Content detection method for six chemical components in standard Jichuan decoction

Abstract

The invention discloses a method for detecting the content of six chemical components in standard Jichuan decoction. The method comprises the following steps: step 1, preparing a reference substance solution; step 2, preparing a test solution; 3, precisely sucking the reference substance solution and the test solution respectively, and injecting into a liquid chromatograph. The detection method established by the invention has the advantages of rapid and accurate analysis, no interference of negative samples, strong specificity, good reproducibility and stability, good peak pattern of the chromatographic peak of the obtained chromatogram, and objective evaluation of the quality of Jichuan decoction.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and in particular to a method for detecting the contents of six chemical components in a standard decoction of Jichuan Jian, which can be used for detecting the contents of six chemical components in a standard decoction of Jichuan Jian and preparations. Background Art

[0002] Classic prescriptions refer to prescriptions recorded in medical books from the Qing Dynasty and before that are currently widely used, have definite efficacy, and have obvious characteristics and advantages.

[0003] Jichuan Decoction is a commonly used prescription for treating Yang deficiency constipation by warming Yang method, which comes from Jingyue Complete Works written by Zhang Jiebin, a famous physician in the Ming Dynasty. The book states: "For any disease involving deficiency and constipation, nitrate and yellow attacking agents must not be used. If the patient has to be relieved, such as typhoid fever and other diseases, but it is Yangming real heat that can be attacked, it is appropriate to use the heat-retention method to relieve it. If the patient's vital energy is already weak, it cannot be purged and the lower part of the body is swollen and blocked, and it is not appropriate to relieve it slowly, only Jichuan Decoction can be used as the main treatment, and there will be no problem."

[0004] "Ancient classic Chinese medicine compound preparations" is a new category in the registration classification of new Chinese medicines. Due to the implementation of simplified registration, targeted technical requirements are stipulated for the research and development of this type of new drugs. Different from the preclinical research-clinical research and "two reports and two batches" requirements and the research and development registration model followed by the research and development of new Chinese medicines in the past, the core of classic Chinese medicine new drugs is "consistency", that is, the preparation method of the classic Chinese medicine reference sample should be consistent with the records of ancient medical books, and the quality of the commercial-scale production preparation should be consistent with the quality of the reference sample. Starting from the "consistency" requirement, it must involve many technical requirements and evaluation methods, such as how to determine the evaluation indicators of consistency? How to evaluate consistency? How to establish a consistent quality control system? Therefore, in order to ensure "inheriting the essence, keeping the integrity and innovating", it is particularly important to establish a series of common key technologies on the premise of meeting the requirements of registration technical regulations. Summary of the invention

[0005] In order to solve the above-mentioned technical problems existing in the prior art, this patent establishes a method for simultaneously determining the content of 6 chemical components in the standard decoction of Jichuan Jian. This detection method can objectively, comprehensively and accurately detect the contents of verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone and neohesperidin, and provide an evaluation method for the quality control of the standard decoction of Jichuan Jian and its preparations.

[0006] The technical solution of the present invention is:

[0007] A method for detecting six chemical components in a standard decoction of Jichuanjian, the method comprising the following steps:

[0008] S1. Prepare mixed reference solution:

[0009] Take verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone, and neohesperidin reference substances, dissolve them in 50% methanol, and prepare a mixed reference substance solution;

[0010] S2. Prepare the test solution:

[0011] Accurately measure the standard decoction to be tested, add water to dissolve, shake well, pass through a polyamide column, and then elute with water and methanol. Collect the methanol eluate, add water to make up the volume, shake well, filter through a microporous filter membrane, and take the filtrate to obtain;

[0012] S3. High performance liquid chromatography detection:

[0013] Accurately pipette the reference solution and the test solution respectively and inject them into the liquid chromatograph;

[0014] The chromatographic conditions are:

[0015] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0016] Mobile phase: Mobile phase A is methanol, mobile phase B is 0.1% formic acid solution;

[0017] Elution conditions: isocratic elution;

[0018] Detection wavelength: 250nm;

[0019] Column temperature: 30°C;

[0020] Flow rate: 0.5-1.0mL / min;

[0021] Injection volume: 10-20uL;

[0022] The theoretical plate number calculated based on the verbascoside peak should not be less than 3000.

[0023] Furthermore, the linear concentration ranges of verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone, and neohesperidin in the reference solution of S1 are 5.3 μg / ml to 132.5 μg / ml, 4.08 μg / ml to 102 μg / ml, 4.4 μg / ml to 110 μg / ml, 30.4 μg / ml to 760 μg / ml, 0.832 μg / ml to 20.8 μg / ml, and 25 μg / ml to 625 μg / ml.

[0024] Furthermore, the concentrations of verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone, and neohesperidin in the reference solution of S1 are 30 ug / mL, 10 ug / mL, 10 ug / mL, 100 ug / mL, 20 ug / mL, and 80 ug / mL, respectively.

[0025] Furthermore, in S2, 5 ml of the standard decoction is taken, 15 ml of water is added to dissolve, the mixture is shaken well, and passed through a 10 g polyamide column, and the column is then washed with 30 ml of water, and then eluted with 50 ml of methanol.

[0026] Furthermore, the volume adjustment in S2 is to add water to the eluent to adjust the volume to 50 ml.

[0027] Furthermore, the chromatographic column in S3 is a YMC-Pack ODS-A column.

[0028] Further, the elution condition in S3 is isocratic elution with a volume ratio of methanol: 0.1% formic acid of 38:62.

[0029] Furthermore, the flow rate in S3 is 0.7 mL / min.

[0030] Beneficial Effects

[0031] The sample detection method established by the present invention has the advantages of fast and accurate analysis, no interference with negative samples, strong specificity, and can realize simultaneous monitoring of the contents of six chemical components from five medicinal materials in the Jichuan Decoction prescription, with good reproducibility and stability. The obtained chromatogram has a better peak shape, and can more effectively control the quality of Jichuan Decoction and its preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the chromatogram of mixed reference;

[0033] Figure 2 is the chromatogram of the test product;

[0034] Figure 3 is the negative sample chromatogram;

[0035] Peaks 1-6 marked in the figure are: verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone, and neohesperidin, respectively. DETAILED DESCRIPTION

[0036] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0037] The embodiments of the present invention are described in detail below with reference to the examples. If no specific conditions are specified in the examples, the reaction is carried out according to conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0038] The instruments and reagents used in the embodiments are as follows:

[0039] Instruments: High performance liquid chromatograph from Shimadzu Corporation, Japan; EL104 electronic analytical balance (Mettler Toledo).

[0040] Drug testing:

[0041] Standard decoction of Jichuanjian: According to the information published in the Key Information Table of Ancient Classic Prescriptions (25 Prescriptions) (Draft for Comments) 2022, "Angelica sinensis 14.92g, Achyranthes bidentata 7.46g, Cistanche deserticola 9.33g, Alismatis rhizome 5.60g, Cimicifuga heracleifolia 2.61g, Fructus Aurantii Immaturus 3.73g, the above medicines are decocted with 300ml of water until about 150ml is decocted". The decoction method is as follows: put the above-mentioned medicinal materials (daily amount) into a medicine pot, add 300ml of water, stir well, make it all soaked, and soak for 20 minutes; heat to boil over high heat, boil over low heat for 30 minutes, pour out about 150ml of the medicinal liquid (pass through a 300-mesh filter cloth), and concentrate the medicinal liquid under reduced pressure to contain 1.0g / mL of the crude drug, which is the standard decoction.

[0042] Verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone, and neohesperidin reference substances were purchased from the China Food and Drug Administration;

[0043] Methanol (analytical grade); formic acid (analytical grade); ultrapure water.

[0044] Embodiment 1 A method for detecting the contents of six chemical components in a standard decoction of Jichuanjian comprises the following steps:

[0045] Step 1: Preparation of reference solution:

[0046] Accurately weigh verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone, and neohesperidin reference substances, place them in a volumetric flask, and add 50% methanol to make reference substance solutions with concentrations of 30ug / mL, 10ug / mL, 10ug / mL, 100ug / mL, 20ug / mL, and 80ug / mL, respectively.

[0047] Step 2: Preparation of test solution:

[0048] Accurately measure 5 ml of the standard decoction, add 15 ml of water to dissolve, shake well, pass through a 10 g polyamide column, then wash the column with 30 ml of water, and then elute with 50 ml of methanol. Collect the eluate before the color band and make up to 50 ml with water, shake well, filter, and take the filtrate as the test solution.

[0049] Step 3, respectively, accurately aspirate 20 μl of the reference solution and the test solution, inject into the liquid chromatograph for determination, and calculate the contents of verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone, and neohesperidin in the test sample by the external standard method.

[0050] The chromatographic conditions were as follows: using octadecyl bonded silica gel as the filler, specifically a YMC-Pack ODS-A column (250×4.6 mm, 5 μm);

[0051] Mobile phase: methanol: 0.1% formic acid (volume ratio 38:62),

[0052] Detection wavelength 250nm,

[0053] Flow rate 0.7ml / min,

[0054] Column temperature: 30℃.

[0055] The number of theoretical plates calculated based on verbascoside should not be less than 3000.

[0056] See the chromatogram of the mixed reference substance for Figure 1 .

[0057] See the chromatogram of the test sample Figure 2 .

[0058] Peaks 1-6 marked in the figure are: verbascoside (peak 1, 12.029 min), ferulic acid (peak 2, 13.366 min), isoferulic acid (peak 3, 15.450 min), naringin (peak 4, 19.524 min), β-ecdysterone (peak 4, 23.637 min), and neohesperidin (peak 5, 15.299 min).

[0059] Example 2 Methodological Investigation:

[0060] 1. Specificity test

[0061] Prepare reference solution and test solution according to the method in Example 1.

[0062] For negative samples, a negative standard decoction lacking medicinal flavor was prepared according to the prescription ingredients and preparation process.

[0063] The negative sample solution, the reference solution and the test solution were respectively taken and tested according to the test conditions of Example 1. The results showed that: at the corresponding retention time of the test component, no corresponding chromatographic peak appeared in the negative sample, the negative sample did not interfere with the determination, and the separation degree R of each test component in the test sample was greater than 1.5. Figure 3 .

[0064] After comparison with negative samples, peak attribution was performed. Verbascoside was derived from Cistanche deserticola; ferulic acid was derived from Angelica sinensis and Cimicifuga heracleifolia; isoferulic acid was derived from Cimicifuga heracleifolia; naringin and neohesperidin were derived from Fructus Aurantii Immaturus, and β-ecdysterone was derived from Achyranthes bidentata.

[0065] 2. Linear relationship

[0066] Accurately weigh verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone and neohesperidin reference substances, place in a volumetric flask, and add 50% methanol to prepare a series of reference substance solutions with concentrations of 5.3μg / ml~132.5μg / ml, 4.08μg / ml~102μg / ml, 4.4μg / ml~110μg / ml, 30.4μg / ml~760μg / ml, 0.832μg / ml~20.8μg / ml, and 25μg / ml~625μg / ml, respectively.

[0067] Accurately pipette the above series of reference substance solutions into liquid chromatograph for determination, and make a linear equation with peak response A as ordinate and reference substance concentration as abscissa.

[0068] Table 1 Experimental results of standard curve of verbascoside (n=3)

[0069]

[0070] The concentration of verbascoside showed a good linear relationship in the range of 5.3 μg / ml to 132.5 μg / ml, Y=19265.4043x+7912.3436, R=0.9999.

[0071] Table 2 Ferulic acid standard curve experimental results (n = 3)

[0072]

[0073] The concentration of ferulic acid showed a good linear relationship in the range of 4.08 μg / ml to 102 μg / ml, Y=68007.5121x+17929.8169, R=0.9999.

[0074] Table 3 Experimental results of isoferulic acid standard curve (n=3)

[0075]

[0076]

[0077] The concentration of isoferulic acid showed a good linear relationship in the range of 4.4 μg / ml to 110 μg / ml, Y=68797.3126x+16691.2263, R=0.9999.

[0078] Table 4 Naringin standard curve experimental results (n = 3)

[0079]

[0080] The concentration of naringin showed a good linear relationship in the range of 30.4 μg / ml to 760 μg / ml, Y=7347.2900x+8691.1811, R=0.9999.

[0081] Table 5 β-ecdysterone standard curve experimental results (n=3)

[0082]

[0083] The concentration of β-ecdysterone showed a good linear relationship in the range of 0.832 μg / ml to 20.8 μg / ml, Y=50199.7542x-3387.2901, R=0.9999.

[0084] Table 6 Experimental results of neohesperidin standard curve (n=3)

[0085]

[0086]

[0087] The concentration of neohesperidin showed a good linear relationship in the range of 25 μg / ml to 625 μg / ml, Y=6626.2296x+4857.4444, R=0.9999.

[0088] 3. Precision test

[0089] The test solution prepared according to the method of Example 1 was injected 6 times in succession, with an injection volume of 20 μL. The RSD of the peak areas of the 6 chemical components was less than 3%, and the results showed that the parallel injection precision of the instrument was good.

[0090] Table 7 Precision test results (n = 6)

[0091]

[0092] 4. Repeatability test

[0093] Five portions of the standard decoction were precisely measured in parallel and prepared into test solutions according to the method in Example 1. The solutions were then measured under the above chromatographic conditions. The RSDs of the contents of the six chemical components were < 3%, indicating that the method had good repeatability.

[0094] Table 8 Repeatability test results (n=5)

[0095]

[0096] 5. Stability test

[0097] The test solution prepared according to the method of Example 1 was sampled and analyzed at different times of 0, 2, 4, 6, and 8 hours to determine its stability. The RSD of the peak areas of the six chemical components was less than 2%. The test results showed that the test solution was stable within 8 hours.

[0098] Table 9 Stability test results

[0099]

[0100] 6. Sample recovery test

[0101] Accurately measure 9 samples of the standard decoction, each in a group of three, each 2.5 ml, and add 4 ml, 5 ml, and 6 ml of the mixed reference solution, respectively, and the test solution prepared according to the method of Example 1.

[0102] Mixed reference solution: ferulic acid 38.8 μg / ml, isoferulic acid 44 μg / ml, verbascoside 53 μg / ml, naringin 46 μg / ml, β-ecdysterone 8.32 μg / ml, neohesperidin 320 μg / ml.

[0103] The sample recovery rate was calculated according to the above chromatographic conditions, and the results are shown in Tables 10 to 15. The test results show that this method has high accuracy and can be used to determine the content of verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone, and neohesperidin in standard decoction.

[0104] Table 10 Verbascum glycosides accuracy test results

[0105]

[0106] Table 11 Ferulic acid accuracy test results

[0107]

[0108] Table 12 isoferulic acid accuracy test results

[0109]

[0110] Table 13 Naringin accuracy test results

[0111]

[0112]

[0113] Table 14β-ecdysterone accuracy test results

[0114]

[0115] Table 15 Neohesperidin accuracy test results

[0116]

[0117] The above experimental results show that the method for detecting the contents of 6 chemical components in the standard decoction of Jichuan Jian provided by the present invention has good stability, high precision and good repeatability, and can objectively evaluate the quality of the standard decoction of Jichuan Jian and preparations, which is of great significance for ensuring clinical efficacy.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting six chemical components in the standard decoction of Jichuanjian, characterized in that: The method comprises the following steps: S1. Prepare mixed reference solution: Take verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone, and neohesperidin reference substances, dissolve them in 50% methanol, and prepare a mixed reference substance solution; S2. Prepare the test solution: Accurately measure the standard decoction to be tested, add water to dissolve, shake well, pass through a polyamide column, and then elute with water and methanol. Collect the methanol eluate, add water to make up the volume, shake well, filter through a microporous filter membrane, and take the filtrate to obtain; S3. High performance liquid chromatography detection: Accurately pipette the reference solution and the test solution respectively and inject them into the liquid chromatograph; The chromatographic conditions are: Chromatographic column: octadecylsilane bonded silica gel chromatographic column; Mobile phase: Mobile phase A is methanol, mobile phase B is 0.1% formic acid solution; Elution conditions: isocratic elution; Detection wavelength: 250nm; Column temperature: 30°C; Flow rate: 0.5-1.0mL / min; Injection volume: 10-20uL; The theoretical plate number calculated based on the verbascoside peak should not be less than 3000.

2. The detection method according to claim 1, characterized in that: The linear concentration ranges of verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone and neohesperidin in the reference solution of S1 are 5.3 μg / ml~132.5 μg / ml, 4.08 μg / ml~102 μg / ml, 4.4 μg / ml~110 μg / ml, 30.4 μg / ml~760 μg / ml, 0.832 μg / ml~20.8 μg / ml and 25 μg / ml~625 μg / ml.

3. The detection method according to claim 1, characterized in that: The concentrations of verbascoside, ferulic acid, isoferulic acid, naringin, β-ecdysterone, and neohesperidin in the reference solution of S1 were 30ug / mL, 10ug / mL, 10ug / mL, 100ug / mL, 20ug / mL, and 80ug / mL, respectively.

4. The detection method according to claim 1, characterized in that: Take 5 ml of the standard decoction in S2, add 15 ml of water to dissolve, shake well, pass through a 10 g polyamide column, then wash the column with 30 ml of water, and finally elute with 50 ml of methanol.

5. The detection method according to claim 4, characterized in that: S2: The volume is made up by adding water to the eluent to make up the volume to 50 ml.

6. The detection method according to claim 1, characterized in that: The chromatographic column described in S3 is a YMC-Pack ODS-A column.

7. The detection method according to claim 1, characterized in that: The elution condition of S3 is isocratic elution with a volume ratio of methanol to 0.1% formic acid of 38:

62.

8. The detection method according to claim 1, characterized in that: The flow rate of S3 is 0.7 mL / min.

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