Determination of Tetrahydroxystilbene Glucoside in Tongbi Huoluo Pills by HPLC

Abstract

The application relates to a method for determining the content of stilbene glucoside in Tongbi Huoluo pills by HPLC, which comprises preparation of a test sample solution: (1) after ultrasonic extraction of the sample solution of the Tongbi Huoluo pills to be detected, centrifugal separation is carried out, and the supernatant is extracted with ethyl acetate to obtain an ethyl acetate extract; (2) the ethyl acetate extract is concentrated to near dryness, dissolved with a solvent allowed by the pharmacopoeia, adsorbed with a macroporous adsorption resin, washed with water and / or eluted with water and an alcohol solution in sequence, and the eluate is collected; after removal of the solvent, an extract of the test sample is obtained. The method can be used for detecting the content of stilbene glucoside in the Tongbi Huoluo pills containing 34 traditional Chinese medicines, has high precision, accuracy and recovery rate, is good in system applicability, durability and repeatability, is simple and feasible, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug detection, and particularly relates to a method for determining the content of stilbene glucoside in Tongbi Huoluo pills by HPLC. BACKGROUND

[0002] "Tongbi Huoluo pills" are traditional Chinese medicine pills developed by Sichuan Yibin County Orthopedic Hospital according to a traditional prescription. The pills are prepared from 50 g of mulberry, 50 g of eucommia ulmoides, 50 g of astragalus membranaceus, 50 g of radix polygoni multiflori, 50 g of prepared rehmannia, 50 g of polygala, 50 g of dog spine, 25 g of radix astragali, 50 g of acanthopanax, 25 g of ophiophagus, 25 g of chaenomeles, 25 g of white peony root, 25 g of rhizoma curculigns, 25 g of cistanche, 25 g of anemarrhena, 25 g of angelica sinensis, 20 g of curcuma, 25 g of radix dipsaci, 25 g of corydalis, 15 g of salvia miltiorrhiza, 15 g of panax notoginseng, 15 g of lycium chinense, 20 g of cuscuta, 15 g of prepared strychnos, 15 g of cortex phellodendri, 15 g of earthworm, 15 g of turtle shell, 15 g of deer antler, 10 g of prepared ephedra, 10 g of asarum, 10 g of scorpion, 10 g of leech, and 10 g of eupolyphaga. The 34 kinds of traditional Chinese medicines are ground, sieved, mixed, and coated to prepare the red-brown water pills. The pills have a slightly sweet and bitter taste, and have the effects of tonifying qi, promoting blood circulation, tonifying kidney and strengthening bones, dispelling wind and removing dampness. The pills are used for the treatment of wind-cold arthralgia, joint pain, and limb numbness. Since 2008, the pills have achieved good therapeutic effects in clinical application, cured a large number of patients with lumbar and leg pain and contusion and injury, and have been highly recognized by doctors and patients.

[0003] Stilbene glucoside contained in radix polygoni multiflori in the prescription of the preparation is one of the effective components of the preparation, and has the effects of anti-aging, neuroprotection, anti-lipid, anti-atherosclerosis, anti-tumor, and anti-tumor metastasis. Stilbene glucoside is used as a quantitative index for detecting radix polygoni multiflori in the Pharmacopeia of the People's Republic of China. In the quality standard for radix polygoni multiflori in the first volume of Chinese Pharmacopeia 2020 edition, the content of stilbene glucoside is not less than 0.70%. However, the prescription of "Tongbi Huoluo pills" developed by Sichuan Yibin County Orthopedic Hospital according to a traditional prescription is complex, and the matrix interference is large. The original standard does not record the determination method of the content of stilbene glucoside. The existing detection method cannot determine the content of stilbene glucoside. In order to further improve the quality of the prescription of "Tongbi Huoluo pills" and meet the requirements of relevant specifications, it is necessary to find a convenient, stable and accurate method for determining the content of stilbene glucoside in "Tongbi Huoluo pills". SUMMARY

[0004] Some methods for determining the content of stilbene glucoside in Radix Polygoni Multiflori in the prior art are also recorded, such as the method recorded in the literature (Su Lijie. Determination of the content of stilbene glucoside in Radix Polygoni Multiflori by HPLC [J]. China's contemporary medicine, 2010, 17(18): 2.) that after the Radix Polygoni Multiflori sample is treated with methanol ultrasonically, it is cooled, filtered, and the initial filtrate is discarded. The solvent is evaporated under reduced pressure, the residue is ultrasonically dissolved with methanol, and the volume is adjusted to obtain a test sample solution. The content of stilbene glucoside is determined by HPLC, which has high accuracy, good repeatability, and high reliability.

[0005] The preparation method of the test sample is recorded in the literature (Yuan Rongxian, Liu Tianxiang, Yang Renhui. Influence of modified processing of Radix Polygoni Multiflori on the content of stilbene glucoside [J]. Family medicine, 2019, 000(009): 29-30.) that the powder of the Radix Polygoni Multiflori decoction piece is added with dilute ethanol, heated to reflux, cooled, the weight loss is made up with dilute ethanol, shaken, filtered with a filter membrane, and the filtrate is taken as the test sample solution.

[0006] The content of stilbene glucoside in raw and four processed products of Radix Polygoni Multiflori is determined and compared by HPLC in the literature (Wang Jianke, Gao Yanming, Chen Huiying. Determination of stilbene glucoside content in raw and four processed products of Radix Polygoni Multiflori [J]. Trace elements and health research, 2004, 21(4): 2.), which records that the powder of raw and processed products of Radix Polygoni Multiflori is dissolved with 50% ethanol. After heating to reflux and cooling, the weight is made up with 50% ethanol, and the filtrate is obtained as the test sample solution.

[0007] The method for determining stilbene glucoside and anthraquinone components in Radix Polygoni Multiflori and Radix Ampelopsis is recorded in the literature (Su Jian, Yuan Zhifang, Zhang Lantong, et al. Determination of stilbene glucoside and anthraquinone components in Radix Polygoni Multiflori and Radix Ampelopsis by RP-HPLC [J]. Chinese herbal medicine, 2007, 38(2): 4.) that the fine powder of Radix Polygoni Multiflori, processed Radix Polygoni Multiflori, and Radix Ampelopsis is dissolved with 50% ethanol, ultrasonically treated for 15 min, cooled, the weight loss is made up with 50% ethanol, filtered, and the filtrate is taken as the test sample solution.

[0008] The patent CN114720600A provides a detection method for stilbene glucoside components in Radix Polygoni Multiflori in bone protection capsules. In the invention, the content of stilbene glucoside in the test sample is not much different when dilute ethanol, 75% ethanol, and 50% methanol are used as extraction solvents. The detection method for stilbene glucoside components in Radix Polygoni Multiflori in bone protection capsules provided by the invention has simple operation, good linear relationship, high precision, stability, and repeatability, high recovery rate of sample addition, accurate and reliable detection results, and meets the requirements of national drug standards.

[0009] It can be seen that the prior art basically uses methanol or ethanol to heat reflux or ultrasonic extraction of test sample solution, the applicant has tried to use the method in the above document to detect the content of stilbene glycoside in Tongbi Huoluo pills, and it is found that whether methanol or ethanol is used for heating reflux or ultrasonic extraction to prepare test sample solution, the content of stilbene glycoside cannot be well detected.

[0010] Based on the technical problems existing in the prior art, the purpose of the present application is to provide an HPLC method for determining the content of stilbene glycoside in Tongbi Huoluo pills, and the accuracy and precision of detection are good.

[0011] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0012] An HPLC method for detecting the content of stilbene glycoside in Tongbi Huoluo pills, comprising preparation of test sample solution:

[0013] (1) After ultrasonic extraction of the sample solution of the Tongbi Huoluo pills to be detected, centrifugation is performed, and the supernatant obtained is extracted with ethyl acetate to obtain an ethyl acetate extract;

[0014] (2) The ethyl acetate extract is concentrated to near dryness, dissolved with a solvent allowed by the pharmacopoeia, adsorbed with a macroporous adsorption resin, washed with water and / or sequentially eluted with water and alcohol solution, and the eluate is collected, and after removal of the solvent, the extract of the detected product is obtained.

[0015] Further, the sample solution of the Tongbi Huoluo pills to be detected is obtained by dissolving the sample of the Tongbi Huoluo pills to be detected with water.

[0016] In the present application, the mass-volume ratio of the sample of the Tongbi Huoluo pills to be detected to water is 1g:(5-8)mL.

[0017] Further, the mass-volume ratio of the sample of the Tongbi Huoluo pills to be detected to water is 1g:6mL.

[0018] The number of ethyl acetate extractions can be selected according to requirements, and can be 1-4 times, of which the extraction number is usually selected to be 1-2 times.

[0019] In the present application, the ultrasonic extraction conditions are: temperature 10-38℃, time 20-45min, ultrasonic frequency 28-50kHz, and ultrasonic power 200-250V.

[0020] Further, the ultrasonic extraction conditions are: temperature 18-30℃, time 30min, ultrasonic frequency 28kHz, and ultrasonic power 220V.

[0021] In the present application, the concentrated mode is selected from at least one of vacuum concentration, nitrogen blow concentration, evaporation concentration, and freeze concentration; further, the concentrated mode is nitrogen blow concentration.

[0022] In the present application, the pharmacopoeia-allowed solvent is selected from at least one of water, ethanol, and ethyl acetate; further, the pharmacopoeia-allowed solvent is water.

[0023] In the present application, the type of the macroporous adsorption resin is selected from at least one of D101, HPD-100, Seplite LX-68, HPD-200A, HPD-722, and HPD-721, and further is D101.

[0024] In the present application, the content of stilbene glycoside contained in Huzhu Huolu Pills is detected.

[0025] In the present application, the chromatographic conditions of HPLC at least include at least one of I-VIII:

[0026] I. Chromatographic column: octadecylsilane bonded chromatographic column;

[0027] II. Mobile phase: organic phase-water;

[0028] III. Column temperature: 30-38 DEG C;

[0029] IV. Flow rate: 0.5-2 mL / min;

[0030] V. Detection wavelength: 315-325 nm;

[0031] VI. Injection volume: 5-15 muL;

[0032] VII. Chromatographic column specification: 3-5 mu m, 4.6 mm*250 mm;

[0033] VIII. Elution mode: gradient elution, as shown in the following table:

[0034]

[0035] Further, the chromatographic conditions of HPLC at least include at least one of I-VIII:

[0036] I. Chromatographic column type: Agilent XDB-C18;

[0037] II. Mobile phase: acetonitrile: water;

[0038] III. Column temperature: 35 DEG C;

[0039] IV. Flow rate: 1 mL / min;

[0040] V. Detection wavelength: 320 nm;

[0041] Injection volume: 10 μL;

[0042] Column size: 5 μm, 4.6 mm x 250 mm;

[0043] Elution mode: Gradient elution, as shown in the following table:

[0044]

[0045] The present application has the following beneficial effects: the proportion of Radix Polygoni Multiflori in "Tongbi Huoluo Pills" is small, and the prescription is complex, and the matrix interference is large. The detection difficulty lies in optimizing the sample processing method and excluding interference. The present application first uses ultrasonic extraction, then uses ethyl acetate extraction, dissolves the residue obtained by concentration and drying with water, and then passes through a column adsorption. The eluent collected with water and ethanol is the test solution. Then, a suitable chromatographic condition is used. The content of stilbene glycoside in the linear concentration range of 22.56-225.6 μg.ml -1 , the linear correlation coefficient R 2 = 0.997, the average recovery rate is 95.6%, the accuracy, precision, durability and system suitability are good, and the internal quality of "Tongbi Huoluo Pills" can be improved and stabilized, and a medicament meeting the standard requirements is provided for the clinic. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 is a linear regression graph of stilbene glycoside;

[0047] Figure 2 is a chromatogram of stilbene glycoside reference solution;

[0048] Figure 3 is a chromatogram of stilbene glycoside test solution;

[0049] Figure 4 is a chromatogram of test solution prepared by the method of Comparative Example 1;

[0050] Figure 5 is a chromatogram of test solution prepared by the method of Comparative Example 2. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solutions and advantages of the present application clearer and more apparent, the present application is further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not intended to limit the present application.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0053] It should be understood that the experimental methods used in the following examples are routine methods unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0054] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a step, a method, or an article that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, step, method, or article.

[0055] Example 1

[0056] Materials and Methods

[0057] 1.1 Reagents and Consumables

[0058] Stilbene glucoside reference substance (94.8%): China Institute for Drug Control; ethyl acetate: analytical pure; D101 macroporous adsorption resin (inner diameter 2.0 cm, column height 6 cm): self-filled.

[0059] 1.2 Instruments and Equipment

[0060] High-performance liquid chromatograph: Agilent 1260; high-performance liquid chromatograph: Shimadzu (SPD-M20A); electronic balance: Mettler-Toledo AG285.

[0061] 1.2.1 Chromatographic Conditions

[0062] Chromatographic column: Agilent XDB-C18 (5 μm x 4.6 mm x 250 mm); mobile phase: acetonitrile: water, gradient elution reference conditions are shown in Table 1 below; flow rate: 1.0 mL / min; column temperature: 35 °C; stilbene glucoside detection wavelength 320 nm; injection volume 10 μL.

[0063] Table 1 Gradient Elution Conditions

[0064]

[0065]

[0066] 1.3 Methods

[0067] 1.3.1 Preparation of Reference Substance Solution

[0068] Accurately weigh 0.0119 g of Stilbene glucoside reference substance (China Institute of Drug Control, 94.8%) into a 10-ml volumetric flask, dissolve in methanol and dilute to the mark, shake well, as the stock solution (concentration of 1128 μg / ml); accurately pipette 2.00 ml of Stilbene glucoside stock solution (1128 μg / ml) into a 10-ml volumetric flask, dissolve in methanol and dilute to the mark, shake well, to obtain Stilbene glucoside (225.6 μg / ml) control solution; accurately pipette 0.1 ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml of Stilbene glucoside stock solution (225.6 μg / ml) into sample bottles, respectively, dilute to 1 ml with methanol, shake well, to prepare a series of control solution with Stilbene glucoside at a concentration of 22.56 μg / ml, 45.12 μg / ml, 90.24 μg / ml, 135.36 μg / ml, 180.48 μg / ml, 225.6 μg / ml per 1 ml.

[0069] 1.3.2 Preparation of test solution

[0070] Accurately weigh about 5 g of sample into a 50-ml centrifuge tube, add 30 ml of water, ultrasonically extract at room temperature for 30 min, ultrasonic frequency is 28 kHz, ultrasonic power is 220 V, centrifuge at 4000 r / min for 5 min, transfer the supernatant to a 50-ml centrifuge tube, add 15 ml of ethyl acetate to the supernatant, vortex extract for 5 min, centrifuge, transfer the ethyl acetate layer to a 50-ml centrifuge tube, repeat the extraction with 15 ml of ethyl acetate once, combine the extract, concentrate to near dryness under nitrogen blow, dissolve the residue in 20 ml of distilled water, pass through a D101 macroporous adsorption resin (inner diameter about 2.0 cm, column height about 8 cm), sequentially elute with 20 ml of water and 40 ml of 80% ethanol, discard the water solution, collect the 80% ethanol eluate, evaporate to dryness on a water bath, dissolve the residue in 5 ml of water, filter through a microporous filter membrane (0.45 μm), to obtain the sample solution.

[0071] 2 Results and analysis

[0072] 2.1 Linear range

[0073] Under the above chromatographic conditions, determine the series of Stilbene glucoside reference solution, take the peak area as the vertical coordinate and the concentration of the control solution as the horizontal coordinate to perform linear regression, to obtain the regression equation of Stilbene glucoside y = 589.98x - 1660.9, the correlation coefficient R = 0.997, the linear concentration range is 22.56 μg / ml-225.6 μg / ml, indicating that the method is linear in this concentration range. 2

[0074] 2.2 Methodology verification

[0075] ​System suitability test: The sample was prepared according to the preparation method of the test solution. 10 μL of the control solution and the test solution were precisely taken, and determined according to the above chromatographic conditions. The theoretical plate number was calculated as 16650 based on the stilbene glycoside, indicating that the system suitability was good and could meet the test requirements.

[0076] 2.3 Durability test

[0077] According to the above method, the test was performed again under the condition of changing the instrument, using Shimadzu (SPD-M20A) liquid chromatograph, chromatographic column Agilent XDB-C18 (5 μm x 4.6 mm x 250 mm), and the rest of the conditions were unchanged. The same mixed control solution and test solution were taken for determination, and the theoretical plate number was calculated as 18066 based on the stilbene glycoside, indicating that the method had good durability.

[0078] 2.4 Precision test

[0079] The sample solution containing 61.312 μg / ml of stilbene glycoside was taken. According to the above chromatographic conditions, 6 times of sample injection were repeated, and the peak area was measured. The RSD of the peak area of stilbene glycoside was 1.4%, and the instrument precision was good.

[0080] 2.5 Accuracy test

[0081] Nine samples with known content were taken, and high, medium and low concentrations of stilbene glycoside control were added respectively. Three test solution samples were prepared for each concentration. The control solution and the test solution were taken according to the above chromatographic conditions, and the recovery rate was calculated. The recovery rate of stilbene glycoside was 95.6%, indicating that the method had good accuracy.

[0082] Table 2: Recovery test results of stilbene glycoside in sample

[0083]

[0084] 2.6 Sample stability test

[0085] 5.0 g of the sample was precisely weighed, and the test solution was prepared according to the method. After 0, 2, 4, 6, 8, 12 and 24 hours, the chromatographic conditions were determined. The RSD of the peak area of stilbene glycoside in the test solution was 1.2%, indicating that the test solution was stable within 24 hours.

[0086] 2.7 Reproducibility test

[0087] 5.0 g of the sample was precisely weighed, and the test solution was prepared according to the method. After 0, 2, 4, 6, 8, 12 and 24 hours, the chromatographic conditions were determined. The RSD of the peak area of stilbene glycoside in the test solution was 1.2%, indicating that the test solution was stable within 24 hours.

[0088] Comparative Example 1

[0089] Comparative Example 1 is different from Example 1 in the preparation method of the test sample solution, and the rest of the methods are the same.

[0090] About 5 g of Tongbi Huoluo Pills was weighed into a 50 mL conical flask, 50 mL of 50% ethanol was accurately added, the weight was determined, heated to reflux for 30 min, cooled, weighed again, the lost weight was made up with 50% ethanol, shaken well, diluted appropriately, the supernatant was filtered with a microporous filter membrane (0.45 μm), and the test sample solution was obtained.

[0091] The detection spectrum of Comparative Example 1 is shown in Figure 4 The results show that the test sample solution prepared by the method of Comparative Example 1 has large chromatographic interference, poor recovery rate, and low sample concentration, and cannot be effectively separated; after the test sample solution is prepared by the method of the application, the content of stibene glycoside is detected, Figure 3 The detection results show that the target peak appears between 15-20 min, and stibene glycoside can be effectively separated.

[0092] Comparative Example 2

[0093] 5 g of Tongbi Huoluo Pills was taken, 8 times of 50% ethanol was added, heated to reflux for extraction for 3 times, 2 hours each time, the extraction liquid was combined, concentrated to 1 / 20 of the total volume, the concentrated liquid was cooled and placed for 8 hours, filtered, and the filtrate was extracted with 1.5 times of ethyl acetate for 3 times, the extraction liquid was combined, and the test sample solution was obtained.

[0094] Figure 5 The results show that the test sample solution prepared by the method of Comparative Example 2 cannot be effectively separated for detecting stibene glycoside, has large chromatographic interference, and cannot accurately quantify the content of stibene glycoside in the sample.

[0095] The foregoing examples are merely illustrative, serving to explain some features of the method described in the application. The appended claims are intended to claim as broad a range as can be conceived, and the examples presented herein are merely illustrative of selected embodiments according to the combination of all possible embodiments. Therefore, it is the intention of the applicant that the appended claims not be limited by the selection of examples illustrating features of the application. Some numerical ranges used in the claims are also intended to include sub-ranges within the range, and variations in the ranges should be interpreted as being covered by the appended claims, if possible.

Claims

1. A HPLC method for detecting the content of styracolside in Tongbi Huoluo pills, characterized in that, The preparation of the sample solution of the Tongbi Huoluo pill includes: (1) The sample solution of the Tongbi Huoluo pill is dissolved in water to obtain a solution; (2) The solution is concentrated to near dryness, dissolved in a solvent allowed by the Pharmacopoeia, and then adsorbed by a macroporous adsorption resin, eluted with water and an alcohol solution in sequence, and collected to obtain an extract of the sample after removal of the solvent; The sample solution of the Tongbi Huoluo pill is dissolved in water to obtain a solution; The type of the macroporous adsorption resin is D101; The HPLC chromatographic conditions include: a chromatographic column: an octadecylsilane-bonded chromatographic column; a mobile phase: an organic phase: water, the organic phase being acetonitrile; a detection wavelength: 315-325 nm; and an elution mode: gradient elution, as shown in the following table 。 2. The HPLC method of claim 1, wherein, The mass-to-volume ratio of the sample solution of the Tongbi Huoluo pill to water is 1 g:(5-8) mL.

3. The HPLC method of claim 2, wherein, The mass-to-volume ratio of the sample solution of the Tongbi Huoluo pill to water is 1 g:6 mL.

4. The HPLC method of claim 1, wherein, The ultrasonic extraction conditions are: a temperature of 10-38℃, a time of 20-45 min, an ultrasonic frequency of 28-50 kHz, and an ultrasonic power of 200-250 V.

5. The HPLC method of claim 4, wherein, The ultrasonic extraction conditions are: a temperature of 18-30℃, a time of 30 min, an ultrasonic frequency of 28 kHz, and an ultrasonic power of 220 V.

6. The HPLC method of claim 1, wherein, The concentration method is at least one selected from vacuum concentration, nitrogen blowing concentration, evaporation concentration, and freeze concentration.

7. The HPLC method according to any one of claims 1 to 6, characterized in that, The method is used for detecting the content of stilbene glycoside contained in the Tongbi Huoluo pill.

8. The HPLC method according to any one of claims 1 to 6, characterized in that, The HPLC chromatographic conditions at least include at least one of the following: a column temperature: 30-38℃; a flow rate: 0.5-2 mL / min; an injection volume: 5-15 μL; and a chromatographic column specification: 3-5 μm, 4.6 mm×250 mm.

9. The HPLC method according to any one of claim 8, characterized in that, The HPLC chromatographic conditions at least include at least one of the following: a chromatographic column type: Agilent XDB-C18; a column temperature: 35℃; a flow rate: 1 mL / min; a detection wavelength: 320 nm; an injection volume: 10 μL; a chromatographic column specification: 5 μm, 4.6 mm×250 mm; and an elution mode: gradient elution, as shown in the following table:

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