The detection wavelength is 294 nm.

[0101] According to the specific embodiment of the present application, preferably, in the content determination of lignans, the chromatographic column uses octadecylsilane bonded silica gel as the filler; and the theoretical plate number is not less than 3800 calculated according to the magnolol peak.

[0102] According to the specific embodiment of the present application, more preferably, the content determination method of lignans comprises the following steps:

[0103] (1) Chromatographic conditions and system suitability test:

[0104] The octadecylsilane bonded silica gel is used as the filler; the methanol-acetonitrile-water (50:20:30) is used as the mobile phase; the detection wavelength is 294 nm; the column temperature is 25℃; and the theoretical plate number is not less than 3800 calculated according to the magnolol peak.

[0105] (2) Preparation of the control solution:

[0106] Take the appropriate amount of magnolol control and honokiol control, and accurately weigh, add methanol to prepare a solution containing 0.3 mg per 1 ml, respectively, namely;

[0107] (3) Preparation of the test solution:

[0108] Take about 1.5-2.0 g of CDD-2101 intermediate powder, accurately weigh, put into a conical flask with a plug, accurately add 25-50 ml of methanol, 70% methanol, ethanol or 70% ethanol, weigh, ultrasonic treatment (power 250 w, frequency 40 KHz) for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, namely;

[0109] (4) Determination method:

[0110] Accurately take 10 μl of the control solution and the test solution, respectively, inject into the liquid chromatograph, determine, take the peak area of magnolol and honokiol measured by the standard curve solution as the vertical coordinate, and the corresponding concentration as the horizontal coordinate, draw the standard curve, calculate the total amount of magnolol and honokiol in the test sample as the percentage content of lignans (calculated as dry product), namely.

[0111] According to the specific embodiment of the present application, preferably, the total fat content determination method comprises the following steps:

[0112] Hydrolyze with 1 mol / L hydrochloric acid as the extraction solvent and CDD-2101 intermediate, add ether or petroleum ether after hydrolysis and heat on the water bath to reflux; evaporate the obtained extract to dryness at 60-100 ℃, weigh the residue after cooling, and calculate the total fat content in the CDD-2101 intermediate.

[0113] According to the specific embodiment of the present application, more preferably, the total fat content determination method comprises the following steps:

[0114] (1) Preparation of the test solution:

[0115] Take 5 g of CDD-2101 intermediate powder, accurately weigh, hydrolyze with 1 mol / L hydrochloric acid, and then put into a Soxhlet extractor, add 150 ml of ether or petroleum ether, heat to reflux extraction on the water bath for 6 hours, and collect the extract;

[0116] (2) Determination method:

[0117] Collect the extract into an evaporating dish that has been dried to a constant weight, evaporate to dryness on the water bath at low temperature, dry at 100 ℃ for 1 hour, move to a desiccator, cool for 30 minutes, accurately weigh, and calculate the percentage content of total fat in the test sample as dry product, namely.

[0118] The technical scheme provided by the present application has the following beneficial effects:

[0119] The method of the present application provides a systematic detection method for various main components in the CDD-2101 intermediate, and determines the contents of nine types of components, which has high sensitivity, strong specificity, good reproducibility, safe and feasible operation, and can efficiently, quickly, comprehensively and accurately control the quality of the traditional Chinese medicine composition, and ensures the safety and effectiveness of the medicine. BRIEF DESCRIPTION OF DRAWINGS

[0120] Fig. 1 is a rutin standard curve in Example 1;

[0121] Fig. 2 is a gallic acid standard curve in Example 2;

[0122] Fig. 3 is a emodin standard curve in Example 3;

[0123] Fig. 4 is an oleanolic acid standard curve in Example 4;

[0124] Fig. 5 is a betaine standard curve in Example 5;

[0125] Fig. 6 is a D-anhydrous glucose standard curve in Example 6;

[0126] Fig. 7 is a serum albumin (bovine) standard curve in Example 7;

[0127] Fig. 8 is a chromatogram of magnolol and honokiol in Example 8 (chromatographic condition five of Comparative Example 4);

[0128] Fig. 9 is a honokiol standard curve in Example 8;

[0129] Fig. 10 is a magnolol standard curve in Example 8;

[0130] Fig. 11 is a total fat standard curve in Example 9;

[0131] Fig. 12 is a total flavonoid determination spectrum in Example 10;

[0132] Fig. 13 is a total flavonoid determination spectrum in Comparative Example 1;

[0133] Fig. 14 is a gallic acid reference substance spectrum in Comparative Example 2;

[0134] Fig. 15 is a total phenol determination spectrum in Comparative Example 2;

[0135] Fig. 16 is a total sugar determination spectrum in Comparative Example 3;

[0136] Fig. 17 is a chromatogram of magnolol and honokiol in Comparative Example 4 (chromatographic condition one);

[0137] Figure 18 is a chromatogram of magnolol and honokiol in Comparative Example 4 (chromatographic condition two);

[0138] Figure 19 is a chromatogram of magnolol and honokiol in Comparative Example 4 (chromatographic condition three);

[0139] Figure 20 is a chromatogram of magnolol and honokiol in Comparative Example 4 (chromatographic condition four). DETAILED DESCRIPTION

[0140] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it should not be understood as limiting the scope of the present application.

[0141] Instruments and reagents

[0142] 1. Instruments

[0143] UV-visible spectrophotometer (model: UV-2700i, Shimadzu, Japan); electronic analytical balance (model: ML204 / 02, Mettler-Toledo Instruments Co., Ltd.); electronic analytical balance (model: XPR205, Mettler-Toledo Instruments Co., Ltd.); ultrasonic processor (model: KQ-500DE, Kunshan Ultrasonic Instruments Co., Ltd.); digital constant temperature water bath (model: HJ-A4, Jiangsu Jin Yi Instruments Technology Co., Ltd.); electric heating constant temperature drying oven (model: HGZF-II-101-2, Shanghai Yuejin Medical Instrument Co., Ltd.); medical centrifuge (model: TDZ5-WS, Xiangyi Centrifuge Instrument Co., Ltd.); electronic universal furnace (Beijing Yongguangming Medical Instrument Co., Ltd.); high performance liquid chromatograph (model: Agilent 1260, Agilent, USA); digestion instrument (model: SPH / SPM, Jinan Alva Instruments Co., Ltd.); Kjeldahl nitrogen determination instrument (model: KN520, Jinan Alva Instruments Co., Ltd.).

[0144] 2. Materials and reagents

[0145] Rutin reference substance (batch number: 100080-201610, content 92.6%, China Institute for Drug Control); Gallic acid reference substance (batch number: 110831-201906, content 91.5%, China Institute for Drug Control); Emodin reference substance (batch number: 110756-201913, content 96.0%, China Institute for Drug Control); Oleanolic acid reference substance (batch number: 110709-201808, content 91.1%, China Institute for Drug Control); D-anhydrous glucose (batch number: 110833-201707, content 99.9%, China Institute for Drug Control); Serum albumin (bovine) (batch number: 140619-201421, China Institute for Drug Control); Magnolol (batch number: 110729-201513, content 99.8%, China Institute for Drug Control); Honokiol (batch number: 110730-201915, content 99.8%, China Institute for Drug Control); Betaine reference substance (batch number: 110894-200503, content 98.5%, China Institute for Drug Control); Linoleic acid reference substance (batch number: 111622-201203, content 99.6%, China Institute for Drug Control); Semen Cannabis Pill intermediate (batch number: A210111410, produced by Peili (Nanning) Pharmaceutical Co., Ltd.).

[0146] Methanol (Merck) and acetonitrile (Merck) were chromatographically pure; water was ultrapure water; and the others were analytically pure.

[0147] Example 1

[0148] This example provides a method for determining the total flavonoid content in a CDD-2101 intermediate, specifically comprising the following steps:

[0149] 1. Preparation of reference substance solution:

[0150] An appropriate amount of rutin reference substance was accurately weighed and measured, and 60% ethanol (i.e. 60 vol% ethanol aqueous solution) was added to prepare a solution containing 0.2 mg per 1 ml, thereby obtaining the solution.

[0151] 2. Preparation of standard curve:

[0152] Accurately pipette 1 ml, 2 ml, 3 ml, 4 ml, 5 ml and 6 ml of the reference substance solution into 25 ml volumetric flasks, respectively, add water to 6.0 ml, add 1 ml of 5% sodium nitrite solution, mix well, stand for 6 minutes, add 1 ml of 10% aluminum nitrate solution, mix well, stand for 6 minutes, add 10 ml of sodium hydroxide test solution, add water to the calibration mark again, shake well, stand for 15 minutes, set up a blank control reagent, immediately determine the absorbance at 500 nm wavelength according to the ultraviolet-visible spectrophotometry, take the absorbance as the vertical coordinate and the concentration as the horizontal coordinate to draw a standard curve.

[0153] 3. Determination method:

[0154] Take 0.2 g of CDD-2101 intermediate powder, accurately weigh, put into a conical flask with a plug, accurately add 25 ml of 60% ethanol, tightly plug, weigh, ultrasonic treat for 30 minutes, take out, cool, weigh again, make up the weight loss with 60% ethanol, shake well, centrifuge (4000 r / min) for 10 minutes, take the supernatant as the test sample solution. Accurately pipette 1 ml of the test sample solution into a 25 ml volumetric flask, according to the method under the preparation of the standard curve, from "add water to 6.0 ml", determine the absorbance according to the method, read the weight of rutin (mg) in the test sample solution from the standard curve, calculate the percentage content of total flavonoids in the test sample as dry product, and the result is shown in Table 1 below.

[0155] Table 1 Determination results of total flavonoids content in intermediate of Maziren pills

[0156] 4. Methodology verification

[0157] (1) Investigation of linear range

[0158] Accurately weigh 11.43 mg of rutin reference substance, put into a 50 ml volumetric flask, add 60% ethanol to dissolve and dilute to the calibration mark, shake well, and the concentration of the reference substance solution is 0.2117 mg / ml.

[0159] Accurately pipette 1 ml, 2 ml, 3 ml, 4 ml, 5 ml and 6 ml of the reference substance solution into 25 ml volumetric flasks, respectively, add water to 6.0 ml, add 1 ml of 5% sodium nitrite solution, mix well, stand for 6 minutes, add 1 ml of 10% aluminum nitrate solution, mix well, stand for 6 minutes, add 10 ml of sodium hydroxide test solution, add water to the calibration mark again, shake well, stand for 15 minutes, set up a blank control reagent, immediately determine the absorbance at 500 nm wavelength according to the ultraviolet-visible spectrophotometry, take the absorbance as the vertical coordinate and the concentration as the horizontal coordinate to draw a standard curve.

[0160] The regression equation of the rutin reference substance is y = 0.0124x - 0.0090 (R 2 = 0.9998).

[0161] The results show that when the rutin concentration is in the range of 8.468 μg / ml-50.81 μg / ml, the concentration has a good linear relationship with the absorbance, which conforms to the Lambert-Beer law. The above test results are shown in Table 2 below, and the standard curve is shown in Figure 1.

[0162] Table 2 Rutin linear range test results

[0163] (2) Precision test

[0164] The same sample solution was taken, and after coloration according to the above determined coloration method, the absorbance was determined for 6 times, the absorbance value was recorded, and the RSD value of the absorbance was calculated, and the results are shown in Table 3 below.

[0165] Table 3 Total flavonoids precision test results

[0166] The results show that the RSD value of the total flavonoids absorbance value is less than 3.0%, which indicates that the instrument precision is good.

[0167] (3) Coloration stability test

[0168] The same sample solution was taken, and according to the above determined coloration method, the absorbance was determined at 10 min, 15 min, 20 min, 25 min, 30 min and 45 min after coloration, and the RSD value of the absorbance was calculated, and the results are shown in Table 4 below.

[0169] Table 4 Total flavonoids stability test results

[0170] The results show that the RSD value of the total flavonoids absorbance value is less than 3.0%, which indicates that the sample solution is stable within 45 minutes after coloration.

[0171] (4) Reproducibility test

[0172] The same batch of sample (batch number: A210111410) was taken, 6 portions of 0.2 g each were precisely weighed, the absorbance was determined according to the above determined determination method, the content of rutin in the sample solution was calculated by the standard curve, and the percentage content of total flavonoids in the sample was calculated based on the dry product, i.e. the percentage content of total flavonoids in the sample was calculated based on the dry product. The results are shown in Table 5 below.

[0173] Table 5 Total flavonoids reproducibility test results

[0174] The results show that the RSD value of the total flavonoids content is less than 3.0%, which indicates that the method has good reproducibility.

[0175] (5) Recovery rate test

[0176] Take 6 dry and clean conical flask, respectively, add rutin reference substance, respectively, add a known amount of sample (total flavonoids content is 11.37%), about 0.1 g, precision weighing, in the stoppered conical flask, according to the above determination method, the determination of absorbance, from the standard curve read rutin weight in test solution, calculate the recovery, see table 6 below.

[0177] Table 6 total flavonoids recovery test results

[0178] The results show that the total flavonoids recovery is between 95% and 105%, and the RSD value is less than 3.0%, indicating that the recovery of the method is good, which meets the requirements of quality standard analysis.

[0179] Example 2

[0180] The present embodiment provides a method for determining the total phenol content in CDD-2101 intermediate, which specifically comprises the following steps:

[0181] 1. Preparation of reference solution: take gallic acid reference substance, precision weighing, add 70% ethanol to prepare a solution containing 25 μg per 1 ml, namely;

[0182] 2. Preparation of standard curve:

[0183] Precisely take 0.2 ml, 0.5 ml, 0.8 ml, 1.0 ml, 1.2 ml, 1.4 ml of reference solution, respectively, in 25 ml volumetric flask, add anhydrous ethanol to 5.0 ml, add 0.3 wt% sodium dodecyl sulfate solution 2.0 ml and 0.6 wt% ferric chloride-0.9 wt% potassium ferricyanide (volume ratio 1:0.9) mixed solution 1.0 ml, mix well, place in the dark for 5 minutes, add 0.1 mol / L hydrochloric acid solution to the mark, shake well, place in the dark for 20 minutes, set blank control reagent, immediately determine the absorbance at 740 nm wavelength by ultraviolet-visible spectrophotometry, take the absorbance as the ordinate and the concentration as the abscissa, draw the standard curve;

[0184] 3. Determination method:

[0185] Take 0.1 g of CDD-2101 intermediate powder, accurately weigh, accurately add 70% ethanol 25 ml, weigh, ultrasonic treatment for 30 minutes, take out, cool, reweigh, make up the weight loss with 70% ethanol, shake well, centrifuge (4000 r / min) for 10 minutes, accurately take 1.0 ml of supernatant, put it into a 10 ml volumetric flask, dilute to the mark with 70% ethanol, shake well, and use it as the test sample solution. Accurately take 1.0 ml of the test sample solution into a 25 ml volumetric flask, according to the method under the preparation of the standard curve, from "add anhydrous ethanol to 5.0 ml", measure the absorbance according to the law, read the weight of gallic acid in the test sample solution (mg) from the standard curve, calculate the percentage content of total phenol in the test sample based on dry product, and get the result. The results are shown in Table 7 below.

[0186] Table 7 Determination results of total phenol content in intermediate of Ma Ziren pills

[0187] 4. Methodology verification

[0188] (1) Investigation of linear range

[0189] Accurately weigh 22.36 mg of gallic acid reference substance into a 50 ml volumetric flask, dissolve and dilute to the mark with 70% ethanol, shake well, and get the reference solution with a concentration of 0.4092 mg / ml. Accurately take 3 ml of the reference solution into a 50 ml volumetric flask, dilute to the mark with 70% ethanol, shake well, and get the reference solution with a concentration of 24.55 μg / ml.

[0190] Accurately take 0.2 ml, 0.5 ml, 0.8 ml, 1.0 ml, 1.2 ml, and 1.4 ml of the reference solution into 25 ml volumetric flasks, respectively, add anhydrous ethanol to 5.0 ml, add 2.0 ml of 0.3 wt% sodium dodecyl sulfate solution and 1.0 ml of 0.6 wt% ferric chloride-0.9 wt% potassium ferricyanide (volume ratio 1:0.9) mixed solution, mix well, place in the dark for 5 minutes, add 0.1 mol / L hydrochloric acid solution to the mark, shake well, place in the dark for 20 minutes, set up a blank control reagent, immediately measure the absorbance at 740 nm wavelength by ultraviolet-visible spectrophotometry. Take the absorbance as the vertical coordinate and the concentration as the horizontal coordinate to draw the standard curve.

[0191] The regression equation of gallic acid reference substance is y=0.7078x-0.0218 (R 2 =0.9980).

[0192] The results show that when the concentration of gallic acid is in the range of 0.1964 μg / ml-1.375 μg / ml, the concentration and absorbance show a good linear relationship, which meets the Lambert-Beer law. The test results are shown in Table 8 below and the standard curve is shown in Figure 2.

[0193] Table 8 Gallic acid linear range investigation results

[0194] (2) Precision test

[0195] Take the same test solution, according to the above-mentioned coloration method coloration, repeated determination of 6 times, record the absorbance value, calculate the RSD value of absorbance, the results are shown in Table 9 below.

[0196] Table 9 Total phenol precision test results

[0197] The results show that the RSD value of total phenol absorbance value is less than 3.0%, indicating that the instrument precision is good.

[0198] (3) Color stability test

[0199] Take the same test solution, according to the above-mentioned coloration method coloration, and after 20 minutes in the dark, respectively, 0 min, 2 min, 5 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 60 min determination of total phenol absorbance, calculate the RSD value of absorbance, the results are shown in Table 10 below.

[0200] Table 10 Total phenol stability test results

[0201] The results show that the RSD value of total phenol absorbance value is less than 3.0%, indicating that the test solution is stable within 60 minutes after coloration.

[0202] (4) Reproducibility test

[0203] Take the same batch of samples, take 6, each 0.1g, accurately weigh, according to the above-mentioned determination method, determination of absorbance, read out the weight of gallic acid in the test solution from the standard curve, calculate, that is. The results are shown in Table 11 below.

[0204] Table 11 Total phenol reproducibility test results

[0205] The results show that the RSD value of total phenol content is less than 3.0%, indicating that the method has good reproducibility.

[0206] (5) Recovery test

[0207] Take 6 dry and clean conical flask, respectively, accurately add the concentration of 0.4092 mg / ml of gallic acid control mother liquor 7 ml, vacuum evaporation of solvent, respectively, add known content of sample (total phenol content is 5.51%), about 0.05 g each, accurately weighed, according to the above determination method, the determination of absorbance, from the standard curve read out the weight of gallic acid in the test solution, calculate the recovery, see table 12.

[0208] Table 12 total phenol recovery test results

[0209] The results show that the total phenol recovery rate is between 95%-105%, RSD value is less than 3.0%, indicating that the recovery of the method is good, in line with the requirements of quality standard analysis.

[0210] Example 3

[0211] The present embodiment provides a method for determining the total anthraquinone content in CDD-2101 intermediate, specifically comprising the following steps:

[0212] 1. Preparation of reference solution:

[0213] Take emodin reference substance, accurately weighed, add methanol to make 0.2 mg per 1 ml solution, that is;

[0214] 2. Preparation of standard curve:

[0215] Accurately take 0.2 ml, 0.5 ml, 1.0 ml, 1.4 ml, 1.8 ml, 2.0 ml of reference solution, respectively, in 10 ml volumetric flask, add 1% magnesium acetate methanol solution to dilute to the mark, shake well, place coloration for 30 minutes, set blank control reagent, according to the ultraviolet-visible spectrophotometry, the determination of absorbance at 508 nm wavelength, with absorbance as the ordinate, concentration as the abscissa, draw the standard curve;

[0216] 3. Determination method:

[0217] Take CDD-2101 intermediate powder 0.4 g, accurately weighed, accurately added methanol 25 ml, weighed, heated to reflux for 1 hour, take out, put cold, make up the weight loss with methanol, shake up, filter, accurately take the filtrate 10 ml, put in flask, evaporate the solvent, add 8 wt% hydrochloric acid solution 15 ml to the residue, ultrasonic treatment for 2 minutes, add chloroform 15 ml, water bath heating reflux for 1 hour, take out, put cold, put in a separatory funnel, wash the container with a small amount of chloroform and put it into the separatory funnel, separate the chloroform layer, the acid solution is extracted with chloroform for 3 times, 20 ml each time, combine the chloroform liquid, evaporate to dryness, add methanol to dissolve and transfer to a 5 ml volumetric flask, add methanol to the mark, shake well, as the test solution;

[0218] Accurately take 1 ml of the test solution, according to the method under the preparation of the standard curve, from "put 10 ml volumetric flask", determine the absorbance according to the method, read the weight of emodin in the test solution from the standard curve (mg), calculate the percentage of total anthraquinone in the test sample as dry product. The results are shown in the following table 13.

[0219] Table 13 Determination results of total anthraquinone content in intermediate of Maziren pill

[0220] 4. Methodology verification

[0221] (1) Investigation of linear range

[0222] Accurately weigh 15.66 mg of emodin reference substance, put it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the mark, shake well, to get a concentration of 0.3007 mg / ml of reference substance stock solution, accurately take 15 ml of reference substance stock solution, put it in a 25 ml volumetric flask, add methanol to dilute to the mark, shake well, to get a concentration of 0.1804 mg / ml of reference substance solution.

[0223] Accurately take 0.2 ml, 0.5 ml, 1.0 ml, 1.4 ml, 1.8 ml, 2.0 ml of the reference substance solution, respectively, put them in 10 ml volumetric flasks, dilute to the mark with 1% magnesium acetate ethanol solution, shake well, stand for color development for 30 minutes, set up a blank control reagent, according to the ultraviolet-visible spectrophotometry, measure the absorbance at 508 nm wavelength, take the absorbance as the vertical coordinate and the concentration as the horizontal coordinate, draw the standard curve.

[0224] The regression equation of emodin reference substance is y=0.0417x+0.0049 (R 2 =0.9998).

[0225] The results show that when the concentration of emodin is in the range of 3.608 μg / ml-36.08 μg / ml, the concentration and absorbance have a good linear relationship, which conforms to the Lambert-Beer law. The test results are shown in Table 14 below, and the standard curve is shown in Figure 3.

[0226] Table 14 Investigation results of linear range of emodin

[0227] (2) Precision test

[0228] The same test sample solution was taken, and the color was developed according to the above-mentioned color development method. The absorbance was determined for 6 times, and the total anthraquinone absorbance value was recorded. The RSD value of the absorbance was calculated, and the results are shown in Table 15 below.

[0229] Table 15 Precision test results of total anthraquinone

[0230] The results show that the RSD value of the total anthraquinone absorbance value is less than 3.0%, which indicates that the instrument precision is good.

[0231] (3) Color stability test

[0232] The same test sample solution was taken, and the color was developed according to the above-mentioned color development method. The absorbance was determined at 10 min, 20 min, 30 min, 35 min, 40 min, 45 min, 60 min, 75 min, 90 min, 120 min, and 150 min after the color was developed, respectively. The RSD value of the total anthraquinone absorbance was calculated, and the results are shown in Table 16 below.

[0233] Table 16 Stability test results of total anthraquinone

[0234] The results show that the total anthraquinone absorbance increases with time, but the RSD value of the absorbance is less than 3.0% within 150 minutes, which indicates that the test sample solution is stable within 150 minutes.

[0235] (4) Reproducibility test

[0236] The same batch of samples was taken, and 6 samples were taken, each weighing 0.4 g. The absorbance was determined according to the above-mentioned determination method. The weight of emodin in the test sample solution was read from the standard curve, and the total anthraquinone content was calculated. The results are shown in Table 17 below.

[0237] Table 17 Reproducibility test results of total anthraquinone

[0238] The results show that the RSD value of the total anthraquinone content is less than 3.0%, which indicates that the method has good reproducibility.

[0239] (5) Recovery test

[0240] Take 6 dry and clean conical flask, respectively, precisely add emodin reference solution with concentration of 0.3007 mg / ml 3.0 ml, evaporate the solvent, and then add sample with known content (total anthraquinone content is 0.4850%) of about 0.2 g, precisely weigh, according to the above determination method, determine the absorbance, read the weight of emodin in the test solution from the standard curve, and calculate the recovery, the results are shown in Table 18.

[0241] Table 18 Total anthraquinone recovery test results

[0242] The results show that the total anthraquinone recovery is between 95%-105%, and the RSD value is less than 3.0%, indicating that the recovery of the method is good, and meets the requirements of quality standard analysis.

[0243] Example 4

[0244] The embodiment provides a method for determining the total triterpene content in CDD-2101 intermediate, which specifically comprises the following steps:

[0245] 1. Preparation of reference solution: take oleanolic acid reference substance, precisely weigh, add ethanol to prepare a solution containing 0.2 mg per 1 ml, and obtain the solution.

[0246] 2. Preparation of standard curve:

[0247] Precisely take 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml and 0.5 ml of the reference solution, respectively, and place them in 15 ml test tubes with stoppers. Evaporate water in a water bath, cool, precisely add 0.2 ml of freshly prepared vanillin glacial acetic acid solution (precisely weigh 0.5 g of vanillin, add glacial acetic acid to dissolve into 10 ml, and obtain the solution), 0.8 ml of perchloric acid, mix, heat in a 70°C water bath for 15 minutes, immediately place in an ice bath for 5 minutes, take out, precisely add 5 ml of glacial acetic acid, shake well, take the corresponding reagent as blank, immediately determine the absorbance at 540 nm wavelength by ultraviolet-visible spectrophotometry, take the absorbance as the vertical coordinate, and the concentration as the horizontal coordinate, and draw the standard curve.

[0248] 3. Determination method:

[0249] Take CDD-2101 intermediate powder 0.2 g, precision weighing, precision 80% ethanol 25 ml, weighing, heating reflux 30 minutes, take out, put cold, with 80% ethanol to make up for the loss of weight, shake up, centrifugal (4000 r / min) 10 minutes, precision pipette supernatant 15 ml, in the evaporating dish, water bath evaporation of ethanol, the residue was dissolved with water 5 ml, add to AB-8 macroporous resin column (column height 12 cm, inner diameter 1.5 cm), the residue in the evaporating dish was dissolved with 30% ethanol 5 ml, add to the same macroporous resin column, add water 30 ml, 60% ethanol 80 ml elution, discard the eluent, eluted with ethanol 150 ml, collect the eluent, evaporated, the residue was dissolved with ethanol and constant volume to 5 ml in a volumetric flask, shake up, obtained. Precision 1.0 ml, in 15 ml test tube, according to the method of preparation of standard curve, from "water bath evaporation", according to the law of determination of absorbance, read from the standard curve of oleanolic acid in the test solution of the weight (mg), the percentage of total triterpenes in the test sample was calculated, obtained. The results are shown in the following table 19.

[0250] Table 19 determination results of total triterpenes in intermediate of Maziren pill

[0251] 4, methodological validation

[0252] (1) Investigation of linear range

[0253] Precision oleanolic acid reference substance 10.47 mg, in 25 ml volumetric flask, add ethanol to dissolve and dilute to the mark, shake up, obtained the concentration of 0.3815 mg / ml of the reference solution, precision 5 ml of the reference solution, in 10 ml volumetric flask, dilute to the mark with ethanol, shake up, obtained the concentration of 0.1908 mg / ml of the reference solution.

[0254] Precision 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml of the reference solution, respectively, in 15 ml test tube with plug, water bath evaporation, cool, precision new preparation of vanillin glacial acetic acid solution (precision oleanolic acid 0.5 g, add glacial acetic acid to dissolve into 10 ml, obtained) 0.2 ml, perchloric acid 0.8 ml, mix well, in 70 DEG C water bath heating 15 minutes, immediately placed in ice bath cooling 5 minutes, take out, precision add glacial acetic acid 5 ml, shake up, with the corresponding reagent as blank, immediately according to the ultraviolet-visible spectrophotometry, at 540 nm wavelength, determination of absorbance, with absorbance as the ordinate, concentration as the abscissa, draw the standard curve.

[0255] The regression equation of oleanolic acid reference substance: y = 0.0623x - 0.0245 (R 2 = 1.0000).

[0256] The results show that when the oleanolic acid concentration is in the range of 3.180 μg / ml-15.90 μg / ml, the concentration has a good linear relationship with the absorbance, which conforms to the Lambert-Beer law. The test results are shown in Table 20 below, and the standard curve is shown in Figure 4.

[0257] Table 20 Investigation results of linear range of oleanolic acid

[0258] (2) Precision test

[0259] The same test sample solution was taken, and the color was developed according to the above-mentioned color development method. The absorbance of total triterpenes was determined for 6 times, and the RSD value of the absorbance was calculated. The results are shown in Table 21 below.

[0260] Table 21 Results of precision test of total triterpenes

[0261] The results show that the RSD value of the absorbance of total triterpenes is less than 3.0%, which indicates that the precision of the instrument is good.

[0262] (3) Color stability test

[0263] The same test sample solution was taken, and the color was developed according to the above-mentioned color development method. The absorbance was determined at 0 min, 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min and 90 min after the color development, respectively. The RSD value of the absorbance of total triterpenes was calculated. The results are shown in Table 22 below.

[0264] Table 22 Results of stability test of total triterpenes

[0265] The results show that the absorbance of total triterpenes decreases with the extension of the placement time, but the RSD value of the absorbance within 60 minutes is less than 3.0%, which indicates that the test sample solution is stable within 60 minutes.

[0266] (4) Reproducibility test

[0267] The same batch of sample was taken, and 6 samples were taken, each weighing 0.2 g. The absorbance was determined according to the above-mentioned determination method. The weight of oleanolic acid in the test sample solution was read from the standard curve, and the total triterpenes content was calculated. The results are shown in Table 23 below.

[0268] Table 23 Results of reproducibility test of total triterpenes

[0269] The results show that the RSD value of the total triterpenes content is less than 3.0%, which indicates that the method has good reproducibility.

[0270] (5) Recovery rate test

[0271] Take 6 dry and clean conical flasks, respectively, accurately add 0.5 ml of oleanolic acid reference substance solution with a concentration of 0.4274 mg / ml, evaporate the solvent, and then respectively add a known amount of sample (total triterpenoid content is 0.2325%), about 0.1 g each, accurately weigh, according to the above determination method, determine the absorbance, read the weight of oleanolic acid in the test solution from the standard curve, and calculate the recovery rate, the results are shown in Table 24 below.

[0272] Table 24 Total Triterpenoid Recovery Test Results

[0273] The results show that the total triterpenoid recovery rate is between 95% and 105%, and the RSD value is less than 3.0%, indicating that the recovery rate of this method is good and meets the requirements of quality standard analysis.

[0274] Example 5

[0275] The present embodiment provides a method for determining the total alkaloid content in CDD-2101 intermediate, which specifically comprises the following steps:

[0276] 1. Preparation of reference solution:

[0277] Take a suitable amount of betaine reference substance, accurately weigh, and add 0.1 mol / L hydrochloric acid to prepare a solution containing 1.5 mg per 1 ml.

[0278] 2. Preparation of standard curve:

[0279] Precisely take 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml of the reference solution, and place them in 10 ml volumetric flasks. Precisely add 2 ml of 2 wt% Reichardt's ammonium salt solution (newly prepared and filtered before use), and then dilute to the mark with 0.1 mol / L hydrochloric acid solution. Shake well, place in a 2°C refrigerator for 2 hours, take out, filter with a 0.22 μm microporous filter, and take the filtrate. Precisely take 4 ml of freshly prepared 2 wt% Reichardt's ammonium salt solution, place it in a 20 ml volumetric flask, dilute to the mark with 0.1 mol / L hydrochloric acid solution, shake well, place in a 2°C refrigerator for 2 hours, take out, filter with a 0.22 μm microporous filter, and take the filtrate as a blank control solution.

[0280] With 0.1 mol / L hydrochloric acid solution as reagent blank, determine the absorbance at 522 nm wavelength according to the ultraviolet-visible spectrophotometry (Chinese Pharmacopoeia 2020 edition four general chapters 0401), take the absorbance difference (the difference between the blank control and the reference) as the vertical coordinate, and the concentration as the horizontal coordinate, to draw the standard curve.

[0281] 3. Determination method:

[0282] Take about 5 g of CDD-2101 intermediate powder, accurately weigh, accurately add 1% hydrochloric acid methanol solution 100 ml, weigh, heat to reflux for 1 hour, cool, weigh again, make up the weight loss with methanol, shake well, filter, accurately take 50 ml of the filtrate, evaporate to dryness, add 0.1 mol / L hydrochloric acid solution to dissolve and dilute to 20 ml in a volumetric flask, shake well, centrifuge (4000 r / min) for 10 minutes, accurately take 10 ml of supernatant, add 1 g of activated carbon, heat in a boiling water bath for 2 minutes, take out, filter into a 20 ml volumetric flask, add 0.1 mol / L hydrochloric acid 6 ml for washing, filter into the volumetric flask together, accurately add 2 wt% reinecke ammonium salt solution 4 ml (newly prepared before use, filtered before use), dilute to the mark with 0.1 mol / L hydrochloric acid solution, shake well, place in a 2°C refrigerator for 2 hours, take out, filter with a 0.22 μm microporous filter membrane, take the filtrate as the test sample solution. With 0.1 mol / L hydrochloric acid solution as reagent blank, according to the ultraviolet-visible spectrophotometry (Chinese Pharmacopoeia 2020 edition four general chapters 0401) at 522 nm wavelength, the absorbance is determined, the absorbance difference (the difference between the blank control and the test sample) is calculated, the weight of betaine in the test sample solution is read from the standard curve (mg), and the percentage of total alkaloids in the test sample is calculated based on the dry product. The results are shown in the following Table 25.

[0283] Table 25 Determination results of total alkaloids in the intermediate of Maziren Pills

[0284] 4. Methodology verification

[0285] (1) Investigation of linear range

[0286] Accurately weigh 69.93 mg of betaine reference substance into a 50 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to dissolve and dilute to the mark, shake well, and obtain a reference solution with a concentration of 1.378 mg / ml.

[0287] Precisely take 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, 5.0 ml of the reference solution, respectively, into 10-ml volumetric flasks, precisely add 2 ml of 2 wt% Rieske ammonium salt solution (freshly prepared and filtered before use), then dilute to the calibration mark with 0.1 mol / L hydrochloric acid solution, shake well, and place in a 2°C refrigerator for 2 hours. Take it out, filter it with a 0.22-μm microporous filter membrane, and take the filtrate, which is obtained. Precisely take 4 ml of freshly prepared 2 wt% Rieske ammonium salt solution into a 20-ml volumetric flask, dilute to the calibration mark with 0.1 mol / L hydrochloric acid solution, shake well, and place in a 2°C refrigerator for 2 hours. Take it out, filter it with a 0.22-μm microporous filter membrane, and take the filtrate, which is used as a blank control solution. Take 0.1 mol / L hydrochloric acid solution as the reagent blank, and determine the absorbance at a wavelength of 522 nm according to the ultraviolet-visible spectrophotometry (General Test Methods 0401 in Chinese Pharmacopoeia 2020 Edition). Take the absorbance difference (the difference between the absorbance of the blank control and the reference) as the vertical coordinate and the concentration as the horizontal coordinate to draw a standard curve. The regression equation of the betaine reference is y = 0.6274x - 0.0465 (R = 0.9987). 2

[0288] The results show that when the concentration of betaine is in the range of 0.1378 mg / ml to 0.6890 mg / ml, the concentration and the absorbance have a good linear relationship, which conforms to the Lambert-Beer law. The test results are shown in Table 26 below, and the standard curve is shown in Figure 5.

[0289] Table 26 Results of betaine linear range test

[0290] (2) Precision test

[0291] Take the same test solution, perform the precipitation reaction according to the method determined above, and repeatedly determine the supernatant after the reaction for 6 times. Record the absorbance values and calculate the RSD value of the absorbance. The results are shown in Table 27 below.

[0292] Table 27 Results of total alkaloid precision test

[0293] The results show that the RSD value of the total alkaloid absorbance is less than 3.0%, indicating that the instrument precision is good.

[0294] (3) Stability test

[0295] Take the same test solution, perform the precipitation reaction according to the method determined above, and determine the absorbance of the supernatant after the reaction at 5 min, 10 min, 15 min, 30 min, 45 min, and 60 min, respectively. Calculate the RSD value of the absorbance. The results are shown in Table 28 below.

[0296] ​Table 28 Total alkaloid stability test results

[0297] The results show that the RSD value of total alkaloid absorbance is less than 3.0%, indicating that the test solution is stable within 60 minutes.

[0298] (4) Repetitive test

[0299] Take the same batch of samples, take 6 parts, each 5 g, accurately weigh, according to the above determined determination method, determine the absorbance, read the weight of betaine in the test solution from the standard curve, calculate, that is. The results are shown in Table 29 below.

[0300] Table 29 Total alkaloid repetitive test results

[0301] The results show that the RSD% value of total alkaloid content is less than 5.0%, indicating that the method has good repeatability.

[0302] (5) Recovery test

[0303] Take 6 dry and clean conical flasks, accurately add betaine reference substance, then add samples with known content (total alkaloid content is 0.6174%) respectively, each 2.5 g, accurately weigh, place in conical flasks with stopper, according to the above determined determination method, determine the absorbance, read the weight of betaine in the test solution from the standard curve, calculate the recovery rate, the results are shown in Table 30 below.

[0304] Table 30 Total alkaloid recovery test results

[0305] As can be seen from the results, the recovery rate of total alkaloid is between 90% and 108%, and the RSD value is less than 5.0%, indicating that the method has good recovery rate.

[0306] Example 6

[0307] The present embodiment provides a method for determining the total sugar content in CDD-2101 intermediate, which specifically comprises the following steps:

[0308] 1. Preparation of reference solution:

[0309] Take D-anhydrous glucose reference substance, accurately weigh, add water to prepare a solution containing 1 mg per 1 ml, and obtain;

[0310] 2. Preparation of standard curve:

[0311] Accurately pipette 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.7 ml of the reference substance solution into 25-ml volumetric flasks, respectively, add 3,5-dinitrosalicylic acid solution 1.5 ml, shake well, heat in a boiling water bath for 5 minutes, cool rapidly with cold water, add water to the calibration mark, shake well, and prepare a blank solution with water 1.0 ml by the same method. Determine by UV-visible spectrophotometry (General Rule 0401). Determine the absorbance at 480 nm, respectively, take the absorbance as the vertical coordinate and the concentration as the horizontal coordinate to draw a standard curve.

[0312] 3. Determination method:

[0313] Take 0.1 g of CDD-2101 intermediate powder, accurately weigh, accurately add water 20 ml, ultrasonic treatment for 30 minutes, cool, centrifuge (4000 r / min) for 10 minutes, accurately pipette 5.0 ml of supernatant, add 6 mol / L hydrochloric acid solution 5 ml, heat in a boiling water bath for 30 minutes, take out, cool, add phenolphthalein indicator 1 drop, neutralize to light red with 5 mol / L sodium hydroxide solution, dilute with water to 25 ml, shake well, accurately pipette 1.0 ml, place in a 25-ml volumetric flask, from "add 3,5-dinitrosalicylic acid 1.5 ml" according to the method, determine the absorbance, read the weight of D-anhydrous glucose in the test solution (mg) from the standard curve, calculate the percentage of total sugar in the test sample as dry product. The results are shown in Table 31 below.

[0314] Table 31 Determination results of total sugar content in intermediate of Ma Ziren pills

[0315] 4. Methodology verification

[0316] (1) Investigation of linear range

[0317] Accurately weigh 51.73 mg of D-anhydrous glucose reference substance, place in a 50-ml volumetric flask, add water to dissolve and dilute to the calibration mark, shake well, and obtain a reference substance solution with a concentration of 1.0336 mg / ml.

[0318] Accurately pipette 0.1 ml, 0.2 ml, 0.3 ml, 0.5 ml, 0.7 ml, 1.0 ml of the reference substance solution into 25-ml volumetric flasks, respectively, add 3,5-dinitrosalicylic acid solution 1.5 ml, shake well, heat in a boiling water bath for 5 minutes, cool rapidly with cold water, add water to the calibration mark, shake well, and prepare a blank solution with water 1.0 ml by the same method. Determine by UV-visible spectrophotometry (General Rule 0401). Determine the absorbance at 480 nm, respectively, take the absorbance as the vertical coordinate and the concentration as the horizontal coordinate to draw a standard curve.

[0319] Regression equation of D-anhydrous glucose control: y = 0.0273x + 0.0500 (R 2 = 0.9990).

[0320] The results showed that when the concentration of D-anhydrous glucose was in the range of 4.134 μg / ml-41.34 μg / ml, the concentration and absorbance showed good linear relationship, which was consistent with Lambert-Beer law. The experimental results were shown in Table 32, and the standard curve was shown in Figure 6.

[0321] Table 32 Results of linear range investigation of D-anhydrous glucose

[0322] (2) Precision test

[0323] The same sample solution was taken, and the color was developed according to the above-mentioned color development method. The absorbance value was recorded after 6 repeated determinations, and the RSD value of absorbance was calculated. The results were shown in Table 33.

[0324] Table 33 Results of precision test of total sugar

[0325] The results showed that the RSD value of total sugar absorbance value was less than 3.0%, which indicated that the precision of the instrument was good.

[0326] (3) Color stability test

[0327] The same sample solution was taken, and the absorbance was determined at 2 min, 3 min, 4 min, 5 min, 10 min, 40 min and 90 min after color development according to the above-mentioned color development method. The RSD value of absorbance was calculated. The results were shown in Table 34.

[0328] Table 34 Results of stability test of total sugar

[0329] The results showed that the RSD value of total sugar absorbance value was less than 3.0%, which indicated that the sample solution was stable within 90 minutes after color development.

[0330] (4) Reproducibility test

[0331] The same batch of sample was taken, and 6 portions of 0.1 g each were precisely weighed and accurately added with water 20 ml. The absorbance was determined according to the above-mentioned determination method. The weight of D-anhydrous glucose in the sample solution was read from the standard curve, and the total sugar content was calculated. The results were shown in Table 35.

[0332] Table 35 Results of reproducibility test of total sugar

[0333] The results showed that the RSD value of total sugar content was less than 3.0%, which indicated that the method had good reproducibility.

[0334] (5) Recovery test

[0335] Take 6 dry and clean conical flask, respectively, add D-anhydrous glucose control product, respectively, add a known amount of sample (total sugar content of 42.19%), about 0.05g, precision weighing, in the stoppered conical flask, according to the above determination method, the determination of absorbance, from the standard curve read the weight of D-anhydrous glucose in the test solution, calculate the recovery, the results are shown in the following table 36.

[0336] Table 36 total sugar recovery test results

[0337] The results show that the recovery rate of total sugar is between 95% and 105%, and the RSD value is less than 3.0%, indicating that the recovery of the method is good and meets the requirements of quality standard analysis.

[0338] Example 7

[0339] The present embodiment provides a method for determining the total protein content in CDD-2101 intermediate, which specifically comprises the following steps:

[0340] 1. Preparation of control solution:

[0341] Take serum albumin (bovine) control product, precision weighing, add water to make a solution containing 0.1mg per 1ml, namely;

[0342] 2. Preparation of standard curve:

[0343] Precisely take 0.1ml, 0.25ml, 0.5ml, 0.8ml, 1.0ml, 1.2ml of control solution, respectively, precisely add 5.0ml of coomassie brilliant blue solution, immediately mix, place for 2 minutes, set blank control reagent, determine according to ultraviolet-visible spectrophotometry (general rule 0401); at 595nm wavelength, respectively determine the absorbance, take absorbance as ordinate, concentration as abscissa, draw standard curve;

[0344] 3. Determination method:

[0345] Take 0.2g of CDD-2101 intermediate powder, precision weighing, precision add 50ml of water, weigh, ultrasonic treatment for 30 minutes to dissolve, take out, cool, add water to make up the weight loss, shake well, centrifuge (4000r / min) for 10 minutes, precisely take 1.0ml of supernatant, according to the method under the preparation of standard curve, from "precisely add 5.0ml of coomassie brilliant blue solution", determine the absorbance according to the method, read the weight of serum albumin (bovine) in the test solution (mg) from the standard curve, calculate the percentage content of total protein in the test sample as dry product, namely. The results are shown in the following table 37.

[0346] Table 37 Results of total protein content determination of Mazirenwan intermediate

[0347] 4. Method validation

[0348] (1) Investigation of linear range

[0349] Accurately weigh 19.01 mg of serum albumin (bovine) control and place it in a 20 ml volumetric flask. Add water to dissolve and dilute to the mark. Shake well to obtain a control stock solution with a concentration of 0.9505 mg / ml. Accurately take 2.0 ml of the control stock solution and place it in a 20 ml volumetric flask. Add water to dilute to the mark. Shake well to obtain a control solution with a concentration of 0.09505 mg / ml.

[0350] Accurately take 0.1 ml, 0.25 ml, 0.5 ml, 0.8 ml, 1.0 ml and 1.2 ml of the control solution and add 5.0 ml of Coomassie brilliant blue solution to each. Mix immediately, stand for 2 minutes, and set up a blank reagent. Determine the absorbance at 595 nm according to the UV-visible spectrophotometry (General rule 0401). Plot the standard curve with absorbance as the vertical coordinate and concentration as the horizontal coordinate.

[0351] The regression equation of the serum albumin (bovine) control is y = 0.0411x + 0.0759 (R 2 = 0.9986).

[0352] The results show that when the concentration of serum albumin (bovine) is in the range of 1.8637 μg / ml to 18.3968 μg / ml, the concentration and absorbance have a good linear relationship, which conforms to the Lambert-Beer law. The above experiment is shown in Table 38 below. The standard curve is shown in Figure 7.

[0353] Table 38 Investigation of linear range of serum albumin (bovine)

[0354] (2) Precision test

[0355] Take the same test sample solution and develop color according to the color development method determined above. Repeat the determination 6 times, record the absorbance values, and calculate the RSD value of absorbance. The results are shown in Table 39 below.

[0356] Table 39 Results of precision test of total protein

[0357] The results show that the RSD value of the absorbance value of total protein is less than 3.0%, indicating that the instrument precision is good.

[0358] (3) Color stability test

[0359] Take the same test solution, according to the above-mentioned coloration method, the absorbance was determined at 2 min, 3 min, 4 min, 5 min, 10 min, 20 min, 30 min after coloration, and the RSD value of the absorbance was calculated. The results are shown in Table 40 below.

[0360] Table 40 Total protein stability test results

[0361] The results show that the RSD value of the total protein absorbance value is less than 3.0%, indicating that the test solution is stable within 30 minutes after coloration.

[0362] (4) Reproducibility test

[0363] Take the same batch of samples, take 6 portions, each 0.2 g, accurately weigh, accurately add water 50 ml, according to the above-mentioned determination method, determine the absorbance, read the concentration of protein in the test solution from the standard curve, calculate, that is. The results are shown in Table 41 below.

[0364] Table 41 Total protein reproducibility test results

[0365] The results show that the RSD value of the total protein content is less than 3.0%, indicating that the method has good reproducibility.

[0366] (5) Recovery test

[0367] Take 6 dry and clean conical flasks, accurately add 2 ml of serum albumin (bovine) control solution with a concentration of 0.9505 mg / ml, then add known amount of sample (total protein content is 2.01%), each about 0.1 g, accurately weigh, place in a conical flask with a plug, according to the above-mentioned determination method, determine the absorbance, read the weight of serum albumin (bovine) in the test solution from the standard curve, calculate the recovery rate, the results are shown in Table 42 below.

[0368] Table 42 Total protein recovery test results

[0369] The results show that the recovery rate of total protein is between 95% and 105%, and the RSD value is less than 3.0%, indicating that the method has good recovery rate and meets the requirements of quality standard analysis.

[0370] Example 8

[0371] The present embodiment provides a method for determining the content of lignans in CDD-2101 intermediates, which specifically comprises the following steps:

[0372] 1. Chromatographic conditions and system suitability test:

[0373] The chromatographic column was Waters XBridge C18 (4.6 mm x 250 mm, 5 μm) with octadecylsilane-bonded silica gel as the filler; methanol-acetonitrile-water (50:20:30) was used as the mobile phase; the detection wavelength was 294 nm; the column temperature was 25 °C; the theoretical plate number should not be less than 3800 calculated by the peak of magnolol.

[0374] 2. Preparation of the reference solution:

[0375] An appropriate amount of magnolol reference substance and honokiol reference substance was precisely weighed, and methanol was added to prepare a solution containing 0.3 mg per 1 ml, thereby obtaining the solution.

[0376] 3. Preparation of the test solution: about 2.0 g of CDD-2101 intermediate powder was precisely weighed, placed in a conical flask with a plug, 25 ml of methanol was precisely added, the weight was determined, ultrasonic treatment (power 250 w, frequency 40 KHz) was performed for 30 minutes, it was cooled, the weight was determined again, methanol was added to make up for the weight loss, it was shaken uniformly, filtered, and the filtrate was obtained, thereby obtaining the test solution.

[0377] 4. Determination method:

[0378] 10 μl of the reference solution and the test solution was precisely taken respectively, injected into the liquid chromatograph, determined, the peak area of magnolol and honokiol determined by the standard curve solution was used as the vertical coordinate, and the corresponding concentration was used as the horizontal coordinate, a standard curve was drawn, the total amount of magnolol and honokiol in the test product was calculated, which was used as the percentage content of lignans (calculated based on the dry product), thereby obtaining the result. The results are shown in Table 43 below.

[0379] Table 43: Determination results of lignan content in intermediate of four batches of Maziren pills

[0380] 5. Methodology verification

[0381] (1) Specificity test

[0382] About 2.0 g of CDD-2101 intermediate powder was precisely weighed, placed in a conical flask with a plug, 25 ml of methanol was precisely added, the weight was determined, ultrasonic treatment (power 250 w, frequency 40 KHz) was performed for 30 minutes, it was cooled, the weight was determined again, methanol was added to make up for the weight loss, it was shaken uniformly, filtered, and the filtrate was obtained, which was used as the test solution.

[0383] 2 g of negative sample powder of Magnolia officinalis was prepared according to the preparation method of the test solution, which was used as the negative sample solution of Magnolia officinalis.

[0384] Take 10 μl of the above-mentioned control solution, sample solution and negative sample solution respectively. Under the above-mentioned chromatographic conditions, the retention time of the peaks of magnolol and honokiol in the sample solution is consistent with that of the control solution, and no peak is observed at the corresponding position in the negative sample, indicating that the method has good specificity. The results are shown in Figure 8. The chromatographic baseline of the sample is smooth, the main peak has good peak shape, and the separation effect from the impurity peak is good.

[0385] (2) Investigation of linear range

[0386] Precisely weigh 7.90 mg of honokiol control and place it in a 25 ml volumetric flask. Add methanol to dissolve and dilute to the mark, and shake to obtain a honokiol control stock solution with a concentration of 0.3154 mg / ml. Precisely weigh 8.36 mg of magnolol control (the content is calculated based on 98.8%) and place it in a 25 ml volumetric flask. Add methanol to dissolve and dilute to the mark, and shake to obtain a magnolol control stock solution with a concentration of 0.3304 mg / ml.

[0387] Precisely measure 0.5 ml, 0.5 ml, 0.5 ml, 1.0 ml, 2.0 ml and 3.0 ml of the above-mentioned honokiol control stock solution and magnolol control stock solution respectively, and place them in the same 50 ml, 25 ml, 10 ml, 10 ml, 10 ml and 10 ml volumetric flasks. Add methanol to dilute to the mark, and shake to obtain the mixed control solution of line 1 to line 6.

[0388] Precisely take 10 μl of the above-mentioned mixed control solution of line 1 to line 6, and inject it into the high performance liquid chromatograph for determination. According to the above-mentioned chromatographic conditions, the peak area integral value is measured. The regression equation of honokiol control is y = 1769 x 10 3 x + 1547.0 (R 2 = 1.0000); and the regression equation of magnolol control is y = 1569 x 10 3 x + 837.0 (R 2 = 1.0000).

[0389] The results show that when the injection amount of honokiol is in the range of 0.03154 μg to 0.9462 μg, and the injection amount of magnolol is in the range of 0.03304 μg to 0.9912 μg, the injection amount and the peak area have a good linear relationship, and the external standard method can be used for determination and calculation. The above-mentioned test results are shown in Tables 44 and 45, and the standard curves are shown in Figures 9 and 10.

[0390] Table 44: Investigation results of honokiol linear range

[0391] Table 45: Investigation results of magnolol linear range

[0392] (3) Precision test

[0393] The precision test solution was prepared by precisely pipetting 10 μl of Magnolia officinalis Rehd. et Wils. extract and Magnolia officinalis Rehd. et Wils. mixed reference solution, and repeating the pipetting 6 times. The peak area was measured. The results are shown in Table 46 below.

[0394] Table 46 Results of precision test

[0395] The results show that the RSD values of the peak areas of Magnolia officinalis Rehd. et Wils. and Magnolia officinalis Rehd. et Wils. are less than 3.0%, indicating that the precision of the instrument is good.

[0396] (4) Stability test

[0397] The same sample solution was taken and injected at 0 h, 2 h, 4 h, 7 h, 12 h and 24 h, respectively. The results are shown in Table 47 below.

[0398] Table 47 Results of stability test

[0399] The results show that the RSD values of the peak areas of Magnolia officinalis Rehd. et Wils. and Magnolia officinalis Rehd. et Wils. are less than 3.0%, indicating that the sample solution is stable within 24 hours.

[0400] (5) Reproducibility test

[0401] The same batch of sample was taken, finely ground, and 6 portions of 2.0 g each were precisely weighed. Methanol 25 ml was added, weighed, and ultrasonically treated for 30 minutes. The solution was taken out, cooled, weighed, and shaken. The solution was filtered, and 10 μl of the filtrate was precisely pipetted for injection. The peak area was measured. The content was calculated according to the standard curve of Magnolia officinalis Rehd. et Wils. and Magnolia officinalis Rehd. et Wils. The results are shown in Table 48 below.

[0402] Table 48 Results of reproducibility test of Magnolia officinalis Rehd. et Wils. and Magnolia officinalis Rehd. et Wils.

[0403] The results show that the RSD values of the contents of Magnolia officinalis Rehd. et Wils. and Magnolia officinalis Rehd. et Wils. are less than 3.0%, indicating that the method has good reproducibility.

[0404] (6) Recovery test

[0405] 6 dry conical flasks were taken, and 1.0 ml of Magnolia officinalis Rehd. et Wils. reference mother liquor (C = 0.3154 mg / ml) and 1.0 ml of Magnolia officinalis Rehd. et Wils. reference mother liquor (C = 0.3304 mg / ml) were precisely added, respectively. The solvent was evaporated on a water bath. Known amounts of samples were taken, each 1.0 g, precisely weighed, and sample solution was prepared according to the sample solution preparation method of the reproducibility test. The peak area was measured by injection. The recovery rate was calculated according to the standard curve of Magnolia officinalis Rehd. et Wils. and Magnolia officinalis Rehd. et Wils. The results are shown in Tables 49 and 50 below.

[0406] Table 49 and Magnolol Spiked Recovery Test Results

[0407] Table 50 Magnolol Spiked Recovery Test Results

[0408] The results show that the average recovery of honokiol is 96.71%, and the RSD value is 2.54%, the average recovery of magnolol is 94.13%, and the RSD value is 2.01%, which meets the requirements of the pharmacopoeia (the content of the to-be-tested component is 0.1%, the recovery is between 90% and 108%, and the RSD value is less than 3.0%), indicating that the recovery of the method is good and meets the requirements of quality standard analysis.

[0409] (7) Intermediate precision test

[0410] The influence of different instrument conditions on the determination results of the sample content was investigated. The same batch of sample was prepared according to the "repeatability test" sample solution preparation method, 10 μl of the control sample solution and the sample solution were precisely taken for sampling, the peak area was determined, and the content was calculated according to the honokiol and magnolol standard curve. The results are shown in Table 51 below.

[0411] Table 51 Intermediate Precision Test Results of Magnolol and Honokiol

[0412] The results show that the contents of magnolol and honokiol in the samples determined by different instruments are stable, and the RSD (%) is less than 3.0%, indicating that the intermediate precision of the method is good.

[0413] Example 9

[0414] The present embodiment provides a method for determining the total fat content in CDD-2101 intermediate, which specifically comprises the following steps:

[0415] 1. Preparation of sample solution:

[0416] 5 g of CDD-2101 intermediate powder was precisely weighed and placed in a Soxhlet extractor, 150 ml of diethyl ether was added, and the extraction was carried out by heating reflux for 6 hours in a water bath, and the extract was collected;

[0417] 2. Determination method:

[0418] The extract was collected into an evaporating dish which had been dried to a constant weight, and was evaporated to dryness on a water bath at low temperature. It was dried at 100°C for 1 hour, then was moved to a desiccator and cooled for 30 minutes. The total fat content in the sample was calculated based on the dried product, and the result is shown in Table 52 below.

[0419] Table 52 Determination Results of Total Fat Content in CDD-2101 Intermediate

[0420] 3. Method validation

[0421] (1) Precision, repeatability test

[0422] Take 6 portions of 6 g of CDD-2101 intermediate powder, precisely weigh, place in a Soxhlet extractor, add 150 ml of diethyl ether, heat to reflux extraction for 6 hours on a water bath, collect the extract, and evaporate to dryness on a water bath, dry at 100°C for 1 hour, move to a desiccator, cool for 30 minutes, precisely weigh, and calculate. The results are shown in Table 53 below.

[0423] Table 53 Precision and repeatability test results of total fat

[0424] As can be seen from the results, the RSD value of the total fat content is less than 5%, indicating that the precision and repeatability of the method are good.

[0425] (2) Linearity range, recovery rate test

[0426] Take 6 portions of 2.5 g of the product powder with known content (total fat content is 0.4090%), precisely weigh, place in a Soxhlet extractor, precisely add a certain amount of linoleic acid reference substance, add 150 ml of diethyl ether, heat to reflux extraction for 6 hours on a water bath, collect the extract, and evaporate to dryness on a water bath, dry at 100°C for 1 hour, move to a desiccator, cool for 30 minutes, precisely weigh, and calculate. The recovery rate test results are shown in Table 54 below, and the linearity range investigation results are shown in Figure 11.

[0427] Table 54 Total fat recovery rate test results

[0428] As can be seen from the results, the RSD value of the total fat recovery rate is less than 5%, indicating that the accuracy of the method is good. In the linearity range investigation, the amount of linoleic acid added and the measured amount showed a good linear relationship, indicating that the accuracy of the total fat measurement is good within the range of 15.80 mg-38.10 mg, i.e., when the sample content is within this range, it has good accuracy.

[0429] Example 10

[0430] The detection wavelength, extraction solvent, and extraction method used in Example 1 were investigated under different conditions, as follows:

[0431] (1) Detection wavelength investigation

[0432] Precisely pipette 2.0 ml of the reference solution and 4.0 ml of the test solution in 25 ml volumetric flasks, respectively, add water to 6.0 ml, add 1 ml of 5% sodium nitrite solution, mix well, stand for 6 minutes, add 1 ml of 10% aluminum nitrate solution, mix well, stand for 6 minutes, add 10 ml of sodium hydroxide test solution, then add water to the mark, shake well, stand for 15 minutes, use the corresponding reagent as blank, carry out spectral determination at a wavelength range of 250-700 nm by ultraviolet-visible spectrophotometry (Chinese Pharmacopoeia 2020, General Test 0401). Take another 4.0 ml of the test solution, add water to the mark in a 25 ml volumetric flask, shake well, and carry out spectral determination at a wavelength range of 250-700 nm without color development to determine whether the background of the test solution itself interferes with the determination. The results are shown in Figure 12.

[0433] The results show that the test solution and the reference solution have corresponding absorption peaks near 500 nm, and the uncolored test solution blank has almost no absorption at this wavelength, indicating that the background of the uncolored test solution does not interfere with the determination, and the method has strong specificity. Therefore, it is proposed to select the sodium nitrite color development method for color development determination, and 500 nm as the determination wavelength.

[0434] (2) Extraction solvent investigation

[0435] Take 8 portions of 0.2 g of CDD-2101 intermediate powder, precisely weigh, and place in a conical flask with a plug, respectively. Precisely add 25 ml of methanol, 70% methanol, 80% ethanol, and 60% ethanol (each solvent is investigated in parallel for 2 portions), tightly plug, weigh, ultrasonic treat for 30 minutes, take out, cool, re-weigh, make up the weight loss with the corresponding solvent, shake well, centrifuge (4000 r / min) for 10 minutes, and take the supernatant as the test solution.

[0436] Precisely pipette 1 ml of each test solution into a 25 ml volumetric flask, add water to 6.0 ml, add 1 ml of 5% sodium nitrite solution, mix well, stand for 6 minutes, add 1 ml of 10% aluminum nitrate solution, mix well, stand for 6 minutes, add 10 ml of sodium hydroxide test solution, then add water to the mark, shake well, stand for 15 minutes, set up a blank control reagent, immediately determine the absorbance at 500 nm wavelength by ultraviolet-visible spectrophotometry (General Test 0401), calculate the content of rutin in the test solution by the standard curve, and calculate the percentage content of total flavonoids in the test solution as dry product. The results are shown in the following Table 55.

[0437] Table 55 Results of total flavonoid extraction solvent investigation

[0438] The results show that the use of 60% ethanol for extraction yields a higher content of total flavonoids, so 60% ethanol is selected as the extraction solvent.

[0439] (3) Extraction method investigation

[0440] Take 4 portions of 0.2 g of CDD-2101 intermediate powder, precisely weigh, and put into conical bottles with stoppers. Precisely add 25 ml of 60% ethanol, tightly seal, weigh, and separately perform ultrasonic treatment and heating reflux for 30 minutes (each factor is investigated in parallel for 2 portions). Take out, cool, re-weigh, make up the weight loss with 60% ethanol, shake well, centrifuge (4000 r / min) for 10 minutes, and take the supernatant as the test sample solution.

[0441] Precisely take 1 ml of each test sample solution, and put into a 25 ml volumetric flask. Add water to 6.0 ml, add 1 ml of 5% sodium nitrite solution, mix well, stand for 6 minutes, add 1 ml of 10% aluminum nitrate solution, mix well, stand for 6 minutes, add 10 ml of sodium hydroxide test solution, and then add water to the calibration mark. Shake well, stand for 15 minutes, set up a blank control reagent, and immediately determine the absorbance at 500 nm wavelength by ultraviolet-visible spectrophotometry (general rule 0401). Calculate the content of rutin in the test sample solution by the standard curve, and calculate the percentage content of total flavonoids in the test sample as dry product. The results are shown in Table 56 below.

[0442] Table 56 Investigation results of total flavonoids extraction method

[0443] The results show that there is no significant difference in the total flavonoid content measured by the two different extraction methods. Therefore, the ultrasonic treatment method which is relatively simple is selected for extraction.

[0444] Comparative Example 1

[0445] This comparative example is used to investigate different detection methods of flavonoid compounds.

[0446] The aluminum trichloride colorimetric method is a commonly used method for detecting flavonoid compounds. This comparative example compares the sodium nitrite colorimetric method and the aluminum trichloride colorimetric method. The results show that the aluminum trichloride colorimetric method has poor specificity and strong background interference, while the sodium nitrite colorimetric method has strong specificity and the background body of the test sample without coloration does not interfere with the determination. Therefore, the sodium nitrite colorimetric method is selected for colorimetric determination.

[0447] The results measured by the sodium nitrite colorimetric method are shown in Example 1, and the results measured by the aluminum trichloride colorimetric method are shown below:

[0448] 1. Preparation of reference solution:

[0449] Take an appropriate amount of rutin reference substance, precisely weigh, and add 60% ethanol to prepare a solution containing 0.4342 mg per 1 ml. Thus, the reference solution is obtained.

[0450] Take an appropriate amount of naringin reference substance, accurately weigh it, and add methanol to make a solution containing 0.3811 mg per 1 ml.

[0451] 2. Preparation of test solution:

[0452] Take 0.5 g of CDD-2101 intermediate powder, accurately weigh it, accurately add 100 ml of 60% ethanol, weigh it, ultrasonicate it for 30 minutes, remove it, let it cool, reweight it, shake it well, centrifuge it (4000 r / min) for 10 minutes, and take the supernatant to obtain it;

[0453] 3. Determination method:

[0454] Accurately measure 0.5 ml of each reference solution and 1.0 ml of the test solution, place them in 10 ml volumetric flasks, add 2 ml of 0.1 mol / L aluminum chloride solution and 3 ml of 1 mol / L potassium acetate solution, then dilute to the mark with 60% ethanol, shake well, and let it sit for 30 minutes. Use the corresponding reagent as a blank and perform spectral measurement in the wavelength range of 250-600 nm according to the UV-visible spectrophotometry method (General Rules 0401 of Part Four of the 2020 Edition of the Chinese Pharmacopoeia). Take another 1.0 ml of the test solution, place it in a 10 ml volumetric flask, dilute to the mark with 60% ethanol, shake well, and perform spectral measurement in the wavelength range of 250-600 nm without aluminum chloride color development to determine whether the background of the test sample itself interferes with the measurement. The results are shown in Figure 13.

[0455] The results showed that the rutin reference had absorption peaks at wavelengths of 273nm and 420nm, respectively, and the naringin reference had a maximum absorption peak at a wavelength of 284nm. The test sample only had an absorption peak near the maximum absorption wavelength of naringin at 284nm, but the uncolored test sample blank also had an absorption peak near this wavelength, indicating that the uncolored background of the test sample itself would interfere with its determination, indicating that the method has poor specificity and is not feasible.

[0456] Comparative Example 2

[0457] This comparative example is used to investigate different detection methods for the content of phenolic compounds.

[0458] The phosphomolybdotungstic acid colorimetric method is a common method for detecting phenolic compounds. This comparative example compared the ferric chloride-potassium ferrocyanide colorimetric method with the phosphomolybdotungstic acid colorimetric method. The results showed that the phosphomolybdotungstic acid colorimetric method is susceptible to interference from other components, while the ferric chloride-potassium ferrocyanide colorimetric method is highly specific and does not interfere with the uncolored background of the test sample. Therefore, the ferric chloride-potassium ferrocyanide colorimetric method was selected for colorimetric determination.

[0459] The results of the ferric chloride-potassium ferrocyanide colorimetric method are shown in Example 2, and the results of the phosphomolybdic acid colorimetric method are shown below:

[0460] 1. Preparation of reference solution:

[0461] Take a certain amount of gallic acid reference substance, accurately weigh, add water to prepare a solution containing 37.15 μg per 1 ml, and obtain it.

[0462] 2. Preparation of test solution:

[0463] Test sample 1: Take 0.5 g of CDD-2101 intermediate powder, accurately weigh, accurately add 50 ml of 60% ethanol, weigh, heat reflux for 30 minutes, take out, cool, supplement weight, shake well, filter, and take the filtrate, and obtain it.

[0464] Test sample 2: Take 1.0 g of CDD-2101 intermediate powder, accurately weigh, accurately add 100 ml of water, weigh, ultrasonic treatment for 1 hour, take out, cool, supplement weight, shake well, centrifuge (4000 r / min) for 10 minutes, take the supernatant, and obtain it.

[0465] 3. Determination method:

[0466] Accurately take 2.0 ml of the reference solution and the test solution respectively, and place them in 25 ml volumetric flasks. Add 1.0 ml of phosphomolybdotungstic acid test solution, and then add 10 ml of water. Dilute to the mark with 29% sodium carbonate solution, shake well, and stand for 30 minutes. Use the corresponding reagent as the blank. Perform spectral determination in the wavelength range of 250-800 nm according to the ultraviolet-visible spectrophotometry (Chinese Pharmacopoeia 2020 edition, General Test 0401). The results are shown in Figures 14 and 15.

[0467] It can be seen from the results that the maximum absorption wavelength of the gallic acid reference substance is about 750 nm, and the maximum absorption wavelength of the test sample is about 650 nm. The difference between the two maximum absorption wavelengths is large, which may be caused by the interference of other components. Therefore, it is considered to replace the color developing method.

[0468] Comparative Example 3

[0469] This comparative example is used to investigate different detection methods of total sugar content.

[0470] The commonly used methods for determining the total sugar content in Chinese Pharmacopoeia 2020 edition are anthrone colorimetry and 3,5-dinitrosalicylic acid color developing method. The results of the two methods are compared in this application. The results show that the absorption peak of anthrone colorimetry is not obvious enough, while the absorption peak of the reference substance and the test sample in 3,5-dinitrosalicylic acid color developing method is more obvious, and the method is more reliable. Therefore, it is proposed to select 3,5-dinitrosalicylic acid color developing method for color developing determination.

[0471] The results measured by 3,5-dinitrosalicylic acid color developing method are shown in Example 6, and the results measured by anthrone colorimetry are shown below:

[0472] Preparation of reference solution

[0473] Take the D-anhydrous glucose reference substance, accurately weigh, add water to make a solution containing 0.062 mg per 1 ml, and obtain it.

[0474] Preparation of test solution

[0475] Test solution 1: Take 1 g of CDD-2101 intermediate powder, accurately weigh, accurately add 100 ml of water, weigh, heat to reflux for 60 minutes, take out, cool, make up the weight, shake well, filter, accurately take 50 ml of the filtrate, add chloroform and shake to extract 4 times, 30 ml each time, discard the chloroform liquid, accurately take 1 ml of the upper layer solution, evaporate to dryness, add water to dissolve and dilute to 100 ml in a volumetric flask.

[0476] Test solution 2: Take 1 g of CDD-2101 intermediate powder, accurately weigh, accurately add 100 ml of water, weigh, heat to reflux for 60 minutes, take out, cool, make up the weight, shake well, filter, accurately take 50 ml of the filtrate, add chloroform and shake to extract 4 times, 30 ml each time, discard the chloroform liquid, accurately take 1 ml of the upper layer solution, evaporate to dryness, add water to dissolve and dilute to 15 ml in a volumetric flask.

[0477] Determination method: accurately take 1 ml of test solution 2, 1 ml of reference solution, and 2 ml of test solution 1, respectively, in a stoppered test tube, add 4.0 ml of 0.2% anthrone-sulfuric acid solution, mix well, quickly place in an ice water bath to cool, then place in a boiling water bath to heat for 10 minutes, take out, place in an ice water bath for 5 minutes, and place at room temperature for 10 minutes. Set up a blank control reagent, and perform spectral determination in the wavelength range of 400-700 nm according to the ultraviolet-visible spectrophotometry (Chinese Pharmacopoeia 2020 edition, four general chapters 0401). The results are shown in Figure 16. As can be seen from the results, the D-anhydrous glucose reference substance has a maximum absorption at 580 nm wavelength, and the diluted 15-fold test solution also has an absorption peak at the corresponding wavelength, therefore the absorbance value at 580 nm wavelength of each spectrum is read, the content of the test solution is calculated according to the concentration of the reference substance, and the results are shown in Table 57 below.

[0478] Table 57 Determination results of total sugar content in intermediate of Maziren Pills

[0479] Comparative example 4

[0480] Magnolol and honokiol were used as index components of lignan components, and content determination research was carried out. Five chromatographic conditions were designed based on the commonly used chromatographic detection methods in existing literature.

[0481] The results of chromatographic condition five determination are shown in Example 8, and the results of chromatographic conditions one to four are shown below:

[0482] 1. Preparation of reference solution

[0483] Accurately weigh Magnolol Reference Substance 7.90 mg into a 25 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, and obtain a Magnolol Reference Substance solution with a concentration of 0.3154 mg / ml.

[0484] Accurately weigh Honokiol Reference Substance 8.36 mg into a 25 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, and obtain a Honokiol Reference Substance solution with a concentration of 0.3304 mg / ml.

[0485] 2. Preparation of test solution

[0486] Take 2 g of CDD-2101 intermediate powder, accurately weigh, add 25 ml of methanol, weigh, ultrasonic treat for 30 minutes, remove, cool, supplement weight, filter, and take the filtrate, and obtain the test solution.

[0487] 3. Chromatographic conditions

[0488] Chromatographic condition one:

[0489] Use octadecylsilane-bonded silica gel as the filler, the chromatographic column is Welch Ultimate XB-C18 (4.6 mm x 250 mm, 5 μm), use methanol-water as the mobile phase, perform gradient elution according to the following table, the flow rate is 1.0 ml / min, and the detection wavelength is 294 nm.

[0490] Chromatographic condition two:

[0491] Use octadecylsilane-bonded silica gel as the filler, the chromatographic column is OSAKA SODA (Osaka Soda) CAPCELL PAK C18 AQ (4.6 mm x 250 mm, 5 μm), use methanol-water (volume ratio 74:26) as the mobile phase, the flow rate is 1.0 ml / min, the detection wavelength is 294 nm, and the column temperature is 25°C.

[0492] Chromatographic condition three:

[0493] Use octadecylsilane-bonded silica gel as the filler, the chromatographic column is Waters Xselect HSS T3 (4.6 mm x 250 mm, 5 μm), use methanol-water as the mobile phase, perform gradient elution according to the following table, the flow rate is 1.0 ml / min, the detection wavelength is 294 nm, and the column temperature is 25°C.

[0494] Chromatographic condition four:

[0495] The chromatographic column was Waters XBridge C18 (4.6 mm*250 mm, 5 μm), octadecylsilane-bonded silica gel was used as the filler, methanol-water was used as the mobile phase, gradient elution was carried out according to the following table, the flow rate was 1.0 ml / min, the detection wavelength was 294 nm, and the column temperature was 25 °C.

[0496] Under the above chromatographic conditions, 10 μl of the control solution and the sample solution were respectively injected. The results are shown in Figures 17-20.

[0497] It can be seen that in the chromatograms of each sample, there are chromatographic peaks at positions corresponding to the honokiol and magnolol control substance chromatographic peaks, but in the sample chromatograms determined by chromatographic conditions one, three and four, there are interfering impurity peaks near the target component peaks, and the separation is poor; in the sample chromatogram determined by chromatographic condition two, the baseline is not flat enough. However, in the sample chromatogram determined by chromatographic condition five (i.e. the chromatographic condition of the present application), the sample chromatogram baseline is flat, the main peak has a good peak shape, and the separation effect from the impurity peaks is good.

Claims

1. A mass balance method for a CDD-2101 intermediate, wherein the raw materials of the CDD-2101 intermediate include the following medicinal materials: hemp seed, rhubarb, magnolia bark, white peony root, and stir-fried aurantium immaturum with bran; the mass balance method includes determining the content of one or more of the following components of the CDD-2101 intermediate: total flavonoids, total phenols, total anthraquinones, total triterpenes, total alkaloids, total sugars, total protein, lignans, and total fat.

2. The mass balance method of CDD-2101 intermediate according to claim 1, wherein: The mass balance method includes one or more of the following methods: (1) The total flavonoids in the CDD-2101 intermediate were determined using a sodium nitrite colorimetric method; (2) The total phenol content in the CDD-2101 intermediate was determined using the ferric chloride-potassium ferrocyanide colorimetric method; (3) The total anthraquinone content in the CDD-2101 intermediate was determined using the magnesium acetate ethanol colorimetric method; (4) After purification using macroporous resin, the total triterpenoid content in the CDD-2101 intermediate was determined using the vanillin glacial acetic acid colorimetric method; (5) Determination of the total alkaloid content in the CDD-2101 intermediate using the retinol ammonium salt colorimetric method; (6) Determination of total sugar content in CDD-2101 intermediate using 3,5-dinitrosalicylic acid colorimetric method; (7) The total protein content in the CDD-2101 intermediate was determined using the Coomassie Brilliant Blue method; (8) Determination of the content of lignans in CDD-2101 intermediates by high performance liquid chromatography; (9) The total fat content in the CDD-2101 intermediate was determined by ether Soxhlet extraction followed by evaporation to dryness.

3. The mass balance method of CDD-2101 intermediate according to claim 2, wherein: The method for determining the content of total flavonoids includes the following steps: The extraction solvent is mixed with the CDD-2101 intermediate, subjected to ultrasonic treatment and / or heating under reflux, and the weight loss is compensated with the extraction solvent; the mixture is centrifuged, and the supernatant is sequentially added with a 5 wt % sodium nitrite solution and a 4 wt % sodium hydroxide solution for reaction; a blank control reagent is set up, and the absorbance is measured at a wavelength of 250-700 nm by UV-visible spectrophotometry; Wherein, the extraction solvent is selected from methanol, 70 vol% methanol aqueous solution, 80 vol% ethanol aqueous solution or 60 vol% ethanol aqueous solution.

4. The mass balance method of CDD-2101 intermediate according to claim 1, wherein: The method for determining the content of total phenols comprises the following steps: The extraction solvent and the CDD-2101 intermediate were mixed and ultrasonically treated, and the weight loss was compensated with the extraction solvent; the solution was centrifuged, the supernatant was collected, and the supernatant was diluted with the extraction solvent. 0.3 wt% sodium lauryl sulfate solution, a solution prepared by mixing 0.6 wt% ferric chloride solution and 0.9 wt% potassium ferricyanide solution in a volume ratio of 1:0.9, and 0.1 mol / L hydrochloric acid solution were then added in sequence for reaction; a blank control reagent was simultaneously set up, and the absorbance was measured at a wavelength of 500-850 nm using UV-visible spectrophotometry; Wherein, the extraction solvent is selected from methanol, ethanol, 70 vol% ethanol aqueous solution or 50 vol% ethanol aqueous solution.

5. The mass balance method of CDD-2101 intermediate according to claim 1, wherein: The method for determining the content of total anthraquinones comprises the following steps: The extraction solvent and the CDD-2101 intermediate were mixed and heated under reflux, and the weight loss was compensated with the extraction solvent; after filtering, the filtrate was evaporated to remove the solvent, and the residue was first ultrasonically treated with 8wt% hydrochloric acid solution, then heated under reflux in a water bath with chloroform, and allowed to stand to separate into chloroform layer and acid layer; the chloroform layer was extracted, and the acid layer was shaken with chloroform, and the chloroform solution was combined and evaporated to dryness; the residue was dissolved in methanol, and after color development with 1wt% magnesium acetate ethanol solution, a blank control reagent was set, and the absorbance was measured at a wavelength of 300-700nm according to UV-visible spectrophotometry; Wherein, the extraction solvent is selected from methanol, 80 vol% ethanol aqueous solution or ethanol.

6. The mass balance method of CDD-2101 intermediate according to claim 1, wherein: The method for determining the content of total triterpenes comprises the following steps: The extraction solvent is mixed with the CDD-2101 intermediate, and the mixture is heated under reflux and / or ultrasonically treated, and the weight loss is compensated with the extraction solvent; the mixture is centrifuged, and the solvent is evaporated from the supernatant. The residue is dissolved in water and / or ethanol and then applied to an AB-8 macroporous resin column. The column is eluted with water and 50-80 vol% ethanol, respectively, and the eluent is discarded. The column is then eluted with ethanol, and the eluent is collected and evaporated to dryness. The residue is dissolved in ethanol to obtain a test solution; To the test solution, add freshly prepared 0.05 g / ml vanillin glacial acetic acid solution and perchloric acid, mix well, heat in a 70°C water bath for 15 min, cool in an ice bath, remove from the water, add glacial acetic acid and shake well; set up a blank control reagent, and measure the absorbance at a wavelength of 400-700 nm using UV-visible spectrophotometry; Wherein, the extraction solvent is selected from 80 vol% ethanol aqueous solution, ethanol or methanol.

7. The mass balance method of CDD-2101 intermediate according to claim 1, wherein: The method for determining the content of total alkaloids comprises the following steps: The extraction solvent was mixed with the CDD-2101 intermediate, heated under reflux, and the weight loss was supplemented with the extraction solvent. The filtrate was evaporated to dryness, and the residue was dissolved in 0.1 mol / L hydrochloric acid solution. The supernatant was centrifuged and added with activated carbon and heated in a boiling water bath for decolorization. After filtration, the filter residue was washed with 0.1 mol / L hydrochloric acid and filtered. 2 wt% retinyl ammonium salt solution was added, and then diluted with 0.1 mol / L hydrochloric acid solution. The mixture was placed at 2 ° C for 60-120 minutes, and the filtrate was taken as the test solution. Using 0.1 mol / L hydrochloric acid solution as blank reagent, the absorbance was measured at a wavelength of 400-700 nm according to UV-visible spectrophotometry; The extraction solvent is selected from 1 vol% hydrochloric acid methanol solution, 1 vol% hydrochloric acid 70 vol% methanol aqueous solution, 1 vol% hydrochloric acid ethanol solution or 1 vol% hydrochloric acid 70 vol% ethanol aqueous solution.

8. The mass balance method of CDD-2101 intermediate according to claim 1, wherein: The method for determining the content of total sugars includes the following steps: Water is used as an extraction solvent and mixed with the CDD-2101 intermediate, subjected to ultrasonic treatment and / or heating under reflux, centrifuged, the supernatant is added with 6 mol / L hydrochloric acid solution, heated in a boiling water bath for 30 minutes, cooled, 1 drop of phenolphthalein indicator solution is added, and the mixture is neutralized with 5 mol / L sodium hydroxide solution until slightly reddish, diluted with water and shaken well; the diluted solution is added with 3,5-dinitrosalicylic acid solution and heated in a boiling water bath at 100°C for 5 minutes, then quickly cooled with cold water, and water is added quantitatively to obtain a test solution; a blank solution is prepared in the same manner with water; and the absorbance is measured at wavelengths of 470-700 nm according to UV-visible spectrophotometry.

9. The mass balance method of CDD-2101 intermediate according to claim 1, wherein: The method for determining the total protein content includes the following steps: Water is used as an extraction solvent and mixed with the CDD-2101 intermediate. The intermediate is dissolved by ultrasonic treatment. After cooling, water is added to make up for the lost weight. The supernatant is collected by centrifugation and a Coomassie brilliant blue solution is added to mix and react. A blank control reagent is set, and the absorbance is measured at wavelengths of 250-700 nm according to ultraviolet-visible spectrophotometry.

10. The mass balance method of CDD-2101 intermediate according to claim 1, wherein: The method for determining the content of lignans comprises the following steps: The extraction solvent was mixed with the CDD-2101 intermediate, and ultrasonic treatment was performed. After cooling, the weight loss was supplemented with the extraction solvent. The subsequent filtrate was used as the test solution and injected into a liquid chromatograph to determine the total amount of magnolol and honokiol as the lignan content. wherein the extraction solvent is selected from methanol, 70 vol% methanol aqueous solution, ethanol or 70 vol% ethanol aqueous solution; Chromatographic conditions include: Chromatographic column: Waters XBridge C18 column, with specifications of 4.6 mm × 250 mm × 5 μm; column temperature 25°C; mobile phase: methanol-acetonitrile-water mixed solution with a volume ratio of 50:20:30; detection wavelength: 294 nm.

11. The mass balance method of CDD-2101 intermediate according to claim 1, wherein: The method for determining the total fat content includes the following steps: 1 mol / L hydrochloric acid is used as an extraction solvent and mixed with the CDD-2101 intermediate. After hydrolysis, ether or petroleum ether is added and the mixture is heated to reflux in a water bath. The resulting extract is evaporated to dryness at 60-100°C. After cooling, the mass of the residue is weighed and the total fat content in the CDD-2101 intermediate is calculated.

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