Method for determining resveratrol in tetrastigma planicaule

Abstract

The invention relates to the technical field of resveratrol detection, in particular to a method for determining resveratrol in tetrastigma planicaule. Comprising the following steps: (1) preparation of a reference substance solution: taking a resveratrol reference substance, and adding methanol to prepare the reference substance solution; (2) preparation of a test solution: taking a tetrastigma planicaule medicinal material, adding methanol, performing ultrasonic treatment, filtering, evaporating filtrate to dryness, adding water into residues for dissolving, performing shaking extraction with ethyl acetate for 2-3 times, combining ethyl acetate solutions, performing vacuum concentration until the solution is dry, performing volume metering with methanol, and filtering to obtain the test solution; and (3) sucking 8-12 microliters of the reference substance solution and 8-12 microliters of the test solution, injecting the solutions into a liquid chromatograph, and performing determination. The method disclosed by the invention has strong tolerance to change of chromatographic conditions, is obviously superior to the prior art, provides a reliable analysis means for quality control of the tetrastigma planicaule medicinal material, and provides methodological reference for determination of resveratrol in other plants at the same time.

Description

Technical Field

[0001] This invention relates to the field of resveratrol detection technology, and in particular to a method for determining resveratrol in *Symplocos buergeriana*. Background Technology

[0002] Resveratrol is a natural polyphenolic compound with various biological activities, widely found in plants such as grapes and Japanese knotweed. Studies have shown that resveratrol has antioxidant, anti-inflammatory, anti-tumor, and cardiovascular protective effects, and has important application value in the fields of medicine and health products. As a traditional Chinese medicine, *Polygonum cuspidatum* has a complex chemical composition, but research on resveratrol within it is limited, and efficient and accurate determination methods are lacking.

[0003] Currently, the main methods for determining resveratrol include high-performance liquid chromatography (HPLC), gas chromatography (GC), and ultraviolet spectrophotometry. Among these, HPLC has become the mainstream technique due to its high sensitivity and selectivity. However, existing methods suffer from low extraction efficiency and poor separation when determining resveratrol in *Symplocos buergeriana*, making it difficult to meet practical detection needs. For example, some methods use single-solvent extraction or isocratic elution, resulting in incomplete extraction of the target component or incomplete separation from other interfering components.

[0004] Therefore, developing an efficient, accurate, and specific method for the determination of resveratrol in *Symplocos buergeriana* is of great significance. This method requires optimization of the extraction process and chromatographic conditions to improve extraction efficiency and separation effect, providing technical support for the quality control and further development and utilization of *Symplocos buergeriana*. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining resveratrol in *Symplocos buergeriana*.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for determining resveratrol in *Symplocos buergeriana*, comprising the following steps:

[0008] (1) Preparation of reference solution: Take resveratrol reference standard and add methanol to prepare reference solution;

[0009] (2) Preparation of test solution: Take the herb of *Symplocos buergeriana*, add methanol and sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate by shaking 2-3 times, combine the ethyl acetate solutions, concentrate under reduced pressure to dryness, make up to volume with methanol, filter, and the test solution is obtained.

[0010] (3) Inject 8-12 μl each of the reference solution and the test solution into the liquid chromatograph and determine the solution; wherein, octadecylsilane-bonded silica gel is used as the stationary phase; acetonitrile is used as mobile phase A and 0.1% formic acid is used as mobile phase B, and the gradient elution program is shown in Table 1:

[0011] Table 1

[0012]

[0013] Preferably, the wavelength is set to 300-320 nm during chromatograph detection in step (3).

[0014] Preferably, the column temperature is set to 33-37°C during chromatograph detection in step (3).

[0015] Preferably, the ratio of the reference herb *Platycarpus stenoptera* in step (1) to methanol is 2g:90-110ml.

[0016] Preferably, the power of the ultrasound in step (1) is 450-550W and the time is 13-17min.

[0017] Preferably, the concentration of the resveratrol reference solution in step (1) is 45–55 μg / ml.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The method for determining resveratrol in *Smilax china* provided by this invention has significant technical advantages. By optimizing the extraction solvent, ultrasonic treatment conditions, and gradient elution program, the extraction efficiency and chromatographic separation effect of resveratrol are significantly improved. Simultaneously, the innovative gradient elution program achieves baseline separation of resveratrol from other components, ensuring the accuracy and repeatability of the detection.

[0020] The technical solution of this invention exhibits excellent performance indicators in methodological validation. It has a wide linear range, a correlation coefficient of 0.9995, and a stable recovery rate of 100.3%. This method is highly tolerant of variations in chromatographic conditions, significantly superior to existing technologies, providing a reliable analytical means for the quality control of *Cynanchum paniculatum* medicinal materials, and also providing a methodological reference for the determination of resveratrol in other plants. Attached Figure Description

[0021] 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 only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 Chromatograms of the test sample and mixed reference standard; Peak 1: protocatechuic acid; Peak 2: protocatechuic aldehyde; Peak 3: leucine; Peak 4: resveratrol;

[0023] Figure 2 Resveratrol standard curve. Detailed Implementation

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] 1. Instruments and reagents

[0027] Agilent 1260 high-performance liquid chromatograph (Agilent Technologies, USA); electronic analytical balance (model: GL224I-1SCN, Sartorius, Germany; XSR205DU, Mettler Toledo Group); rotary evaporator (model: RV10, Aika (Guangzhou) Instrument Equipment Co., Ltd.); CNC ultrasonic cleaner (KQ2200E, Kunshan Ultrasonic Instrument Co., Ltd.); digital display precision constant temperature water bath (DK-98-ⅡA, Tianjin Test Instrument Co., Ltd.).

[0028] Resveratrol reference standard (batch number 111535-201703, content calculated as 99.4%, China National Institutes for Food and Drug Control); Paclitaxel reference standard (batch number 2415698, content calculated as 98.0%, Shanghai Anpu Cuishi Standard Technical Service Co., Ltd.); Protocatechuic acid reference standard (batch number 110809-202207, content calculated as 97.5%, China National Institutes for Food and Drug Control); Protocatechuic aldehyde reference standard (batch number 110810-202210, content calculated as 99.9%, China National Institutes for Food and Drug Control); 16 batches of *Smilax china* slices and 3 batches of *Spatholobus suberectus* (YP2025JK1004, YP2025JK1005, YP2025JK1006) slices, batch numbers and places of origin are shown in Table 2; Acetonitrile (chromatographic grade), water (ultrapure water), and other reagents were all analytical grade.

[0029] Table 2. Batch numbers and places of origin for *Smilax china* and *Spatholobus suberectus*.

[0030]

[0031]

[0032] 2 methods

[0033] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and 0.1% formic acid as mobile phase B. Gradient elution was performed according to the specifications in Table 1. The detection wavelength was 310 nm, the flow rate was 0.8 ml / min, the column temperature was 35 °C, and the theoretical plate number calculated based on the resveratrol peak should not be less than 5000.

[0034] Preparation of the reference solution: Take an appropriate amount of resveratrol reference standard, accurately weigh it, and add methanol to prepare a solution containing 50 μg per ml.

[0035] Preparation of the test solution: Take 2.0 g of *Smilax china* herb and place it in an Erlenmeyer flask. Add 100 ml of methanol and sonicate for 15 minutes. Filter, evaporate the filtrate to dryness, dissolve the residue in 15 ml of water, and extract twice with 20 ml of ethyl acetate each time. Combine the ethyl acetate extracts, concentrate to dryness under reduced pressure, and dilute to 5 ml with methanol. Filter to obtain the test solution. Separately, accurately weigh an appropriate amount of resveratrol reference standard and prepare a solution containing 50 μg per ml with methanol.

[0036] The assay involves precisely pipetting 10 μl of each test solution and injecting it into the liquid chromatograph for determination.

[0037] Example 2

[0038] 1. Investigation of the extraction solvent (all subsequent analyses are based on the method in Example 1)

[0039] Accurately weigh 2g of *Smilax china* herb and place it in a stoppered conical flask. Add 50ml each of solvents (water, 50% methanol, 70% methanol, and methanol) to the flask. Sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 15ml of water, and extract twice with 20ml of ethyl acetate each time. Combine the ethyl acetate extracts, concentrate to dryness under reduced pressure, and dilute to 5ml with methanol. Filter to obtain the final product. Separately, accurately weigh an appropriate amount of resveratrol reference standard and prepare a solution containing 50μg per ml with methanol. Accurately inject 10μl each of the reference solution and the test solution, determine the peak area, and calculate the content. The results are shown in Table 3.

[0040] Table 3. Investigation of extraction solvents

[0041]

[0042] The results showed that methanol extraction yielded a higher resveratrol content, therefore methanol was chosen as the extraction solvent.

[0043] 2. Investigation on the amount of extraction solvent used

[0044] Accurately weigh 2g of *Smilax china* herb and place it in a stoppered conical flask. Add methanol (50ml, 100ml, and 150ml) to the flasks respectively, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 15ml of water, and extract twice with ethyl acetate (20ml each time). Combine the ethyl acetate extracts, concentrate to dryness under reduced pressure, and dilute to 5ml with methanol. Filter to obtain the final product. Separately, accurately weigh an appropriate amount of resveratrol reference standard and prepare a solution containing 50μg per ml with methanol. Accurately inject 10μl each of the reference solution and the test solution, determine the peak area, and calculate the content. The results are shown in Table 4.

[0045] Table 4. Investigation of Extraction Solvent Dosage

[0046]

[0047]

[0048] The results showed that the resveratrol content was low when the solvent volume was 50 ml, and there was no significant difference in resveratrol content when the solvent volumes were 100 ml and 150 ml. To ensure complete extraction and to save solvent, it is recommended that the extraction solvent volume be 100 ml.

[0049] 3. Examination of ultrasound time

[0050] Accurately weigh 2g of *Smilax china* herb and place it in a stoppered conical flask. Add 100ml of methanol and sonicate for 15min, 30min, and 60min respectively. Filter, evaporate the filtrate to dryness, dissolve the residue in 15ml of water, and extract twice with 20ml of ethyl acetate each time. Combine the ethyl acetate extracts, concentrate to dryness under reduced pressure, and dilute to 5ml with methanol. Filter to obtain the final product. Separately, accurately weigh an appropriate amount of resveratrol reference standard and prepare a solution containing 50μg per ml with methanol. Accurately inject 10μl each of the reference solution and the test solution, determine the peak area, and calculate the content. The results are shown in Table 5.

[0051] Table 5. Examination of ultrasound time

[0052]

[0053] The results showed that there was no significant difference in resveratrol content after 15 min, 30 min, and 60 min of sonication. Considering the need to save time, it is recommended to sonicate for 15 min.

[0054] 4. Examination of extraction methods

[0055] Accurately weigh 2g of *Smilax china* herb and place it in a stoppered conical flask. Add 100ml of methanol, sonicate and reflux for 15min each, filter, evaporate the filtrate to dryness, dissolve the residue in 15ml of water, and extract twice with 20ml of ethyl acetate each time. Combine the ethyl acetate extracts, concentrate to dryness under reduced pressure, and dilute to 5ml with methanol. Filter to obtain the final product. Separately, accurately weigh an appropriate amount of resveratrol reference standard and prepare a solution containing 50μg per ml with methanol. Accurately inject 10μl each of the reference solution and the test solution, determine the peak area, and calculate the content. The results are shown in Table 6.

[0056] Table 6. Examination of Extraction Methods

[0057]

[0058] The results showed that there was no significant difference in resveratrol content between ultrasonic and reflux extraction for 15 min, so ultrasonic extraction for 15 min was chosen.

[0059] 5. Investigation of extraction solvents

[0060] Accurately weigh 2g of *Smilax china* herb and place it in a stoppered conical flask. Add 100ml of methanol and sonicate for 15min each time. Filter, evaporate the filtrate to dryness, dissolve the residue in 15ml of water, and extract twice with 20ml each time using chloroform, ether, and ethyl acetate. Combine the chloroform, ether, and ethyl acetate extracts, concentrate to dryness under reduced pressure, and dilute to 5ml with methanol. Filter to obtain the final product. Separately, accurately weigh an appropriate amount of resveratrol reference standard and prepare a solution containing 50μg per ml using methanol. Accurately inject 10μl of both the reference solution and the test solution, determine the peak area, and calculate the content. The results are shown in Table 7.

[0061] Table 7. Investigation of extraction solvents

[0062]

[0063] The results showed that ethyl acetate extraction yielded a high content of resveratrol, therefore ethyl acetate was chosen as the extraction solvent.

[0064] 6. Examination of the number of extractions

[0065] Accurately weigh 2g of *Smilax china* herb and place it in a stoppered conical flask. Add 100ml of methanol and sonicate for 15min. Filter, evaporate the filtrate to dryness, dissolve the residue in 15ml of water, and extract with ethyl acetate 1, 2, 3, and 4 times, 20ml each time. Combine the ethyl acetate extracts, concentrate to dryness under reduced pressure, and dilute to 5ml with methanol. Filter to obtain the final product. Separately, accurately weigh an appropriate amount of resveratrol reference standard and prepare a solution containing 50μg per ml with methanol. Accurately inject 10μl of both the reference solution and the test solution, determine the peak area, and calculate the content. The results are shown in Table 8.

[0066] Table 8. Examination of the number of extractions

[0067]

[0068] The results showed that there was no significant difference in resveratrol content after extraction 1, 2, 3, and 4 times. To ensure complete extraction and to save solvent, it is recommended to extract twice.

[0069] 7. Investigation on the amount of extraction solvent used

[0070] Accurately weigh 2g of *Smilax china* herb and place it in a stoppered conical flask. Add 100ml of methanol and sonicate for 15min. Filter the mixture, evaporate the filtrate to dryness, dissolve the residue in 15ml of water, and extract twice with 10ml, 20ml, and 30ml of ethyl acetate respectively. Combine the ethyl acetate extracts, concentrate to dryness under reduced pressure, and dilute to 5ml with methanol. Filter the mixture to obtain the final product. Separately, accurately weigh an appropriate amount of resveratrol reference standard and prepare a solution containing 50μg per ml with methanol. Accurately inject 10μl of both the reference solution and the test solution, determine the peak area, and calculate the content. The results are shown in Table 9.

[0071] Table 9. Investigation of Extraction Solvent Dosage

[0072]

[0073] The results showed that there was no significant difference in resveratrol content when extracted with 10ml, 20ml, and 30ml of ethyl acetate. However, it was found during the experiment that the sample was easily emulsified and difficult to separate when extracted with 10ml of ethyl acetate. Therefore, it is recommended that the extraction solvent volume be 20ml.

[0074] In summary, the method for preparing the test sample is as follows: Take 2.0g of *Symplocos buergeriana* herb, place it in an Erlenmeyer flask, add 100ml of methanol, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, add 15ml of water to dissolve the residue, extract twice with ethyl acetate, 20ml each time, combine the ethyl acetate extracts, concentrate under reduced pressure to dryness, make up to 5ml with methanol, filter, and the sample is obtained.

[0075] Example 3

[0076] 1. Specificity test (all subsequent tests are based on the method in Example 1)

[0077] Use the blank extraction solvent as the negative control solution. Inject 10 μl each of the negative control solution, the test solution, and the reference solution into the liquid chromatograph, and analyze under the chromatographic conditions suitable for the system's suitability. Results are shown in the table below. Figure 1 .

[0078] The results showed that the negative sample had no corresponding chromatographic peak at the corresponding position of the analyte in the test sample, indicating that the negative sample did not interfere with the detection and that the method has good specificity.

[0079] 2. Examination of the linear range

[0080] Weigh 10.03 mg (99.4%) of resveratrol reference standard and place it in a 20 mL volumetric flask. Dissolve and dilute to the mark with methanol, and shake well to obtain a resveratrol reference standard stock solution with a concentration of 0.4985 mg / mL. Accurately pipette 1.0 mL, 1.0 mL, 1.0 mL, 1.0 mL, 1.0 mL, 1.0 mL, and 1.0 mL of the above resveratrol reference standard stock solution into volumetric flasks of 200 mL, 100 mL, 50 mL, 25 mL, 10 mL, and 5 mL, respectively. Dilute to the mark with methanol and shake well to obtain the reference solutions for curves 1 to 6. Inject 10 μL of each reference solution for curves 1 to 6 into the liquid chromatograph for determination.

[0081] The peak area was measured under the above chromatographic conditions. A standard curve was plotted with the injection volume (μg) on ​​the x-axis and the peak area (mAU) on the y-axis. The regression equation was calculated as follows:

[0082] The regression equation for resveratrol is: Y = 10012X + 13.905 (r = 0.9995, n = 6).

[0083] The results showed that when the injection amount of resveratrol was in the range of 0.0249 μg to 0.9970 μg, there was a good linear relationship between the injection amount and the peak area. Detailed results are shown in Table 10, and the standard curve is shown below. Figure 2 .

[0084] Table 10. Investigation of the linear range of resveratrol

[0085]

[0086] 3. Precision test

[0087] Take the reference solution, inject it 6 times, measure the peak area, and calculate the RSD value. The results are shown in Table 11.

[0088] Table 11 Precision Test Results

[0089]

[0090] The results showed that the RSD value of the resveratrol peak area was less than 3.0%, indicating that the instrument had good precision.

[0091] 4. Stability Test

[0092] Take the test solution and inject it at 0h, 8h, 12h, 20h, 24h and 28h respectively. Measure the peak area and calculate the RSD value. The results are shown in Table 12.

[0093] Table 12 Stability Test Results

[0094]

[0095] The results showed that the RSD value of the resveratrol peak area was less than 3.0%, indicating that the test solution had good stability within 28 hours.

[0096] 5 Repeatability Test

[0097] Take 6 portions of *Symplocos buergeriana* powder, each approximately 2g, and accurately weigh them. Prepare the test solution according to the preparation method of the test solution. Accurately inject 10μl of each test solution into the sample, determine the peak area, calculate the content and RSD value. The results are shown in Table 13.

[0098] Table 13 Results of Repeatability Tests

[0099]

[0100] The results showed that the RSD value of resveratrol content was less than 3.0%, indicating that the method had good reproducibility.

[0101] 6 Recovery rate test

[0102] Take 6 portions of the powder with known content, each approximately 1 g, accurately weigh them, and accurately add an appropriate amount of reference standard to each. Follow the preparation method for the test solution, accurately pipetting 10 μl of each solution. Measure the peak area using the chromatographic conditions described above, and calculate the recovery rate using the following formula. The results are shown in Table 14.

[0103]

[0104] Table 14 Resveratrol Recovery Test

[0105]

[0106] The results show that the average recovery rate of resveratrol was 100.3%, and the RSD value was less than 3%, indicating that the method has good accuracy.

[0107] 7. Durability Test

[0108] Accurately weigh approximately 2g of the powder and prepare the test solution according to the method for preparing the test solution. Under the established chromatographic conditions, use different brands of columns of the same specifications, with minor variations in column temperature and flow rate to determine the sample content and examine the robustness of the chromatographic conditions. Accurately inject 10μl each of the reference solution and the test solution into the sample for determination, calculate the content and RSD value, and the results are shown in Table 15.

[0109] Table 15 Durability Tests

[0110]

[0111] Column 1: CAPCELL PAK C18 MGIII (4.6 × 250 mm, 5 μm)

[0112] Column 2: YMC Hydrosphere C18 (4.6 × 250 mm, 5 μm)

[0113] Column 3: Welch Ultimate Plus C18 (4.6 × 250 mm, 5 μm)

[0114] The results show that the RSD value of resveratrol content is less than 3.0% when the chromatographic conditions are slightly varied and different brands of chromatographic columns are used, indicating that the method has good robustness.

[0115] 8. Intermediate Precision Test

[0116] The effect of different instruments on the precision was investigated. The *Phyllostachys edulis* herb was used, and a test solution was prepared according to the method for preparing the test solution. 10 μl of each of the reference solution and the test solution were precisely pipetted into the solution, and the peak area was measured. The content was calculated, and the results are shown in Table 16.

[0117] Table 16 Intermediate Precision Test

[0118]

[0119] Instrument 1: Agilent 1260 Infinity

[0120] Instrument 2: Agilent 1200

[0121] The results showed that the RSD values ​​of the index components in the samples measured under different instruments were less than 3.0%, indicating that the intermediate precision of this method was good.

[0122] 9 Samples were tested

[0123] Take 16 batches of *Pinellia ternata* slices, about 2g each, accurately weigh them, prepare the test solution according to the preparation method of the test solution, and determine the results under the above chromatographic conditions. The results are shown in Table 17.

[0124] Table 17 Results of the content determination of *Pinellia ternata* slices in 16 batches (n=2)

[0125]

[0126]

[0127] As shown in the table above, the resveratrol content of 16 batches of *Biandan Teng* (a type of vine) slices ranged from 0.010 mg / g to 0.152 mg / g.

[0128] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining the amount of resveratrol in Polygonum perfoliatum Houtt, comprising the steps of: It comprises the following steps: (1) Preparation of the control solution: take the control sample of white wine alcohol, and add methanol to prepare the control solution; (2) Preparation of the sample solution: take the medicine of flat pole, and add methanol to prepare the sample solution; (3) Take 8-12 μl of the control solution and the sample solution, and inject them into the liquid chromatograph to determine, wherein the filling agent is octadecylsilane bonded silica gel, the mobile phase A is acetonitrile, the mobile phase B is 0.1% formic acid, and the gradient elution program is shown in Table 1: Table 1 2. The assay method according to claim 1, characterized by In step (3), the wavelength is set to 300-320 nm when the chromatograph is detected.

3. The assay method according to claim 1, characterized by, In step (3), the column temperature is set to 33-37 ℃ when the chromatograph is detected.

4. The assay method according to claim 1, characterized by, In step (1), the ratio of the flat pole control medicine to methanol is 2 g: 90-110 ml.

5. The assay method according to claim 1, characterized by, In step (1), the power of the ultrasonic is 450-550 W, and the time is 13-17 min.

6. The assay method according to claim 1, characterized by, In step (1), the concentration of the white wine alcohol control sample solution is 45-55 μg / ml.

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