Method for detecting content of effective components in seven-ingredient gastrodia elata medicinal liquor

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for detecting the content of effective components in seven-ingredient gastrodia elata medicinal liquor, the seven-ingredient gastrodia elata medicinal liquor is prepared from gastrodia elata, codonopsis pilosula, kadsura longepedunculata, poria cocos, fructus lycii, pericarpium citri reticulatae and radix polygoni multiflori preparata, has the effects of tonifying the kidney and soothing the nerves, and is used for neurasthenia, insomnia and dreaminess and the like. The method for detecting the content of the effective components of the seven-ingredient rhizoma gastrodiae medicinal liquor comprises the step of determining the content of gastrodin in a rhizoma gastrodiae medicinal material, the content of stilbene glucoside in a radix polygoni multiflori preparata medicinal material and the content of deoxyschizandrin in a kadsura longepedunculata medicinal material by adopting a high performance liquid chromatography. According to the method, a quantitative analysis method of three main active components is established through high performance liquid chromatography, a quality control system of the seven-ingredient gastrodia elata medicinal liquor is perfected, the adopted detection method is scientific and reasonable, high in specificity, high in accuracy and good in reproducibility, the quality of the seven-ingredient gastrodia elata medicinal liquor can be comprehensively and effectively controlled, and the curative effect and safety of the product are ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis and detection, and particularly relates to a detection method for effective component content of Qiwentiannama medicinal liquor. BACKGROUND

[0002] Qiwentiannama medicinal liquor has the effects of benefiting kidney and tranquilizing mind, and is commonly used for various diseases such as neurasthenia and insomnia and dreaminess. The prescription of Qiwentiannama medicinal liquor is composed of seven Chinese medicinal materials, i.e., Gastrodia elata, Radix Codonopsis, Fructus Schisandrae, Poria cocos, Fructus Lycii, Pericarpium Citri Reticulatae and Radix Polygoni Multiflori Preparata. Gastrodia elata is the main medicinal material of Qiwentiannama medicinal liquor, which has the functions of calming wind and stopping convulsion and suppressing liver yang, and is used for treating insomnia and neurasthenia. Radix Polygoni Multiflori Preparata and Radix Codonopsis are the minister medicinal materials, which have the functions of tonifying qi and essence and consolidating the foundation, and are used for enhancing the efficacy of the monarch medicinal material and tonifying liver and kidney. Fructus Schisandrae, Poria cocos, Fructus Lycii and Pericarpium Citri Reticulatae are the auxiliary medicinal materials, which have the functions of soothing heart, invigorating spleen, nourishing kidney and regulating qi, and are used for treating and relieving symptoms and preventing side effects. Pericarpium Citri Reticulatae is the ministerial medicinal material, which has the functions of guiding medicinal materials to the meridian and regulating the whole prescription, and is used for ensuring the absorption and targeting of the medicinal efficacy.

[0003] Qiwentiannama medicinal liquor has complex components. The existing quality evaluation method only depends on the content determination of a single compound, i.e., hesperidin, which can partially reflect the product quality, but ignores the interaction and overall effect among the components, resulting in the problems of insufficient systematic evaluation and stability evaluation and inability to comprehensively reflect the material basis of medicinal efficacy. By formulating the content detection methods for gastrodin in Gastrodia elata, astringin in Radix Polygoni Multiflori Preparata and schisantherin A in Fructus Schisandrae, the quality, clinical efficacy and safety of Qiwentiannama medicinal liquor are comprehensively ensured. Therefore, the present application provides a content determination method for gastrodin, astringin and schisantherin A in Qiwentiannama medicinal liquor. SUMMARY

[0004] The present application provides a detection method for effective component content of Qiwentiannama medicinal liquor, which is used to solve the problems mentioned in the background.

[0005] The present application provides a detection method for effective component content of Qiwentiannama medicinal liquor. Qiwentiannama medicinal liquor is composed of Gastrodia elata, Radix Codonopsis, Fructus Schisandrae, Poria cocos, Fructus Lycii, Pericarpium Citri Reticulatae and Radix Polygoni Multiflori Preparata. The content detection method is to determine the content of any one or more of Gastrodia elata, Radix Polygoni Multiflori Preparata and Fructus Schisandrae by high performance liquid chromatography, and the content detection indexes include any one or more of gastrodin, astringin and schisantherin A.

[0006] Optionally, the content determination method for gastrodin in Qiwentiannama medicinal liquor comprises the following steps:

[0007] Chromatographic analysis conditions: The chromatographic column is packed with alkyl-bonded silica gel; the mobile phase consists of an organic phase (A) and an aqueous phase (B), with gradient elution. The elution conditions are: 0-30 min, mobile phase A 2%-4%; column temperature 20-40℃, detection wavelength 220-320 nm; flow rate 0.5-1.5 mL / min.

[0008] Preparation of reference solution: Accurately weigh gastrodin reference standard, add acetonitrile-water (2:98) to prepare a solution containing 50 μg per mL.

[0009] Preparation of the test solution: Take an appropriate amount of the Seven-Flavor Gastrodia Wine, accurately measure 25 mL, evaporate to dryness in a water bath, add an appropriate amount of water to dissolve, transfer to a 25 mL volumetric flask, dilute to the mark with water, shake well, filter through a microporous membrane (0.22 μm), and collect the filtrate to obtain the test solution.

[0010] Assay: Accurately pipette 10-20 μL of the reference solution and the test solution into the liquid chromatograph and determine.

[0011] The alkyl-bonded silica gel described in this invention is used as a filler, including but not limited to ethane, butyl, heptyl, octyl, cyclohexyl, dodecyl, and octadecyl-bonded silica gels. In some embodiments, octadecyl-bonded silica gel is used as the filler.

[0012] The organic phase of the mobile phase described in this invention includes, but is not limited to, methanol, acetonitrile, ethanol, isopropanol, and tetrahydrofuran. In some embodiments, the organic phase is methanol, acetonitrile, or ethanol; in some specific embodiments, the organic phase is acetonitrile.

[0013] The aqueous phase of the mobile phase described in this invention includes, but is not limited to, water, formic acid aqueous solution, acetic acid aqueous solution, and phosphoric acid aqueous solution. In some embodiments, the aqueous phase is water, formic acid aqueous solution, and phosphoric acid aqueous solution; in some specific embodiments, the aqueous phase is phosphoric acid aqueous solution; in some more specific embodiments, the concentration of the phosphoric acid aqueous solution is 0.1-1.0% phosphoric acid solution; in some more specific embodiments, it is 0.1% phosphoric acid solution.

[0014] The column temperature described in this invention is 20-40℃, the detection wavelength is 220-320nm, and the flow rate is 0.5-1.5mL / min. In some embodiments, the temperature is 30℃, the detection wavelength is 220nm, and the flow rate is 0.8ml / min.

[0015] Optionally, the method for determining the stilbene glycoside content in Seven-Flavor Gastrodia Wine includes the following steps:

[0016] Chromatographic analysis conditions: The column was packed with alkyl-bonded silica gel; the mobile phase consisted of an organic phase (A) and an aqueous phase (B) ((24-26):(76-74)); the column temperature was 20-40℃; the detection wavelength was 220-320nm; and the flow rate was 0.5-1.5mL / min.

[0017] Preparation of reference solution: Accurately weigh 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standard, add methanol to prepare a solution containing 0.5 mg per mL.

[0018] Preparation of the test solution: Take an appropriate amount of the Seven-Flavor Gastrodia Wine, filter it, and take the filtrate to obtain the test solution.

[0019] Assay: Accurately pipette 10-20 μL of the reference solution and the test solution into the liquid chromatograph and determine.

[0020] The alkyl-bonded silica gel described in this invention is used as a filler, including but not limited to ethane, butyl, heptyl, octyl, cyclohexyl, dodecyl, and octadecyl-bonded silica gels. In some embodiments, octadecyl-bonded silica gel is used as the filler.

[0021] The organic phase of the mobile phase described in this invention includes, but is not limited to, methanol, acetonitrile, ethanol, isopropanol, and tetrahydrofuran. In some embodiments, the organic phase is methanol, acetonitrile, or ethanol; in some specific embodiments, the organic phase is acetonitrile.

[0022] The aqueous phase of the mobile phase described in this invention includes, but is not limited to, water, aqueous formic acid solution, aqueous acetic acid solution, and aqueous phosphoric acid solution. In some embodiments, the aqueous phase is water, aqueous formic acid solution, and aqueous phosphoric acid solution; in some specific embodiments, the aqueous phase is water.

[0023] The column temperature described in this invention is 20-40℃, the detection wavelength is 220-320nm, and the flow rate is 0.5-1.5mL / min. In some embodiments, the temperature is 30℃, the detection wavelength is 320nm, and the flow rate is 1.0ml / min.

[0024] Optionally, the method for determining the schisandrin A content in Qiwei Tianma medicinal wine includes the following steps:

[0025] Chromatographic analysis conditions: The chromatographic column was packed with octadecyl bonded silica gel; the mobile phase consisted of an organic phase (A) and an aqueous phase (B) ((67-73):(33-27)); the column temperature was 20-40℃; the detection wavelength was 220-320nm; and the flow rate was 0.5-1.5mL / min.

[0026] Chromatographic column: The alkyl-bonded silica gel used in this invention is the packing material, including but not limited to ethane, butyl, heptyl, octyl, cyclohexyl, dodecyl, and octadecyl-bonded silica gels. In some embodiments, octadecyl-bonded silica gel is used as the packing material.

[0027] Preparation of reference solution: Accurately weigh schisandrin A reference standard, add methanol to prepare a solution containing 50 μg per mL;

[0028] Preparation of the test solution: Take an appropriate amount of the Seven-Flavor Gastrodia Wine, filter it, and take the filtrate to obtain the test solution;

[0029] Assay: Accurately pipette 10-20 μL of the reference solution and the test solution into the liquid chromatograph and determine.

[0030] The alkyl-bonded silica gel described in this invention is used as a filler, including but not limited to ethane, butyl, heptyl, octyl, cyclohexyl, dodecyl, and octadecyl-bonded silica gels. In some embodiments, octadecyl-bonded silica gel is used as the filler.

[0031] The organic phase of the mobile phase described in this invention includes, but is not limited to, methanol, acetonitrile, ethanol, isopropanol, and tetrahydrofuran. In some embodiments, the organic phase is methanol, acetonitrile, or ethanol; in some specific embodiments, the organic phase is acetonitrile.

[0032] The aqueous phase of the mobile phase described in this invention includes, but is not limited to, water, formic acid aqueous solution, acetic acid aqueous solution, and phosphoric acid aqueous solution. In some embodiments, the aqueous phase is water, formic acid aqueous solution, and phosphoric acid aqueous solution; in some specific embodiments, the aqueous phase is phosphoric acid aqueous solution; in some more specific embodiments, the concentration of the phosphoric acid aqueous solution is 0.1-1.0% phosphoric acid solution; in some more specific embodiments, it is 0.1% phosphoric acid solution.

[0033] The column temperature described in this invention is 20-40℃, the detection wavelength is 220-320nm, and the flow rate is 0.5-1.5mL / min. In some embodiments, the temperature is 30℃, the detection wavelength is 220nm, and the flow rate is 1.0mL / min.

[0034] The beneficial effects of the method for detecting the content of effective components in the Seven-Flavor Gastrodia elata medicinal wine provided by this invention are as follows:

[0035] 1. This invention adds the HPLC content determination of gastrodin to the original quality standards. This method has high accuracy, strong specificity, good reproducibility, and convenient operation, thus strengthening the quality control of the main medicinal herb, Gastrodia elata.

[0036] 2. This invention also includes the HPLC content determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside. This method has high accuracy, strong specificity, good reproducibility, and is easy to operate, which is beneficial to product quality control.

[0037] 3. This invention also adds an HPLC method for the determination of schisandrin A content. This method has high accuracy, strong specificity, good reproducibility, and is easy to operate. It can effectively control the quality of the Seven-Flavor Gastrodia Wine and ensure the clinical efficacy of the product. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 The chromatogram for the gastrodin specificity test provided by this invention;

[0040] Figure 2 Chromatogram of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside specificity test provided by the present invention;

[0041] Figure 3 The chromatogram for the specificity test of schisandrin A provided by the present invention. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are also within the scope of protection of the present invention.

[0043] In all embodiments, all original reagent materials are commercially available. Experimental methods not specifically described are conventional methods and conditions well known in the art, or are performed according to the conditions recommended by the instrument manufacturer. See Tables 1, 2, 3, and 4, where Table 1 lists the instruments and equipment used in this invention, Table 2 lists the reagents used in this invention, Table 3 lists the sample information used in this invention, and Table 4 lists the reference standards used in this invention.

[0044] Table 1

[0045] Instrument name Model Manufacturer Ultra-high performance liquid chromatograph LC-20ADXR Shimadzu High performance liquid chromatograph LC-2050 Shimadzu High performance liquid chromatograph LC-2050C Shimadzu Electronic analytical balance ME55 Mettler-Toledo Instruments (Shanghai) Co., Ltd. Ultra-pure water machine KNT-VF-16 Hefei Koinet Water Treatment Equipment Co., Ltd. Numerical control ultrasonic cleaner KQ-250DE Kunshan Ultrasonic Instruments Co., Ltd. Digital constant-temperature water bath HH-6 Shanghai Kuntian Laboratory Instruments Co., Ltd.

[0046] Table 2

[0047] Reagent name Grade Manufacturer Acetonitrile Chromatographic grade Mcgill Reagent (Shanghai) Co., Ltd. Methanol Chromatographic grade Mcgill Reagent (Shanghai) Co., Ltd. Methanol Analytical pure Luoyang Haohua Chemical Reagent Co., Ltd. Phosphoric acid Chromatographic grade Tianjin Kemio Chemical Reagent Co., Ltd. Ultra-pure water First-grade water Laboratory self-made

[0048] Table 3

[0049]

[0050]

[0051] Table 4

[0052]

[0053] The technical solution provided by the present invention will be described in detail below with reference to specific embodiments.

[0054] Example 1

[0055] Investigation on the determination method of gastrodin content in Seven-Flavor Gastrodia Wine

[0056] In an embodiment of the present invention, the method for determining the gastrodin content in the Seven-Flavor Gastrodia Wine includes the following specific steps:

[0057] S1. Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid water was used as mobile phase B, with gradient elution performed according to the specifications in Table 5; the detection wavelength was 220 nm; the column temperature was 30 ℃; and the flow rate was 0.8 mL / min. The theoretical plate number calculated based on the gastrodin peak was not less than 6000.

[0058] Table 5

[0059] Time (min) Mobile phase A (%) Mobile phase B (%) 0 2 98 30 4 96

[0060] S2. Preparation of reference solution: Accurately weigh gastrodin reference standard, add acetonitrile-water (2:98) to prepare a solution containing 50 μg per 1 mL.

[0061] S3. Preparation of the test solution: Take an appropriate amount of the Seven-Flavor Gastrodia Wine, accurately measure 25 mL, evaporate to dryness in a water bath, add an appropriate amount of water to dissolve, transfer to a 25 mL volumetric flask, dilute to the mark with water, shake well, filter through a microporous membrane (0.22 μm), and take the filtrate to obtain the test solution.

[0062] S4. Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0063] (1) Specificity test

[0064] Following step S3 in Example 1, prepare the test solution, gastrodin reference solution, and negative control solution for gastrodia elata (lacking the medicinal material). Accurately inject 10 μL of each solution into the liquid chromatograph and determine the results. (See attached figure).Figure 1 .

[0065] The results showed that the test solution and the reference solution had chromatographic peaks at the corresponding retention times, while the negative control solution lacking Gastrodia elata did not have chromatographic peaks at the corresponding retention times as the test solution and the reference solution. This indicates that the method of the present invention is specific.

[0066] (2) Linear Examination

[0067] Accurately weigh gastrodin reference standard, dissolve and dilute it with an appropriate amount of acetonitrile-water (2:98) mixed solution to prepare solutions containing 4.9647 μg, 24.8235 μg, 49.647 μg, 99.294 μg, 151.014 μg, and 198.588 μg of gastrodin per ml. Accurately pipette 10 μL of blank solution and reference solution of each concentration into the liquid chromatograph and record the peak area. Plot a standard curve with peak area as the ordinate (y) and injection concentration as the abscissa (x).

[0068] The results showed that the linear regression equation for gastrodin was: y = 24364x + 1967.5, R0 2 =1. This indicates a good linear relationship between the concentration of gastrodin and the peak area in the range of 0-198.588 μg / mL. The results of calculating the content using the measured peak area of ​​the sample and the external standard method were similar; therefore, the external standard method was used to calculate the content.

[0069] (3) Instrument precision

[0070] Accurately pipette 10 μL of the gastrodin reference solution and inject it six times consecutively, recording the retention time and peak area. The results showed that the RSD of the peak area of ​​the gastrodin reference solution was less than 1.5%, indicating good instrument precision. The results are shown in Table 6.

[0071] Table 6

[0072]

[0073] (4) Repeatability test

[0074] Six test solutions were prepared in parallel according to the test solution preparation method in step S3 of Example 1. 10 μL of each solution was injected, and the content and RSD were determined. The results showed that the test solutions were within the range of sample size, and the method had good repeatability. See Table 7 for details.

[0075] Table 7

[0076]

[0077] (5) Intermediate precision test

[0078] Six test solutions were prepared by different subjects at different times according to the test solution preparation method in step S3 of Example 1, and the results were measured. The intermediate precision of the method was good, as detailed in Table 8.

[0079] Table 8

[0080]

[0081] (6) Stability test

[0082] Take 25 ml of this product (batch number: 250301) and operate according to the specified preparation and determination method of the test solution. Inject the sample and determine the results at 0, 3, 6, 9, 12 and 24 hours after preparation. The results showed that the peak area RSD of gastrodin was less than 1.5%, and the test solution had good stability within 24 hours. See Table 9 for details.

[0083] Table 9

[0084]

[0085]

[0086] (7) Accuracy test

[0087] The accuracy of the method was investigated by a spiking recovery test. An appropriate amount of gastrodin reference standard was accurately weighed and mixed with acetonitrile-water (2:98) to prepare a solution containing 53.451 μg per 1 mL. Then, 3 mL of the reference standard solution was accurately pipetted into a 50 mL volumetric flask and diluted with acetonitrile-water (2:98) to prepare a reference standard stock solution of 3.20706 μg / mL.

[0088] Accurately pipette 12.5 mL each of the reference stock solution and the test solution into the same evaporating dish, evaporate to dryness in a water bath (prepare 6 in parallel), dissolve in water and dilute to a 25 mL volumetric flask, filter, and collect the filtrate.

[0089] The recovery rates were 100.9%–104.1%, with an RSD of 1.1%, indicating good accuracy of the method. See Table 10 for details.

[0090] Table 10

[0091]

[0092] (8) Durability test

[0093] By fine-tuning the flow rate, acidity, column temperature, and chromatographic column, the gastrodin content was observed to see if there were any significant changes. Results: The results of the method of the present invention were basically consistent under different flow rates, different acidities, different column temperatures, and different chromatographic column conditions, indicating that the method has good robustness. See Table 11 for details.

[0094] Table 11

[0095]

[0096]

[0097] Example 2

[0098] Investigation on the determination method of stilbene glycosides in Seven-Flavor Gastrodia Wine

[0099] In an embodiment of the present invention, the method for determining the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in the Seven-Flavor Gastrodia elata medicinal wine comprises the following specific operating steps:

[0100] S1. Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-water (25:75) was used as the mobile phase; the detection wavelength was 320 nm; the column temperature was 30 °C; and the flow rate was 1.0 mL / min. The theoretical plate number, calculated based on the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside peak, should be no less than 2000.

[0101] S2. Preparation of reference solution: Accurately weigh 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standard, add methanol to prepare a solution containing 0.5 mg per mL.

[0102] S3. Preparation of the test solution: Take an appropriate amount of the Seven-Flavor Gastrodia Wine, filter it, and take the filtrate to obtain the test solution.

[0103] S4. Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0104] (1) Specificity test

[0105] Following step S3 in Example 2, prepare the test solution, the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference solution, and the negative control solution for Polygonum multiflorum (He Shou Wu) without prepared medicinal material. Accurately inject 10 μL of each of the above solutions into the liquid chromatograph for determination. Results are shown in [Figure 1]. Figure 2 .

[0106] The results showed that the test solution and the reference solution had chromatographic peaks at the corresponding retention times, while the negative control solution lacking processed Polygonum multiflorum did not have chromatographic peaks at the corresponding retention times, unlike the test solution and the reference solution. This indicates that the method of the present invention is specific.

[0107] (2) Linear Examination

[0108] Accurately weigh 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standard, dissolve and dilute with methanol to prepare solutions containing 4.895 μg, 24.475 μg, 48.95 μg, 97.9 μg, and 195.8 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per mL. Accurately pipette 10 μL of the blank solution and each concentration of the reference standard solution and inject them into the liquid chromatograph, recording the peak areas. Plot a standard curve with peak area as the ordinate (y) and injection concentration as the abscissa (x).

[0109] The results showed that the linear regression equation for 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside was: y = 39173x - 15465, R0 2 =1. This indicates a good linear relationship between the concentration of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside and the peak area in the range of 0-195.8 μg / mL. The results of calculating the content using the measured peak area of ​​the sample by substituting it into the standard curve and by using the external standard method were not significantly different; therefore, the external standard method was used to calculate the content.

[0110] (3) Instrument precision

[0111] Accurately pipette 10 μL of the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference solution and inject it six times consecutively, recording the retention time and peak area. The results showed that the RSD of the peak area of ​​the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference solution was less than 1.5%, indicating good instrument precision. The results are shown in Table 12.

[0112] Table 12

[0113]

[0114] (4) Repeatability test

[0115] Six test solutions were prepared in parallel according to the test solution preparation method in step S3 of Example 2. 10 μL of each solution was injected, and the content and RSD were determined. The results showed that the test solutions were within the range of sample size, and the method had good repeatability. See Table 13 for details.

[0116] Table 13

[0117]

[0118] (5) Intermediate precision test

[0119] Six test solutions were prepared by different subjects at different times according to the test solution preparation method in step S3 of Example 1, and the results were measured. The intermediate precision of the method was good, as detailed in Table 14.

[0120] Table 14

[0121]

[0122] (6) Stability test

[0123] Take 25 ml of this product (batch number: 250301) and operate according to the specified method for preparation and determination of the test solution. Inject the sample and determine the results at 0, 3, 6, 9, 12 and 24 hours after preparation. The peak area RSD of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside is less than 1.5%. The test solution has good stability within 24 hours. See Table 15 for details.

[0124] Table 15

[0125]

[0126] (7) Accuracy test

[0127] The accuracy of the method was investigated by spiking recovery test. An appropriate amount of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standard was accurately weighed and added to methanol to prepare a solution containing 0.466 μg per 1 mL.

[0128] Accurately pipette 0.4 mL of the reference solution into a 10 mL volumetric flask, and dilute the test solution to 10 mL. Prepare six parallel aliquots, shake well, filter, and collect the filtrate.

[0129] The recovery rate was 99.3%–100.7%, and the RSD was 0.5%, indicating that the method has good accuracy. See Table 16 for details.

[0130] Table 16

[0131]

[0132] (8) Durability test

[0133] By fine-tuning the flow rate, mobile phase ratio, column temperature, and chromatographic column, we observed whether there were significant changes in the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside. Results: The results obtained by the method of the present invention under different flow rates, mobile phase ratios, column temperatures, and chromatographic column conditions were basically consistent, indicating that the method has good robustness. See Table 17 for details.

[0134] Table 17

[0135]

[0136] Example 3

[0137] Investigation on the determination method of schisandrin A content in Seven-Flavor Gastrodia Wine

[0138] In an embodiment of the present invention, the method for determining the schisandrin A content in the Seven-Flavor Gastrodia Wine includes the following specific steps:

[0139] S1. Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-0.1% phosphoric acid solution (70:30) was used as the mobile phase; the detection wavelength was 220 nm; the column temperature was 30 °C; and the flow rate was 1.0 mL / min. The theoretical plate number, calculated based on the schisandrin A peak, should not be less than 3000.

[0140] S2. Preparation of reference solution: Accurately weigh schisandrin A reference standard, add methanol to prepare a solution containing 50 μg per mL.

[0141] S3. Preparation of the test solution: Take an appropriate amount of the Seven-Flavor Gastrodia Wine, filter it, and take the filtrate to obtain the test solution.

[0142] S4. Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0143] (1) Specificity test

[0144] Following step S3 in Example 3, prepare the test solution, schisandrin A reference solution, and negative control solution for Schisandra chinensis lacking raw material. Accurately inject 10 μL of each solution into the liquid chromatograph and determine the results. (See attached figure). Figure 3 .

[0145] The results showed that the test solution and the reference solution had chromatographic peaks at the corresponding retention times, while the negative control solution lacking Schisandra chinensis root had no chromatographic peaks at the corresponding retention times compared to the test solution and the reference solution. This indicates that the method of the present invention is specific.

[0146] (2) Linear Examination

[0147] Accurately weigh schisandrin A reference standard, dissolve and dilute it in methanol to prepare solutions containing 5.6715 μg, 28.3575 μg, 56.715 μg, 113.43 μg, and 226.86 μg of schisandrin A per mL. Accurately pipette 10 μL of the blank solution and each concentration of the reference standard solution into the liquid chromatograph, and record the peak areas. Plot a standard curve with peak area as the ordinate (y) and injection concentration as the abscissa (x).

[0148] The results showed that the linear regression equation for schisandrin A was: y = 65214x - 34857, R0 2=0.9999. This indicates a good linear relationship between schisandrin A concentration and peak area in the range of 0–226.86 μg / mL. The results of calculating the content using the measured peak area of ​​the sample and the external standard method were similar; therefore, the external standard method was used to calculate the content.

[0149] (3) Instrument precision

[0150] Accurately pipette 10 μL of schisandrin A reference solution and inject it 6 times consecutively, recording the retention time and peak area. The results showed that the RSD of the peak area of ​​schisandrin A reference solution was less than 1.5%, indicating good instrument precision. The results are shown in Table 18.

[0151] Table 18

[0152]

[0153] (4) Repeatability test

[0154] Six test solutions were prepared in parallel according to the test solution preparation method in step S3 of Example 1. 10 μL of each solution was injected, and the content and RSD were determined. The results showed that the test solutions were within the range of sample size, and the method had good repeatability. See Table 19 for details.

[0155] Table 19

[0156]

[0157] (5) Intermediate precision test

[0158] Six test solutions were prepared by different subjects at different times according to the test solution preparation method in step S3 of Example 3, and the results were measured. The intermediate precision of the method was good, as detailed in Table 20.

[0159] Table 20

[0160]

[0161] (6) Stability test

[0162] Take 25 mL of this product (batch number: 250301) and accurately measure it. Follow the preparation and determination method of the test solution as specified in the instructions. Inject the sample and determine the results at 0, 3, 6, 9, 12 and 24 hours after preparation. The peak area RSD of schisandrin A was less than 1.5%, and the test solution showed good stability within 24 hours. See Table 21 for details.

[0163] Table 21

[0164]

[0165] (7) Accuracy test

[0166] The accuracy of the method was examined by a spiking recovery test. An appropriate amount of schisandrin A reference standard was accurately weighed and added to methanol to prepare a solution containing 52.636 μg per 1 mL.

[0167] Accurately pipette 0.2 mL of the reference solution into a 10 mL volumetric flask, and dilute the test solution to 10 mL. Prepare six parallel aliquots, shake well, filter, and collect the filtrate.

[0168] The recovery rate was 92.1%–98.3%, with an RSD of 2.5%, indicating good accuracy of the method. See Table 22 for details.

[0169] Table 22

[0170]

[0171] (8) Durability test

[0172] By fine-tuning the flow rate, mobile phase ratio, column temperature, and chromatographic column, the schisandrin A content was observed to see if there were any significant changes. Results: The results of the method of the present invention were basically consistent under different flow rates, different mobile phase ratios, different column temperatures, and different chromatographic column conditions, indicating that the method has good robustness. See Table 23 for details.

[0173] Table 23

[0174]

[0175] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0176] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0177] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the content of effective components in a seven-ingredient Gastrodia elata medicinal wine, wherein the seven-ingredient Gastrodia elata medicinal wine is composed of Gastrodia elata, Codonopsis pilosula, Schisandra chinensis, Poria cocos, Lycium barbarum, Citrus reticulata peel, and processed Polygonum multiflorum, characterized in that, The content detection method uses high performance liquid chromatography to detect the content of one or more of Gastrodia elata, processed Polygonum multiflorum, and Schisandra chinensis. The content detection indicators include one or more of gastrodin, stilbene glycoside, and schisandrin.

2. The detection method according to claim 1, characterized in that, The method for determining the gastrodin content is as follows: using octadecylsilane-bonded silica gel as the packing material; using acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution is performed; the detection wavelength is 220 nm; the column temperature is 30 ℃; the flow rate is 0.8 mL / min; the theoretical plate number calculated based on the gastrodin peak is not less than 5000; the gradient elution is as follows: 0-30 min, mobile phase A 2% → 4%.

3. The detection method according to claim 2, characterized in that, The steps of the method for detecting the content of gastrodin include: Preparation of reference solution: Accurately weigh gastrodin reference standard, add acetonitrile-water solution with a volume ratio of 2:98 to prepare a solution containing 50 μg per mL; Preparation of the test solution: Take an appropriate amount of the Seven-Flavor Gastrodia Wine, accurately measure 25 mL, evaporate to dryness in a water bath, add an appropriate amount of water to dissolve, transfer to a 25 mL volumetric flask, dilute to the mark with water, shake well, filter through a microporous membrane with a pore size of 0.22 μm, and take the filtrate to obtain the test solution. Determination: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine.

4. The detection method according to claim 1, characterized in that, The method for determining the stilbene glycoside content is as follows: using octadecylsilane-bonded silica gel as the filler; using an acetonitrile aqueous solution with a volume ratio of 25:75 as the mobile phase; using a detection wavelength of 320 nm; a column temperature of 30 °C; and a flow rate of 1.0 mL / min.

5. The detection method according to claim 4, characterized in that, The method for detecting the stilbene glycoside content includes the following steps: Preparation of reference solution: Accurately weigh 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standard, add methanol to prepare a solution containing 0.5 mg per mL; Preparation of the test solution: Take an appropriate amount of the Seven-Flavor Gastrodia Wine, filter it, and take the filtrate to obtain the test solution; Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine.

6. The detection method according to claim 1, characterized in that, The method for determining the schisandrin A content is as follows: octadecylsilane-bonded silica gel is used as the packing material; acetonitrile-0.1% phosphoric acid aqueous solution is used as the mobile phase, wherein the volume ratio of acetonitrile to 0.1% phosphoric acid is 70:30; the detection wavelength is 220 nm, the column temperature is 30 °C, and the flow rate is 1.0 mL / min.

7. The detection method according to claim 6, characterized in that, The detection of schisandrin A content includes the following steps: Preparation of reference solution: Accurately weigh schisandrin A reference standard, add methanol to prepare a solution containing 50 μg per mL; Preparation of the test solution: Take an appropriate amount of the Seven-Flavor Gastrodia Wine, filter it, and take the filtrate to obtain the test solution; Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine.

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