Method for simultaneously detecting the contents of five ingredients in a laxative Chinese medicine

Abstract

The present invention discloses a method for simultaneously detecting the contents of five components of a laxative Chinese medicine, wherein the laxative Chinese medicine is made of five Chinese medicinal materials, namely, Atractylodes macrocephala, Herba Taraxaci, Fructus Aurantii Immaturus, Semen Cassiae, and Apricot Kernel, and the five components are caffeic acid, chicoric acid, Atractylodes lactone I, Auricularia cassiae, and Chrysophanol. The present invention successfully establishes the method by exploring the detection conditions, and can detect the contents of multiple components by one extraction and loading, thus realizing the multi-index quantification of Chinese medicine. Combining with the fingerprint qualitative method established in the early stage, it will be more conducive to establishing a comprehensive, traceable, and highly correlated full-process quality control system for Chinese medicine.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis and relates to a method for simultaneously detecting the contents of five components of a laxative Chinese medicine. Background Art

[0002] Zhishu Runchang Granule is a new Chinese medicine of Class 1 under development by Jianmin Pharmaceutical Group Co., Ltd. This product is made of five Chinese medicinal materials: Atractylodes macrocephala, Taraxacum mongolicum, Citrus aurantium, Cassia seed, and Apricot kernel, and is used to treat constipation caused by heat accumulation in children. CN 109342579A discloses part of the prescription of this product, and also discloses a method for HPLC fingerprint detection thereof.

[0003] HPLC fingerprint can comprehensively reflect the types of chemical components of traditional Chinese medicine, and can realize the comprehensive evaluation and effective control of the intrinsic quality of traditional Chinese medicine. However, the disadvantage of this method is that it can only qualitatively identify the chemical components by the number of peaks to determine the presence or absence of the components, and it is difficult to quantitatively analyze the chemical components to determine the amount of the components. Therefore, HPLC fingerprint still has great limitations in evaluating the quality of medicinal materials and the consistency of various links in the production of traditional Chinese medicine.

[0004] If a method for determining the content of multiple chemical components of traditional Chinese medicine can be established and combined with the traditional Chinese medicine fingerprint, so as to conduct qualitative and quantitative analysis of the traditional Chinese medicine components, it will be more conducive to establishing a comprehensive, traceable, and highly correlated quality control system for traditional Chinese medicine. Summary of the invention

[0005] The purpose of the present invention is to provide a method for simultaneously detecting the contents of five components, namely, caffeic acid, chicoric acid, atractylodes lactone I, chrysophanol and chrysophanol, in view of the defect of a single content determination index of a laxative Chinese medicine, which can be used for all-round control of the quality of Chinese medicine.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for simultaneously detecting the contents of five ingredients of a laxative Chinese medicine, wherein the laxative Chinese medicine is made of five Chinese medicinal materials, namely, Atractylodes macrocephala, Taraxacum officinale, Citrus aurantium, Cassia seed, and Apricot kernel, wherein the five ingredients are caffeic acid, chicoric acid, Atractylodes lactone I, chrysophanol, and chrysophanol. The detection method comprises the following steps:

[0008] (1) After the sample to be tested is ground into powder, it is first ultrasonically extracted with chloroform, then ultrasonically extracted with methanol, the chloroform and methanol extracts are combined, the organic solvent is evaporated and then adsorbed on a macroporous resin column, first eluted with water until the solution is clear, then eluted with ethyl acetate for 3-5 times the column volume, the ethyl acetate eluate is collected, concentrated and evaporated, and then re-dissolved with methanol to prepare the test solution;

[0009] (2) The sample solution is tested on a high performance liquid chromatograph, wherein the high performance liquid chromatograph contains an ultraviolet detector, the detection wavelength of the ultraviolet detector is set to 240-250nm, and the stationary phase of the high performance liquid chromatograph is C 18 Chromatographic column, mobile phase A is 0.05-0.5% phosphoric acid aqueous solution, mobile phase B is acetonitrile containing 0.5-3% isopropanol, gradient elution; flow rate 0.5-2ml / min; column temperature 25-35℃;

[0010] (3) Determine the contents of the five components based on the peak time and peak area.

[0011] Preferably, the procedure of the gradient elution is:

[0012]

[0013] Preferably, the macroporous adsorption resin is of model LSA-5B, with a particle size of 0.8 mm.

[0014] Preferably, the chromatographic column is Shiseido SPOLAP-C18, 4.6×250 mm, 5 μm.

[0015] Preferably, the set detection wavelength is 245 nm.

[0016] Preferably, the mobile phase A is a 0.1% phosphoric acid aqueous solution.

[0017] Preferably, the mobile phase B is acetonitrile containing 1% isopropanol.

[0018] Preferably, the flow rate is 1 ml / min and the column temperature is 30°C.

[0019] Preferably, the traditional Chinese medicine is in the form of granules.

[0020] The percentages involved in the present invention are not volume ratios. For example, 0.1% phosphoric acid aqueous solution means that every 100ml of solution contains 0.1ml of phosphoric acid; acetonitrile containing 1% isopropanol means that every 100ml of acetonitrile contains 1ml of isopropanol.

[0021] The beneficial effects of the present invention are:

[0022] (1) The present invention has successfully established a method for simultaneously detecting the contents of five components of a traditional Chinese medicine by exploring the detection conditions. The traditional Chinese medicine is a laxative medicine composed of five traditional Chinese medicinal materials: Atractylodes macrocephala, Taraxacum officinale, Citrus aurantium, Cassia seed, and Apricot kernel. The five components are caffeic acid, chicoric acid, atractylodes lactone I, chrysophanol, and chrysophanol.

[0023] (2) The content determination method established by the present invention has the advantages of high precision, accuracy, and good repeatability.

[0024] (3) The present invention can detect the content of multiple components through one extraction and sampling, realizing the multi-index quantification of traditional Chinese medicine. Combined with the fingerprint qualitative method established in the early stage, it will be more conducive to establishing a comprehensive, traceable, and highly correlated quality control system for traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Chromatogram of the mixed reference solution tested according to the chromatographic conditions of CN 109342579A.

[0026] Figure 2 : Chromatogram of the mixed reference solution detected under improved chromatographic conditions.

[0027] Figure 3 : Chromatogram of the sample solution detected according to the extraction method of CN 109342579A.

[0028] Figure 4 : Chromatogram of the sample solution detected according to the improved extraction method. DETAILED DESCRIPTION

[0029] The present invention is described in detail below through specific examples.

[0030] Test drug:

[0031] The Zhishu Intestinal Runny Granules used in the following examples were provided by Jianmin Pharmaceutical Group Co., Ltd.

[0032] Prescription: 266.67g of Citrus aurantium; 1000g of Atractylodes macrocephala; 400g of Taraxacum mongolicum; 200g of Bitter Apricot kernel; 266.67g of Cassia seed

[0033] Preparation method: Add water to the above five flavors and boil them for extraction. Concentrate the extract to a relative density of 1.06-1.08, wait until it cools to room temperature, add ethanol for precipitation, take the supernatant after the precipitation and concentrate it under reduced pressure into a thick paste. Add powdered sugar, dextrin, fruit essence and sucralose to the thick paste, mix well, granulate, and make 1000g granules.

[0034] Instruments and reagents:

[0035] Waters e2695 high performance liquid chromatograph series (Waters Technologies, Inc., USA, including quaternary pump, autosampler, column oven, Waters 2489 UV detector, Thermo Fisher Scientific Chromeleon 7 network workstation); AB204-E electronic balance (Shanghai Mettler-Toledo Instrument Co., Ltd.).

[0036] Caffeic acid reference substance (for content determination, purchased by China Food and Drug Inspection Institute, batch number 110885-201703), chicoric acid reference substance (for content determination, purchased by China Food and Drug Inspection Institute, batch number: 111752-201703), atractylodes lactone I reference substance (for content determination, purchased by China Food and Drug Inspection Institute, batch number: 111975-201501), aurantium dulcis reference substance (for content determination, purchased by China Food and Drug Inspection Institute, batch number: 111900-202006), chrysophanol reference substance (for content determination, purchased by China Food and Drug Inspection Institute, batch number: 110796-201922).

[0037] Acetonitrile and methanol were of chromatography grade, phosphoric acid, isopropanol, chloroform, and ethyl acetate were of analytical grade, and water was ultrapure water.

[0038] Example 1 Exploration of Chromatographic Conditions

[0039] (1) Preparation of mixed reference solution: Accurately weigh appropriate amounts of caffeic acid, chicoric acid, atractylodes lactone I, nerolidinone, and chrysophanol standards to prepare a mixed solution containing 20ug, 50ug, 10ug, 30ug, and 15ug of caffeic acid, chicoric acid, atractylodes lactone I, nerolidinone, and chrysophanol per 1ml, respectively.

[0040] (2) The mixed reference solution was tested according to the chromatographic conditions of CN 109342579A. Chromatographic column: Agilent ZORBAX SB-C 18 , 4.6×250mm, 5μm; detection wavelength: 280nm; injection volume 10μl; mobile phase A is acetonitrile, mobile phase B is buffer salt (0.8g potassium dihydrogen phosphate, 1.0g sodium dodecyl sulfate, 1ml glacial acetic acid, dissolved in water and diluted to 1000ml, pH 3.0), gradient elution; flow rate: 1ml / min; column temperature: 30℃. Gradient elution program:

[0041] Elution time Buffer salt phase Acetonitrile phase 0~7min 92％ 8％ 7~20min 92→80％ 8→20％ 20~36min 80％ 20％ 36~55min 80→52％ 20→48％ 55~70min 52→38％ 48→62％

[0042] The chromatogram of the mixed reference solution obtained is shown in Figure 1 .

[0043] from Figure 1 It can be seen from the results that the mixed reference solution has peaks at around 57min and 67min, which correspond to chrysophanol and chrysophanol respectively after comparison with the single reference chromatograms. Caffeic acid, chicoric acid and atractylodes lactone I in the mixed reference solution have no peaks, indicating that the mobile phase of CN 109342579A cannot achieve simultaneous detection of five detection components.

[0044] (3) The improved chromatographic conditions are as follows: Chromatographic column: Shiseido SPOLAP-C18 , 4.6×250mm, 5μm; detection wavelength: 245nm; injection volume 10μl; mobile phase A is 0.1% phosphoric acid aqueous solution, mobile phase B is acetonitrile containing 1% isopropanol, gradient elution; flow rate: 1ml / min; column temperature: 30℃. Gradient elution program:

[0045] Elution time Mobile phase A Mobile phase B 0~5min 85→80％ 15→20％ 5~15min 80→78％ 20→22％ 15~30min 78→62％ 22→38％ 30~38min 62％ 38％ 38~50min 62→45％ 38→55％ 50~55min 45→15％ 55→85％ 55~75min 15→10％ 85→90％

[0046] The chromatogram of the mixed reference solution obtained is shown in Figure 2 .

[0047] from Figure 2 It can be seen from the results that the mixed reference solution has peaks at around 12min, 28min, 52min, 56min and 69min. After comparison with the chromatograms of single reference products, they correspond to caffeic acid, chicoric acid, atractylodes lactone I, chrysophanol and chrysophanol, respectively. All five components in the mixed reference solution were detected.

[0048] Example 2 Sample Preparation Method Exploration

[0049] On the basis of determining the chromatographic conditions, the present invention further explores the sample preparation method.

[0050] (1) Prepare the test sample solution according to the method of CN 109342579A: grind the Zhishu Runchang granules to be tested, take 1 g, weigh accurately, add 50 ml of 50% methanol accurately, weigh the weight, ultrasonicate for 40 minutes (power 250 W, frequency 40 kHz), let cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, accurately measure 20 ml of the filtrate, evaporate to dryness, add 25 ml of water to dissolve the residue, pass it through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm, dry column), elute with 25 ml of water, collect the eluate, transfer it to a 25 ml volumetric flask, add water to the scale, shake well, and obtain.

[0051] The above test solution was tested according to the improved chromatographic conditions of Example 1. The results are shown in Figure 3 .

[0052] from Figure 3 It can be seen from the results that the sample solution has peaks at around 12min, 28min, 52min and 56min, corresponding to caffeic acid, chicoric acid, atractylodes lactone I and chrysanthemum oleoresin, respectively, while chrysophanol (69min) fails to peak and the peak shape of atractylodes lactone I (52min) is very poor.

[0053] (2) Improved sample solution preparation method: Take the product under the item of loading difference, mix well, take an appropriate amount, grind finely, take about 1.5g, accurately weigh, place in a 50ml stoppered conical flask, accurately add 25ml chloroform, stopper, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, add 25ml 50% methanol to the residue, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, combine the two parts of the filtrate, evaporate the organic solvent by rotary evaporation, and then apply it to a macroporous adsorption resin column (resin model LSA-5B, particle size 0.8mm), first elute with water to remove impurities, and after the solution is clarified, elute with ethyl acetate for 5 times the column volume, collect the ethyl acetate eluate, concentrate and evaporate to dryness, and then make up to 50ml with 50% methanol.

[0054] The above test solution was tested according to the improved chromatographic conditions of Example 1. The results are shown in Figure 4 .

[0055] from Figure 4 From the results, we can see that the peaks appear around 12min, 28min, 52min, 56min, and 69min, which correspond to caffeic acid, chicoric acid, atractylodes lactone I, chrysophanol, and chrysophanol, respectively. All five components can be detected. Figure 3 and Figure 4 It can also be seen from the comparison that Figure 4 The peak area of ​​aurantin increased and the peak shape of atractylodes lactone Ⅰ improved.

[0056] The specific HPLC chromatographic data of the two sample extraction methods are as follows:

[0057]

[0058] Example 3 Methodological Investigation and Verification

[0059] 1. Test methods

[0060] Preparation of test solution: Take the product under the item of filling amount difference, mix well, take appropriate amount, grind finely, take about 1.5g, accurately weigh, put it in a 50ml stoppered conical flask, accurately add 25ml chloroform, seal it, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, add 25ml 50% methanol to the residue, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, combine the two parts of the filtrate, evaporate the organic solvent by rotary evaporation, and then apply it to a macroporous adsorption resin column (resin model LSA-5B, particle size 0.8mm), first elute with water to remove impurities, and after the solution is clarified, elute with ethyl acetate for 5 times the column volume, collect the ethyl acetate eluate, concentrate and evaporate to dryness, and then make up to 50ml with 50% methanol.

[0061] Preparation of reference solution: Accurately weigh appropriate amounts of caffeic acid, chicoric acid, atractylodes lactone I, nerolidinone, and chrysophanol standards to prepare mixed reference solutions containing 20ug, 50ug, 10ug, 30ug, and 15ug of caffeic acid, chicoric acid, atractylodes lactone I, nerolidinone, and chrysophanol per 1ml, respectively.

[0062] Instrument: Waters e2695 high performance liquid chromatograph series (Waters Technology Co., Ltd., USA, including quaternary pump, autosampler, column oven, Waters 2489 UV detector, Thermo Fisher Scientific Chromeleon 7 network workstation)

[0063] Chromatographic column: Shiseido SPOLAP-C 18 , 4.6×250mm, 5μm

[0064] Mobile phase: 0.1% phosphoric acid aqueous solution as mobile phase A: acetonitrile (containing 1% isopropanol) as mobile phase B;

[0065] Gradient elution program:

[0066] Elution time Mobile phase A Mobile phase B 0~5min 85→80％ 15→20％ 5~15min 80→78％ 20→22％ 15~30min 78→62％ 22→38％ 30~38min 62％ 38％ 38~50min 62→45％ 38→55％ 50~55min 45→15％ 55→85％ 55~75min 15→10％ 85→90％

[0067] Column temperature: 30°C

[0068] Detection wavelength: 245nm

[0069] Injection volume: 10ul

[0070] Flow rate: 1ml / min

[0071] Calculation method:

[0072]

[0073] C 对 is the concentration of the reference solution (mg / ml)

[0074] A 对 is the peak area of ​​the reference solution

[0075] A 样 is the sample peak area

[0076] W 样 The sample weight (g)

[0077] 2. Test results

[0078] (1) Linear

[0079] Weigh 10.11 mg of caffeic acid, 25.08 mg of chicoric acid, 5.14 mg of atractylodes lactone I, 15.02 mg of citrus cassiae, and 7.51 mg of chrysophanol standard respectively, place them in a 100 ml volumetric flask, add 50% methanol to dissolve and dilute to the mark, shake well. Accurately pipette 0.5, 1.0, 2.0, 3.0, 4.0, and 10.0 ml of the above reference solution respectively, place them in a 10 ml volumetric flask, dilute to the mark with water, shake well, and measure according to the above chromatographic conditions. The results are shown in the table below. Linear regression is performed on the injection volume (X: ug) with the peak area integral value (Y: Mau*min) to obtain the corresponding regression equation. The value of the linear regression coefficient r should be no less than 0.9990. The results are shown in Table 1.

[0080] Table 1 Standard curves and correlation coefficients of five substances

[0081]

[0082]

[0083] (2) Repeatability

[0084] Six sample solutions of the same concentration were prepared and tested. One injection was made for each solution. The relative standard deviation of the six content determination results was required to be no greater than 2.0%. The results are shown in Table 2.

[0085] Table 2 Repeatability test results of five substances content determination

[0086] sample Caffeic acid Chicoric acid Atractylodes lactone Ⅰ Cassia aurantium Chrysophanol RSD(%) 1.34 1.17 1.95 1.06 1.92

[0087] The results showed that the RSD values ​​of each component were within the required range, indicating that the repeatability of the detection method was good.

[0088] (3) Accuracy

[0089] Take Zhishu Runchang Granules (batch 220701) with known content. The content is based on the repeatability result. Take about 0.75g, a total of 6 portions, accurately weigh, and place in stoppered conical bottles respectively. Accurately add 50ml of reference solution with the same content as the test sample, weigh the weight, place in a 50ml stoppered conical bottle, accurately add 25ml of chloroform, seal, and ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, shake, filter, and add 25ml of the residue 50% methanol, ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, cool, shake, filter, combine the two parts of the filtrate, rotary evaporate the organic solvent and then apply it to a macroporous adsorption resin column (resin model LSA-5B, particle size 0.8mm), first wash with water to remove impurities, and then elute with ethyl acetate for 5 times the column volume after the solution is clarified, collect the ethyl acetate eluate, concentrate and evaporate, and then use 50% methanol to make up to 50ml to obtain the test solution. By adding known amounts of each component, and then determining the ratio of the measured results of the known components in the added sample to the theoretical value, the recovery rate is calculated, expressed as a percentage, and the recovery rate is required to be between 95.0% and 105.0%. The results are shown in Table 3.

[0090] Table 3 Recovery results of five substances

[0091] sample Caffeic acid Chicoric acid Atractylodes lactone Ⅰ Cassia aurantium Chrysophanol Average recovery rate (%) 97.84 99.36 96.87 99.76 100.15

[0092] The results showed that this method had good accuracy in determining relevant substances.

[0093] (4) Detection limit

[0094] Weigh 10.04 mg of caffeic acid, 12.25 mg of chicoric acid, 10.08 mg of atractylodes lactone I, 16.02 mg of chrysophanol standard, and place them in a 500 ml volumetric flask. Add 50% methanol to dissolve and dilute to the mark, and shake well. Accurately pipette 1.0, 2.0, 1.0, 1.0, and 1.0 ml of the above reference solution, place them in a 1000 ml volumetric flask, add 50% methanol to the mark, shake well, and measure according to the above chromatographic conditions. If the peak height is 3 times higher than the baseline noise, the solution is the detection limit of the substance. The results are shown in the table below.

[0095] Table 4 Detection limit determination results of five substances

[0096]

[0097] The results showed that the minimum detection concentration of each component was 0.020 μg / ml, and the S / N value of each component peak in the detection limit solution was greater than 3, which met the requirements for content determination.

[0098] (5) Sample measurement

[0099] Take 6 batches of Xiaoer Zhishu Tongbian Granules to prepare the test solution, accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph, and determine and calculate the content. The results are shown in the table below.

[0100] Table 5 Content of the components to be tested in 6 batches of samples

[0101] batch Caffeic acid Chicoric acid Atractylodes lactone Ⅰ Cassia aurantium Chrysophanol 220501 0.0127％ 0.0375％ 0.0063％ 0.0083％ 0.0166％ 220502 0.0122％ 0.0370％ 0.0060％ 0.0080％ 0.0167％ 220701 0.0124％ 0.0372％ 0.0061％ 0.0078％ 0.0165％ 220801 0.0131％ 0.0367％ 0.0062％ 0.0079％ 0.0163％ 220802 0.0124％ 0.0372％ 0.0064％ 0.0082％ 0.0162％ 220803 0.0127％ 0.0373％ 0.0061％ 0.0080％ 0.0164％ .

Claims

1. A method for simultaneously detecting the contents of five ingredients of a laxative Chinese medicine, wherein the laxative Chinese medicine is made of five Chinese medicinal materials: Atractylodes macrocephala, Taraxacum officinale, Citrus aurantium, Cassia seed, and Apricot kernel, and characterized in that: The five ingredients are caffeic acid, chicoric acid, atractylodes lactone I, chrysophanol and chrysophanol. The method comprises the following steps: (1) After the sample to be tested is finely ground, it is first ultrasonically extracted with chloroform, then ultrasonically extracted with methanol, the chloroform and methanol extracts are combined, the organic solvent is evaporated and then adsorbed on a macroporous resin column, first eluted with water until the solution is clear, then eluted with ethyl acetate for 3-5 times the column volume, the ethyl acetate eluate is collected, concentrated and evaporated, and then re-dissolved with methanol to prepare the test solution; (2) The sample solution is tested on a high performance liquid chromatograph, wherein the high performance liquid chromatograph contains an ultraviolet detector, the detection wavelength of the ultraviolet detector is set to 240-250nm, and the stationary phase of the high performance liquid chromatograph is C 18 Chromatographic column, mobile phase A is 0.1% phosphoric acid aqueous solution, mobile phase B is acetonitrile containing 1% isopropanol, gradient elution; flow rate 0.5-2ml / min; column temperature 25-35℃; (3) Determine the contents of the five components according to the peak time and peak area. The procedure of the gradient elution is:

2. The method according to claim 1, characterized in that: The model of the macroporous resin column is LSA-5B, and the particle size is 0.8 mm.

3. The method according to claim 1, characterized in that: The chromatographic column is Shiseido SPOLAP-C18, 4.6×250 mm, 5 μm.

4. The method according to claim 1, characterized in that: The detection wavelength is set to 245 nm.

5. The method according to claim 1, characterized in that: The flow rate was 1 ml / min and the column temperature was 30°C.

6. The method according to claim 1, characterized in that: The traditional Chinese medicine is in the form of granules.

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