Method for measuring contents of tenuifolin and sinapic acid in hydrolysate of traditional Chinese medicine polygala tenuifolia by dual wavelengths

Abstract

The invention discloses a dual-wavelength method for determining the content of tenuifolin and sinapic acid in hydrolysate of traditional Chinese medicine polygala tenuifolia, and belongs to the technical field of analysis of traditional Chinese medicine components. The method comprises the following steps: respectively injecting a test solution and a mixed reference solution into a high performance liquid chromatograph for determination by adopting a diode array detector; chromatographic conditions are as follows: a 0.05 vol% formic acid acetonitrile solution is taken as a mobile phase A, a 0.05 vol% formic acid aqueous solution is taken as a mobile phase B, and gradient elution is carried out; the gradient elution procedure is 0-10 min, 80% B to 70% B, and the detection wavelength is 320 mm; in 10-20 min, 70% B to 50% B, and the detection wavelength is 192 mm; the preparation process of the test solution comprises the following steps: extracting by using 70 vol% ethanol with the use amount of 20 mL / g polygala tenuifolia powder, carrying out ultrasonic extraction for 60 minutes, filtering, drying by distillation, hydrolyzing, adjusting the pH value, extracting by using water-saturated n-butyl alcohol, drying by distillation, and redissolving to obtain the test solution.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine component analysis, and particularly relates to a method for determining the contents of tenuifolin and sinapic acid in traditional Chinese medicine polygala. BACKGROUND

[0002] Traditional Chinese medicine polygala has a long history. The 1953 edition of Chinese Pharmacopoeia includes polygala, and stipulates that the base is polygala Polygala tenuifolia Willd., and the base was increased to polygala Polygala sibirica L. in the 1977 edition of Chinese Pharmacopoeia, and has been used to date. The 2020 edition of Chinese Pharmacopoeia includes 33 traditional Chinese medicine containing polygala. The “Kai Xian San”, “Dihuang Yinzi” and “Guyin Jian” containing polygala in the prescription have been included in the “Ancient Classical Prescription Catalogue (First Batch)” published by the State Administration of Traditional Chinese Medicine. Since the 1953 edition of Chinese Pharmacopoeia includes polygala and two preparations of polygala tincture and polygala flow extract, the quality standard of polygala has been continuously improved.

[0003] According to the prior art, the determination methods of polygala content are mainly divided into two categories. The first method is to directly determine part of oligosaccharide ester components and uranolone components in polygala medicinal materials. The second method is to indirectly determine polygalic acid hydrolysate (including polygalasapogenin and polygalic acid) or alkali hydrolysate (such as tenuifolin) to evaluate the total saponin content of polygala. At present, the 2025 edition of Chinese Pharmacopoeia adopts the two methods to jointly detect 3,6'-disinapoyl sucrose and tenuifolin in polygala. However, this process is relatively cumbersome, and polygala has many components, and the content of some components cannot completely reflect the quality information of the medicinal materials.

[0004] In the previous characteristic spectrum experiment combined with mass spectrometry identification of chemical components, it is found that the main components of polygala medicinal materials include oligosaccharide ester components and triterpenoid saponin components, and the peak area of uranolone components is relatively low. Through in-depth analysis of the chemical structure of polygala, it is found that most of the oligosaccharide ester components contain sinapoyl groups, and these components can generate sinapic acid after alkali hydrolysis; and the triterpenoid saponin components are converted into tenuifolin and organic acid after alkali hydrolysis.

[0005] Therefore, the present patent proposes a new method which can simultaneously determine the contents of tenuifolin and sinapic acid. The establishment of this method can reflect the contents of total sucrose ester components and total saponin components to a certain extent; at the same time, the sample pretreatment scheme is optimized, which can effectively improve the quality control level of polygala medicinal materials, and provide reliable technical support for the clinical application of polygala. SUMMARY

[0006] The application provides a method for determining the contents of polygalasaponin 38 and sinapinic acid in polygalae radix hydrolysate by dual-wavelength determination, which improves the accuracy and efficiency of traditional Chinese medicine component analysis, reduces the use of solvents by optimizing the extraction method, is more economical and environmentally friendly, and has important significance for traditional Chinese medicine quality control, efficacy evaluation and modernization research of traditional Chinese medicine. The application provides a method for determining the contents of polygalasaponin 38 and sinapinic acid in polygalae radix hydrolysate by dual-wavelength determination, which improves the accuracy and efficiency of traditional Chinese medicine component analysis, reduces the use of solvents by optimizing the extraction method, is more economical and environmentally friendly, and has important significance for traditional Chinese medicine quality control, efficacy evaluation and modernization research of traditional Chinese medicine. Preparation of the control solution: sinapinic acid and polygalasaponin 38 standard samples are prepared into a mixed control solution of the standard samples.

[0007] Preparation of the test solution: the test solution is prepared by using the test sample.

[0008] The test solution and the mixed control solution are injected into a high-performance liquid chromatograph for determination by using a diode array detector.

[0009] Chromatographic conditions: octadecylsilane-bonded silica gel is used as the filler; 0.03-0.07vol% formic acid acetonitrile solution is used as the mobile phase A, and 0.03-0.07vol% formic acid aqueous solution is used as the mobile phase B for gradient elution; the gradient elution program is as follows: 0-10min, 75-85%B→65-75%B, the detection wavelength is 320mm.

[0010] 10-20min, 65-75%B→45-55%B, the detection wavelength is 192mm.

[0011] The column temperature is 25-35℃, and the flow rate is 0.5-1.5ml / min.

[0012] The preparation process of the test solution is as follows: 65-75vol% ethanol is used for extraction, the ethanol amount is 15-25mL / g of polygalae radix powder, ultrasonic extraction is performed for 45-90min, filtration is performed, evaporation is performed, 5-15wt% sodium hydroxide solution is added to the residue, heating reflux treatment is performed for 1.5-2.5h, the amount of the sodium hydroxide solution is 1.5-3.0 times the volume of the filtrate; dilute hydrochloric acid is used to adjust the pH value to 3-5, water-saturated n-butanol is used for extraction for 2-3 times, the amount of the extraction agent is 1.5-3.0 times the volume of the filtrate, the n-butanol liquid (extraction liquid) is combined, evaporation is performed, and the residue is dissolved in 65-75vol% ethanol and constant volume is performed.

[0013] The preparation process of the mixed reference solution is as follows: the reference substances sinapinic acid and polygalaxn are weighed and placed in a brown volumetric flask, and 65-75 vol% (preferably 70 vol%) of an ethanol solution is added to prepare a mixed reference solution with a concentration of 0.20 mg / mL (sinapinic acid) and 0.50 mg / mL (polygalaxn).

[0014] Specifically, the preparation process of the test sample solution is as follows: 1 g of polygalaxn powder is placed in a conical flask, which is tightly capped, 20 mL of 70 vol% ethanol is added, and ultrasonic treatment is performed for 1 h, and then the weight is determined again, and the weight loss is made up with 70 vol% ethanol, and then it is shaken and filtered. 10 mL of the filtrate is measured, and then it is evaporated to dryness, 20 mL of 10 wt% sodium hydroxide solution is added to the residue, and then it is heated to reflux at 100°C for 2 h, and then it is cooled. The pH value is adjusted to 3-5 with dilute hydrochloric acid, and then it is extracted with n-butanol saturated with water twice, each time with 20 mL, and then the n-butanol liquid is combined and evaporated to dryness, and then the residue is dissolved in an appropriate amount of 70 vol% ethanol, and then it is transferred to a 25 m volumetric flask, and then it is diluted and shaken, and then it is filtered through a 0.22 μm microporous filter to obtain the test sample solution. The ultrasonic treatment conditions are as follows: power 160 W, frequency 40 kHz.

[0015] Preferably, the chromatographic conditions are as follows: octadecylsilane-bonded silica gel is used as the filler; 0.05 vol% formic acid acetonitrile solution is used as the mobile phase A, and 0.05 vol% formic acid aqueous solution is used as the mobile phase B, and gradient elution is performed; and the gradient elution program is as follows: 0-10 min, 80% B→70% B, and the detection wavelength is 320 mm.

[0016] 10-20 min, 70% B→50% B, and the detection wavelength is 192 mm.

[0017] The flow rate is 1 ml / min, and the injection volume is 2 uL.

[0018] The chromatographic column is selected from Waters Atlantis T3 (4.6×250 mm, 5 μm), Agilent Eclipse XDB C 18 (4.6×250 mm, 5 μm), or Agilent Zorbax SB C 18 (4.6×250 mm, 5 μm), etc. The chromatographic column has little effect on the results of the present patent.

[0019] Under the optimized conditions, the linear regression equation of sinapinic acid is Y=9432.2X+37.814, and the linear regression equation of polygalaxn is Y=1212.9X+34.494. The retention time of sinapinic acid is 8.077 min, and the retention time of polygalaxn is 18.151 min.

[0020] Advantages of the present patent: (1) This method optimizes the extraction method of Polygala tenuifolia hydrolysis products by systematically optimizing key parameters of sample pretreatment (extraction solvent, amount of extraction solvent, extraction time, pH range, amount of hydrolysis solvent, hydrolysis time, number of extractions and resolution solvent), making it more economical and environmentally friendly.

[0021] (2) This method uses dual-wavelength determination combined with gradient elution technology to achieve rapid and accurate determination of the content of polygala saponins and sinapic acid in the hydrolysis products of the traditional Chinese medicine Polygala tenuifolia. It transforms the two methods in the pharmacopoeia into one method, which can reflect the metabolic level of polygala triterpenoid saponins and oligosaccharide esters. Moreover, the reference standard is cheaper, which can save a certain amount of time and money. This method solves the problem of the limitation of a single indicator and the cumbersome operation caused by the coexistence of multiple components in the quality evaluation of polygala tenuifolia.

[0022] (3) It has high accuracy, low detection limit and good reproducibility. Attached Figure Description

[0023] Figure 1 This is a chromatogram of the hydrolysis products of Polygala tenuifolia; Figure 2 This is a linear relationship graph of sinapic acid; Figure 3 This is a linear relationship graph of saponins from Polygala tenuifolia. Detailed Implementation

[0024] The scope of protection of this invention is not limited to the following embodiments. Those skilled in the art can make adjustments to one or more steps, and all such adjustments are within the scope of protection of this application without departing from the essence of this invention.

[0025] (1) Preparation of test solution Accurately weigh approximately 1g of Polygala tenuifolia powder (passed through a No. 3 sieve), place it in a conical flask and seal tightly. Accurately add 20mL of 70% ethanol, weigh, and sonicate (power 160W, frequency 40kHz) for 1 hour. Cool, weigh again, and make up the weight loss with 70% ethanol. Shake well, filter, and accurately measure 10mL of the filtrate. Evaporate to dryness, add 20mL of 10% sodium hydroxide solution to the residue, heat under reflux at 100℃ for 2 hours, cool, and adjust the pH to 3-5 with dilute hydrochloric acid (approximately 18.6mL). Extract twice with 20mL of water-saturated n-butanol each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue with an appropriate amount of 70% ethanol, transfer to a 25mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the final product.

[0026] (2) Preparation of reference solution Accurately weigh appropriate amounts of sinapic acid and polygala saponins (reference standards), place them in a brown volumetric flask, and add 70% ethanol solution to prepare a solution with a concentration of 0.20 mg / mL. -1 and 0.50 mg·mL-1 Mixed control solution.

[0027] (3) Determination method The obtained test sample solution and control solution were injected into a high performance liquid chromatograph for determination by using a diode array detector (DAD detector), and corresponding chromatograms were obtained; the determination conditions of the high performance liquid chromatograph met the following: Chromatographic column: octadecylsilane-bonded silica gel, 4.6*250 mm, 5 μm.

[0028] Chromatographic column temperature: 25-35 °C.

[0029] Mobile phase: 0.05 vol% formic acid acetonitrile solution (A); 0.05 vol% formic acid aqueous solution (B).

[0030] Gradient elution: 0-10 min, 80%B→70%B, 10-20 min, 70%B→50%B.

[0031] Determination wavelength: 0-10 min, 320 mm; 10-20 min, 192 mm.

[0032] Flow rate of mobile phase: 1.0 mL·min -1 .

[0033] Injection amount: 2 uL.

[0034] I. Investigation on preparation method of test sample solution 1. Investigation on extraction solvent The test sample solution was prepared according to the operation method for extracting polygalasaponin in 2025 Chinese Pharmacopoeia, and the influence of extraction solvent (70% methanol, 70% ethanol, 95% ethanol) on the contents of sinapinic acid and polygalasaponin was investigated. The results showed that the content of sinapinic acid was 70% methanol > 70% ethanol > 95% ethanol, the content of polygalasaponin was 70% ethanol > 70% methanol > 95% ethanol, and the total content of 70% ethanol extract was higher. Considering the green chemistry concept, 70% ethanol was selected as the extraction solvent. The data are shown in Table 1.

[0035] Table 1 Results of investigation on extraction solvent

[0036] 2. Investigation on amount of extraction solvent The test sample solution was prepared by the method for extracting polygalasaponins in 2025 Chinese Pharmacopoeia, and the extraction solvent was changed to 70% ethanol. The effects of the solvent amount (10, 20, 50 mL) on the contents of sinapinic acid and polygalasaponins were investigated. The results showed that with the increase of the extraction solvent amount, the content of sinapinic acid slightly decreased, the content of polygalasaponins slightly increased, and the total content had little difference. Considering the actual operation difficulty and solvent saving, the extraction solvent amount was selected as 20 mL. The data are shown in Table 2.

[0037] Table 2 Results of the investigation of the extraction solvent amount

[0038] 3. Investigation of extraction time The test sample solution was prepared by the method for extracting polygalasaponins in 2025 Chinese Pharmacopoeia, and the extraction solvent was changed to 70% ethanol. The solvent amount was changed to 20 mL. The effects of the ultrasonic time (15, 30, 45, 60 min) on the contents of sinapinic acid and polygalasaponins were investigated. The results showed that with the increase of the extraction time, the content of sinapinic acid changed little, and the content of polygalasaponins increased. The extraction time was selected as 60 min. The data are shown in Table 3.

[0039] Table 3 Results of the investigation of the extraction time

[0040] 4. Investigation of pH value The test sample solution was prepared by the method for extracting polygalasaponins in 2025 Chinese Pharmacopoeia, and the extraction solvent was changed to 70% ethanol. The solvent amount was changed to 20 mL. The ultrasonic time was unchanged. The effects of the pH value range (2-5) on the contents of sinapinic acid and polygalasaponins were investigated. The results showed that with the increase of the pH value, the content of sinapinic acid changed little, and the content of polygalasaponins increased. Therefore, the pH value range was selected as 3-5. The data are shown in Table 4.

[0041] Table 4 Results of the investigation of the pH value

[0042] 5. Investigation of the amount of hydrolysis solvent The test sample solution was prepared by the method for extracting polygalasaponins in 2025 Chinese Pharmacopoeia, and the extraction solvent was changed to 70% ethanol. The solvent amount was changed to 20 mL. The ultrasonic time was unchanged. The pH value range was changed to 3-5. The effects of the amount of 10 wt% sodium hydroxide solution as the hydrolysis solvent (10, 20, 50 mL) on the contents of sinapinic acid and polygalasaponins were investigated. The results showed that when the amount of the hydrolysis solvent was 20 mL and 50 mL, the contents of sinapinic acid and polygalasaponins had little difference, and the total content was consistent. Therefore, the amount of the hydrolysis solvent was selected as 20 mL. The data are shown in Table 5.

[0043] Table 5 Results of hydrolysis solvent amount investigation

[0044] 6. Investigation of hydrolysis time Referring to the method for extracting polygalasaponin in 2025 edition of Chinese Pharmacopoeia, the test sample solution was prepared, the extraction solvent was changed to 70% ethanol, the solvent amount was changed to 20 mL, the ultrasonic time was unchanged, the pH value range was changed to 3-5, the amount of 10 wt% sodium hydroxide solution was 20 mL, and the influence of reflux hydrolysis time (1, 2, 3 h) on the contents of sinapinic acid and polygalasaponin was investigated. The results showed that when the hydrolysis time was 2 h, sinapinic acid and polygalasaponin could be completely hydrolyzed, and the total content was the highest, so the hydrolysis time was unchanged. The data are shown in Table 6.

[0045] Table 6 Results of hydrolysis time investigation

[0046] 7. Investigation of extraction times Referring to the method for extracting polygalasaponin in 2025 edition of Chinese Pharmacopoeia, the test sample solution was prepared, the extraction solvent was changed to 70% ethanol, the solvent amount was changed to 20 mL, the ultrasonic time was unchanged, the pH value range was changed to 3-5, the amount of 10 wt% sodium hydroxide solution was 20 mL, the reflux time was unchanged, and the influence of extraction times (1, 2, 3 times) on the contents of sinapinic acid and polygalasaponin was investigated. The results showed that when the extraction times were 2 and 3 times, the contents of sinapinic acid and polygalasaponin were basically consistent, and considering the saving of solvent, the extraction times were selected to be 2 times. The data are shown in Table 7.

[0047] Table 7 Results of extraction times investigation

[0048] 8. Investigation of redissolving solvent Referring to the method for extracting polygalasaponin in 2025 edition of Chinese Pharmacopoeia, the test sample solution was prepared, the extraction solvent was changed to 70% ethanol, the solvent amount was changed to 20 mL, the ultrasonic time was unchanged, the pH value range was changed to 3-5, the amount of 10 wt% sodium hydroxide solution was 20 mL, the reflux time was unchanged, the extraction times were changed to 2 times, and the influence of redissolving solvent (methanol, anhydrous ethanol, 70% ethanol) on the contents of sinapinic acid and polygalasaponin was investigated. The results showed that the contents of sinapinic acid in the test sample solutions redissolved by the three kinds of solvents were relatively similar, the content of polygalasaponin was 70% ethanol > methanol > anhydrous ethanol, and considering the green chemistry concept, 70% ethanol was selected as the redissolving solvent. The data are shown in Table 8.

[0049] Table 8 Results of redissolving solvent investigation

[0050] 9. Determination of the preparation method for the test solution Accurately weigh approximately 1g of Polygala tenuifolia powder (passed through a No. 3 sieve), place it in a conical flask and seal tightly. Accurately add 20mL of 70% ethanol, weigh, and sonicate (160W power, 40kHz frequency) for 1 hour. Cool, weigh again, and make up the weight loss with 70% ethanol. Shake well, filter, and accurately measure 10mL of the filtrate. Evaporate to dryness, add 20mL of 10wt% sodium hydroxide solution to the residue, heat under reflux at 100℃ for 2 hours, cool, and adjust the pH to 3-5 with dilute hydrochloric acid (approximately 18.6 mL). Extract twice with 20mL of water-saturated n-butanol each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue with an appropriate amount of 70% ethanol, transfer to a 25mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the final product.

[0051] II. Methodological Validation (1) Preparation of reference solution Accurately weigh the standards of Polygala tenuifolia saponins and sinapic acid, place them in a brown volumetric flask, and add 70% ethanol solution to prepare solutions with a concentration of 1.535 mg / mL. -1 and 3.974 mg·mL -1 Prepare a series of reference standard stock solutions. Accurately pipette different volumes of the reference standard stock solution, dilute with 70% aqueous ethanol solution to prepare a series of solutions of different concentrations, and determine according to chromatographic conditions.

[0052] (2) Preparation of test solution Accurately weigh approximately 1g of Polygala tenuifolia powder (passed through a No. 3 sieve), place it in a conical flask and seal tightly. Accurately add 20mL of 70% ethanol, weigh, and sonicate (power 160 W, frequency 40 kHz) for 1 hour. Cool, weigh again, and make up the weight loss with 70% ethanol. Shake well, filter, and accurately measure 10mL of the filtrate. Evaporate to dryness, add 20mL of 10wt% sodium hydroxide solution to the residue, heat under reflux at 100℃ for 2 hours, cool, and adjust the pH to 3-5 with dilute hydrochloric acid (approximately 18.6mL). Extract twice with 20mL of water-saturated n-butanol each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue with an appropriate amount of 70% ethanol, transfer to a 25mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the final product.

[0053] (3) Linear relationship Plotting the reference concentration X on the x-axis and the peak area integral Y on the y-axis, the regression equation for sinapic acid was obtained as Y = 9432.2X + 37.814, R0. 2 =0.9997, linear range is 0.024-0.768 mg·mL -1 The regression equation for saponins from Polygala tenuifolia is Y = 1212.9X + 34.494, R0 2=0.9999, linear range is 0.062-1.987 mg·mL -1 See attached Figures 2-3 .

[0054] (4) Precision test The test solution was prepared by taking Polygala tenuifolia powder according to the test solution preparation method of the present invention. The sample was injected continuously for analysis 6 times under the aforementioned chromatographic conditions. The peak area was measured and the content was calculated. The results are shown in Table 9. The average contents of sinapic acid and Polygala tenuifolia saponins were 0.324% and 3.65%, respectively, and the RSDs were 1.26% and 1.99%, respectively, indicating that the instrument precision was good.

[0055] Table 9 Precision Test Results

[0056] (5) Repeatability test Using the method for preparing test solutions of Polygala tenuifolia powder, six test solutions were prepared in parallel. The solutions were analyzed under the aforementioned chromatographic conditions, the peak areas were measured, and the contents were calculated. The results are shown in Table 10. The average contents of sinapic acid and Polygala tenuifolia saponins were 0.320% and 3.55%, respectively, with RSDs of 2.57% and 0.64%, respectively, indicating that the method has good repeatability.

[0057] Table 10 Repeatability Test Results

[0058] (6) Stability test The test solution was prepared by taking Polygala tenuifolia powder according to the test solution preparation method of the present invention. The solution was analyzed at 0, 4, 8, 12, 24, 48 and 72 h under the aforementioned chromatographic conditions. The peak area was measured, the content was calculated and determined. The results are shown in Table 11. The average contents of sinapic acid and Polygala tenuifolia saponins were 0.322% and 3.66%, respectively, and the RSDs were 1.52% and 2.20%, respectively. This indicates that the test solution has good stability and the detection within 72 h has no significant effect on the results.

[0059] Table 11 Stability Test Results

[0060] (7) Spiking recovery test Take 0.5g of Polygala tenuifolia powder and prepare the test solution according to the method of the present invention. When redissolving, add the amount of each reference standard at 50%, 100%, and 150% of the content of sinapic acid and Polygala tenuifolia saponins in 0.25g of Polygala tenuifolia powder, respectively. Make three parallel samples for each concentration to prepare the test solution. Analyze it according to the aforementioned chromatographic conditions, determine the peak area, and calculate the content. The results are shown in Table 12. The average recoveries of sinapic acid and Polygala tenuifolia saponins are 97.19% and 98.77%, respectively, and the RSDs are 2.84% and 4.45%, respectively, indicating that the determination method has good accuracy.

[0061] Table 12 Results of the recovery test

[0062] (8) Durability test 8.1 Examination of Flow Velocity Polygala tenuifolia powder was prepared according to the method for preparing the test solution of this invention, and the solutions were subjected to different flow rates (0.9, 1.0, 1.1 mL·min). -1 According to the chromatographic conditions of this invention, the contents of sinapic acid and polygala tenuifolia saponins were determined, and the results are shown in Table 13. The average contents of sinapic acid and polygala tenuifolia saponins were 0.306% and 3.61%, respectively, and the RSDs were 0.46% and 2.35%, respectively, indicating that the flow rate has little effect on the detection results and has good durability.

[0063] Table 13 Results of the flow velocity investigation

[0064] 8.2 Investigation of column temperature Polygala tenuifolia powder was prepared according to the method for preparing the test solution of this invention. The sample was injected and determined at different column temperatures (25, 30, 35℃) according to the chromatographic conditions of this invention. The contents of sinapic acid and polygala tenuifolia saponins were used as evaluation indicators. The results are shown in Table 14. The average contents of sinapic acid and polygala tenuifolia saponins were 0.303% and 3.57%, respectively, and the RSDs were 0.56% and 3.69%, respectively. This indicates that the column temperature has little effect on the detection results and has good robustness.

[0065] Table 14 Results of Column Temperature Investigation

[0066] 8.3 Examination of the chromatographic column Polygala tenuifolia powder was prepared according to the method for preparing the test solution of this invention. Chromatographic conditions according to this invention were applied using different types of columns of the same specification [Waters Atlantis T3 (4.6 × 250 mm, 5 μm), Agilent Eclipse XDB C] 18 (4.6×250mm, 5μm), Agilent Zorbax SB C18 The results of the (4.6×250mm, 5μm) injection determination are shown in Table 15. The average contents of sinapic acid and polygala tenuifolia saponins were 0.294% and 3.54%, respectively, with RSDs of 3.99% and 3.88%, respectively, indicating that the chromatographic column has little impact on the detection results and has good durability.

[0067] Table 15 Results of Column Chromatography

[0068] 8.4 Investigation of the concentration of formic acid in the aqueous phase Polygala tenuifolia powder was prepared according to the method for preparing the test solution of this invention. The powder was then injected and analyzed under the chromatographic conditions of this invention at different aqueous phase formic acid concentrations (0, 0.05%, 0.10%). The contents of sinapic acid and polygala tenuifolia saponins were used as evaluation indicators. The results are shown in Table 16. The average contents of sinapic acid and polygala tenuifolia saponins were 0.297% and 3.57%, respectively, with RSDs of 2.12% and 3.37%, respectively. The aqueous phase formic acid concentration had a relatively small effect on the content results, but the baseline was not stable enough at concentrations of 0 and 0.1%, with 0.05% being the most suitable.

[0069] Table 16 Results of the investigation on mobile phase acid concentration

[0070] III. Batch Consistency Verification Fifteen batches of Polygala tenuifolia powder were prepared according to the method for preparing the test solution of this invention. The contents of sinapic acid and Polygala tenuifolia saponins in the powder were determined according to the chromatographic conditions of this invention. The results are shown in Table 17. The contents of each component varied among different samples. The sinapic acid content ranged from 0.160% to 0.467%, with an average of 0.339% and a mean ± 30% range of 0.24% to 0.44%. The Polygala tenuifolia saponin content ranged from 0.95% to 3.64%, with an average of 2.81% and a mean ± 30% range of 1.98% to 3.67%. It is recommended to calculate based on the dried product. The sinapic acid content (C...) is... 11 H 12 O5) must not be less than 0.20%, containing polygala tenuifolia saponins (C 36 H 56 O 12 () shall not be less than 2.0%.

[0071] Two batches of samples were randomly selected and extracted according to the 2025ChP test solution preparation method for comparison. The results showed that the sinapic acid content increased slightly after the revised method, and the content of polygala tenuifolia saponins fluctuated within ±10%. Moreover, the revised method is more environmentally friendly, saves time and solvents.

[0072] Table 17 Results of Content Determination of Polygala tenuifolia Powder

[0073] IV. Comparison of this method with existing methods The existing extraction method for Polygala tenuifolia is as follows: Take about 1g of the powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 70% methanol, weigh it, sonicate it (power 400W, frequency 40kHz) for 1 hour, cool it, weigh it again, replenish the lost weight with 70% methanol, shake it well, filter it, accurately measure 25ml of the filtrate, place it in a round-bottom flask, evaporate it to dryness, add 50ml of 10wt% sodium hydroxide solution to the residue, heat it under reflux for 2 hours, cool it, adjust the pH value to 4-5 with hydrochloric acid, extract it 3 times with water-saturated n-butanol, 50ml each time, combine the n-butanol solutions, recover the solvent to dryness, add an appropriate amount of methanol to dissolve the residue, transfer it to a 25ml volumetric flask, add methanol to the mark, shake it well, and the product is obtained.

[0074] Table 18 Technical Comparison and Advantages Summary

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the content of urushi-alcohol and sibiricin in the water hydrolysate of Chinese medicine polygala, characterized in that, The method comprises: Preparation of the reference solution: prepare a mixed reference solution of the standard by taking sinapinic acid and polygalaxin as the standard; Preparation of the test solution: prepare the test solution by using the test sample; Determine the test solution and the mixed reference solution by injecting them into a high performance liquid chromatograph with a diode array detector; Chromatographic conditions: use octadecylsilane-bonded silica gel as the filler; use 0.03-0.07vol% formic acid acetonitrile solution as the mobile phase A, and use 0.03-0.07vol% formic acid water solution as the mobile phase B for gradient elution; the gradient elution program is as follows: 0-10min, 75-85%B→65-75%B, the detection wavelength is 320mm; 10-20min, 65-75%B→45-55%B, the detection wavelength is 192mm; The column temperature is 25-35℃; the flow rate is 0.5-1.5ml / min; The preparation process of the test solution is as follows: extract by using 65-75vol% ethanol, the amount of ethanol is 15-25mL / g of the polygala powder, ultrasonic extraction for 45-90min, filtration, evaporation, add 5-15wt% sodium hydroxide solution to the residue, heat reflux treatment for 1.5-2.5h, the amount of sodium hydroxide solution is 1.5-3.0 times of the volume of the filtrate; adjust the pH value to 3-5 by using dilute hydrochloric acid, extract 2-3 times by using n-butanol saturated with water, the amount of the extracting agent for each extraction is 1.5-3.0 times of the volume of the filtrate, combine the n-butanol liquids, evaporate, dissolve the residue in 65-75vol% ethanol and make up the volume.

2. The method of claim 1, wherein, The preparation process of the mixed reference solution is as follows: take the reference sinapinic acid and polygalaxin, put them into a brown volumetric flask, and add 65-75vol% ethanol solution to prepare a mixed reference solution with a concentration of 0.20mg / mL and 0.50mg / mL.

3. The method of claim 1, wherein, The preparation process of the test solution is as follows: take 1g of the polygala powder, put it into a conical flask, tightly seal it, add 20mL of 70vol% ethanol, ultrasonic treatment for 1h, cool, re-weigh, make up the weight loss with 70vol% ethanol, shake well, filter; take 10mL of the filtrate, evaporate, add 20mL of 10wt% sodium hydroxide solution to the residue, heat reflux at 100℃ for 2h, cool, adjust the pH value to 3-5 by using dilute hydrochloric acid, extract 2 times by using n-butanol saturated with water, each time 20mL, combine the n-butanol liquids, evaporate, add appropriate amount of 70vol% ethanol to the residue to dissolve, transfer to a 25m volumetric flask, make up the volume, shake well, pass through a 0.22μm microporous filter, and obtain the test solution.

4. The method of claim 1, wherein, Chromatographic conditions: use octadecylsilane-bonded silica gel as the filler; use 0.05vol% formic acid acetonitrile solution as the mobile phase A, and use 0.05vol% formic acid water solution as the mobile phase B for gradient elution; the gradient elution program is as follows: 0-10min, 80%B→70%B, the detection wavelength is 320mm; 10-20min, 70%B→50%B, the detection wavelength is 192mm; The flow rate is 1ml / min, and the injection amount is 2uL.

5. The method of claim 1, wherein, The chromatographic column is selected from Waters Atlantis T3 (4.6 x 250 mm, 5 μm), Agilent Eclipse XDB C 18 (4.6 x 250 mm, 5 μm), or Agilent Zorbax SB C 18 (4.6 x 250 mm, 5 μm).

6. The method of claim 4, wherein, The linear regression equation of the sinapinic acid is Y=9432.2X+37.814; and the linear regression equation of the polygalasaponin is Y=1212.9X+34.

494.

7. The method of claim 4, wherein, The retention time of the sinapinic acid is 8.077 min, and the retention time of the polygalasaponin is 18.151 min.

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