Method for detecting ziyuglycoside II in ovateleaf holly bark medicinal material and application of ziyuglycoside II

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting ziyuglycoside II in an ovateleaf holly bark medicinal material and application of the method. Comprising the following steps: preparation of a reference substance solution, preparation of a test solution, liquid chromatography detection and data analysis, and the data analysis comprises calculation of the content of ziyuglycoside II in the ovateleaf holly bark medicinal material. Wherein the liquid chromatography conditions are as follows: a chromatographic column filler is octadecylsilane chemically bonded silica; the mobile phase is acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B), and 38%-40% A isocratic elution is adopted, or 0-10 min, 35%-40% A; in 10-45 min, 40%-46% A gradient elution is carried out. The method for detecting the content of ziyuglycoside II in the ovateleaf holly bark medicinal material is established and is applied to whole-process detection of ziyuglycoside II in the ovateleaf holly bark medicinal material and a preparation thereof, and an analysis means is provided for quality control of the ovateleaf holly bark medicinal material and the preparation thereof.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and particularly relates to a detection method and application of sanguisorba officinalis saponin II in the medicinal material Jiubiying. Background Art

[0002] Jiubiying (Suibiying) is derived from the dried bark of Ilex rotunda Thunb. (Aquifoliaceae). It has a bitter taste and cold properties, and can clear away heat and toxins, reduce swelling and relieve pain, and stop bleeding and promote tissue regeneration. It is primarily distributed in Nanning and Jinxiu, Guangxi. Yao medicine practitioners often use Jiubiying powder for internal use to treat epigastric pain, or combine it with water chestnuts and sanguisorba officinalis to treat gastric and duodenal ulcers. Jiubiying extract has shown therapeutic effects in mice with ulcerative colitis, and its active ingredients are syringin, pedunculate hollyside, and sanguisorba officinalis saponin II. The 2020 edition of the Chinese Pharmacopoeia uses syringin and pedunculin as content determination indicators for scutellaria baicalensis. Chinese invention patent application CN104764828A discloses a method for constructing a fingerprint of scutellaria baicalensis and a detection method thereof, comprising the following steps: (1) preparation of a reference solution; (2) preparation of a test solution; (3) determination: accurately aspirating the test solution and the reference solution respectively, injecting them into a high-performance liquid chromatograph for determination, and obtaining a fingerprint of scutellaria baicalensis consisting of 13 common characteristic peaks. It does not involve sanguisorbaside II as a content determination indicator, and the detection wavelength range of the invention is 288-292 nm. Sanguisorbaside II has no absorption within this wavelength range, so sanguisorbaside II cannot be detected. The literature "Determination and comparative analysis of the content of sanguisorbaside I and sanguisorbaside II in Sanguisorba officinalis slices from different origins" (Wang Xue, Dai Yanpeng, Xu Bo, et al.. Central South Pharmacy, 2018, 16(03):400-404) determined the content of sanguisorbaside I and sanguisorbaside II in Sanguisorba officinalis slices from different regions. The results showed that the content of sanguisorbaside I in 18 batches of Sanguisorba officinalis slices from 7 provinces was 3.1%-7.7%, and the content of sanguisorbaside II was 0.16%-0.87%. There were differences in the content of sanguisorbaside I and sanguisorbaside II in Sanguisorba officinalis slices from different origins. However, this method only detected sanguisorbaside II in Sanguisorba officinalis. Sanguisorbaside II is the active ingredient of Jiubiying for the treatment of ulcerative colitis. Establishing a method to accurately detect sanguisorbaside II in Jiubiying medicinal materials and their preparations can ensure the quality stability of Jiubiying medicinal materials and their preparations. Summary of the Invention

[0003] In view of the existing technology, the present invention provides a method for detecting Sanguisorba officinalis saponin II in the medicinal material of Jiubiying and its application. By comparing the HPLC chromatographic conditions of various Sanguisorba officinalis saponins II, the best method for determining the content of Sanguisorba officinalis saponin II in the medicinal material of Jiubiying is selected, and the method is applied to the whole process detection of Sanguisorba officinalis saponin II in the medicinal material of Jiubiying and its preparations, providing an analytical means for the quality control of the medicinal material of Jiubiying and its preparations, such as Kujie tablets.

[0004] The present invention is achieved through the following technical solutions: The first aspect of the present invention relates to a method for detecting sanguisorbaside II in the medicinal material of Scutellaria baicalensis, which comprises the following steps: preparation of a reference solution, preparation of a test solution, liquid chromatography detection and data analysis, wherein the data analysis comprises calculating the content of sanguisorbaside II in the medicinal material of Scutellaria baicalensis; wherein the conditions adopted for the liquid chromatography detection include: the chromatographic column filler is octadecylsilane bonded silica gel; the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, and an isocratic elution of 38% A to 40% A is adopted, or a gradient elution of 0-10 min, 35%→40% A; 10-45 min, 40%→46% A is adopted.

[0005] Preferably, the conditions adopted for the liquid chromatography detection further include: a mobile phase flow rate of 0.80-1.20 mL / min, a column temperature of 30-35° C., and a detection wavelength of 203-210 nm.

[0006] Preferably, the chromatographic column is selected from Diamonsil® C 18 (250 × 4.6 mm, 5 μm) column or InnoC 18 -AQ (250 × 4.6 mm, 5 μm) column.

[0007] Further preferably, the chromatographic column is Diamonsil® C 18 (250 × 4.6 mm, 5 μm) column.

[0008] Preferably, the preparation of the test solution comprises the following steps: taking the powder of the medicinal material Jiubing, accurately weighing it, placing it in a container, accurately adding the extraction solvent, sealing it, weighing it, cooling it after extraction, weighing it again, making up the lost weight with the extraction solvent, shaking it evenly, filtering it, and obtaining the solution.

[0009] Further preferably, the concentration of the test solution is 20-120 mg / mL, preferably 100 mg / mL.

[0010] Further preferably, the injection volume of the test solution in liquid chromatography detection is 10-30 μL, preferably 10 μL.

[0011] Further preferably, the extraction solvent is a methanol aqueous solution, and the concentration of the methanol aqueous solution is greater than or equal to 70% by volume of methanol; preferably 100% methanol solution.

[0012] Further preferably, the extraction is carried out by ultrasonic extraction or heating reflux extraction, and the extraction time is 40-60 minutes, preferably 50 minutes.

[0013] Preferably, the preparation of the reference solution comprises the following steps: taking a Sanguisorba officinalis saponin II reference substance, placing it in a container, accurately adding an extraction solvent, sealing it, weighing it, cooling it after extraction, weighing it again, making up the lost weight with the extraction solvent, shaking it evenly, and filtering it to obtain the solution.

[0014] Further preferably, the solvent in the preparation of the reference solution is a methanol aqueous solution, and the concentration of the methanol aqueous solution is greater than or equal to 70% by volume of methanol.

[0015] The second aspect of the present invention relates to the application of the above detection method in the quality detection of samples containing salvia miltiorrhiza, wherein the samples containing salvia miltiorrhiza are selected from salvia miltiorrhiza medicinal materials, salvia miltiorrhiza decoction pieces, salvia miltiorrhiza extracts, salvia miltiorrhiza intermediates or salvia miltiorrhiza preparations.

[0016] The beneficial effects of the present invention are as follows: (1) a method for detecting the content of Sanguisorba officinalis saponin II in the medicinal material of Jiubijing is established, and the method is applied to the whole process detection of Sanguisorba officinalis saponin II in the medicinal material of Jiubijing and its preparations, providing an analytical means for the quality control of the medicinal material of Jiubijing and its preparations; (2) the methodological investigation of the content detection method provided by the present invention is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : HPLC chromatograms of sanguisorbaside II under different elution conditions; the elution conditions of AE correspond to conditions I to V, respectively, and peak 1 is sanguisorbaside II; Figure 2 : HPLC diagram of the separation of Sanguisorba officinalis saponin II by different chromatographic columns; A is Inno C 18 -AQ (250×4.6mm, 5μm) column, B is Diamonsil® C 18 (250 × 4.6 mm, 5 μm) column, peak 1 is sanguisorbaside II; Figure 3 : HPLC spectra of Sanguisorbaside II at different column temperatures, where A is 30°C, B is 35°C, C is 40°C, and peak 1 is Sanguisorbaside II; Figure 4 : HPLC spectra of Sanguisorbaside II at different wavelengths, where A is 195 nm, B is 203 nm, and C is 210 nm; Peak 1 is Sanguisorbaside II; Figure 5 : HPLC graphs of Sanguisorbaside II detected by different extraction methods, where A is ultrasound, B is reflux, and peak 1 is Sanguisorbaside II; Figure 6: HPLC graphs of sanguisorbaside II detected by different extraction solvents, where A is ethanol, B is 100% methanol, C is 70% methanol, D is 50% methanol, and E is 30% methanol. Peak 1 is sanguisorbaside II; Figure 7 : HPLC chart of the adaptability investigation of the isocratic elution system for Sanguisorba officinalis saponin II; wherein A is the blank control, B is the reference substance of Sanguisorba officinalis saponin II, C is the test substance of Jiubiying medicinal material, and peak 1 is Sanguisorba officinalis saponin II; Figure 8 : Standard curve of isocratic elution of Sanguisorba officinalis saponin II; Figure 9 : HPLC chart of the adaptability investigation of the gradient elution system for Sanguisorba officinalis saponin II; wherein A is the blank control, B is the reference substance of Sanguisorba officinalis saponin II, C is the test substance of Jiubiying medicinal material, and peak 1 is Sanguisorba officinalis saponin II; Figure 10 : Standard curve of gradient elution of Sanguisorba officinalis saponin II.

[0018] Note: Figure 1-Figure 7 、 Figure 9 The solvent peak or non-Sanguisorbaside II peak in the chromatogram is relatively high. In order to make the chromatographic peak of the target component more intuitive and clear, a local spectrum is provided. This spectrum does not affect the determination of the target peak Sanguisorbaside II and the accuracy of the analysis results. DETAILED DESCRIPTION

[0019] To illustrate the purpose, technical solutions and advantages of the present invention, the present invention is further described in detail below with reference to specific examples. In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further described below with reference to specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work all fall within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same. The present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all common commercial products in the field of this technology, and the purity is all above 98%.

[0020] The instruments and equipment used in the following experimental examples are shown in Table 1.

[0021] Table 1: Laboratory instruments and equipment

[0022] The materials and reagents used in the following experimental examples are shown in Table 2.

[0023] Table 2: Experimental materials and reagents

[0024] Example 1: Optimization of chromatographic conditions The preparation method of the test sample in this embodiment is: Weigh approximately 0.5 g of Jiubiying medicinal material powder (passed through a No. 3 sieve), accurately weigh it, add 25 mL of methanol, weigh it, and perform ultrasonic treatment for 40 min (power 300 W, frequency 45 kHz). Let it cool, weigh it again, make up the weight loss with methanol, and filter it to obtain the product.

[0025] 1.1 Investigation of different elution gradients Using Diamonsil® C 18 A 250×4.6 mm, 5 μm chromatographic column was used with acetonitrile (A) and 0.1% phosphoric acid water (B) as the mobile phase. The column temperature was 30℃, the detection wavelength was 203 nm, and the injection volume was 20 μL. The separation of sanguisorbaside II in Jiubiying was investigated under different gradient elution or isocratic elution conditions, and the chromatographic detection conditions for sanguisorbaside II in Jiubiying were optimized.

[0026] Condition I: isocratic elution, 45% A, 55% B; Condition II: isocratic elution, 40% A, 60% B; Condition III: isocratic elution, 38% A, 62% B; Condition IV: gradient elution, program: 0-10 min, 35%→40% A; 10-45 min, 40%→46% A; Condition V: Gradient elution, program: 0-10 min, 35%→40% A; 10-60 min, 40%→50% A.

[0027] See the results Figure 1 AE in the analysis, among which, when condition I, i.e., 45% A, was used for isocratic elution, sanguisorbaside II could not be separated from the front peak. When 40% and 38% A were used for isocratic elution, sanguisorbaside II was well separated, with peak elution times of 34.83 min and 47.73 min, respectively. Therefore, 38% A or 40% A elution could be selected for isocratic elution. When gradient elution was used, the separation of sanguisorbaside II in conditions IV and V both met the requirements. Under condition IV, i.e., 0-10 min, 35%→40% A; 10-45 min, 40%→46% A, the separation of sanguisorbaside II was 3.486, and the separation time was short. This gradient elution condition was preferred.

[0028] 1.2 Investigation of different chromatographic columns Diamonsil® C 18(250 × 4.6 mm, 5 μm) column and Inno C 18 The separation of sanguisorba officinalis saponin II in Jiubica oleracea was investigated using a HPLC-AQ column (250×4.6mm, 5μm) with acetonitrile (A) and 0.1% phosphoric acid water (B) as the mobile phase, column temperature at 30℃, detection wavelength at 203 nm, injection volume of 20 μL, and isocratic elution (40% A, 60% B).

[0029] The results are as follows Figure 2 As shown. Figure 2 The results showed that chromatographic columns from different manufacturers with octadecylsilane bonded silica gel as filler can be used to detect Sanguisorba officinalis saponin II.

[0030] 1.3 Investigation of column temperature Using Diamonsil® C 18 The separation of sanguisorba officinalis saponin II in Jiubiying was investigated using a 250×4.6 mm, 5 μm column with acetonitrile (A) and 0.1% phosphoric acid water (B) as the mobile phase, detection wavelength at 203 nm, injection volume of 20 μL, and isocratic elution (40% A, 60% B). The column temperatures were 30℃, 35℃, and 40℃.

[0031] The separation and chromatogram of Sanguisorba officinalis saponin II determined at different column temperatures are shown in Table 3 and Figure 3 .

[0032] Table 3: Investigation of different column temperatures

[0033] When the column temperature is 30℃-35℃, the separation degree of Sanguisorbaside II is greater than 1.5, and the chromatographic peak shape is good. When the column temperature is 40℃, Sanguisorbaside II does not appear. Therefore, the column temperature for Sanguisorbaside II detection should be 30℃-35℃. 1.4 Detection wavelength investigation Using Diamonsil® C 18 The chromatographic column (250×4.6 mm, 5 μm) was used with acetonitrile (A) and 0.1% phosphoric acid water (B) as the mobile phase. The column temperature was 30℃, the injection volume was 20 μL, and the elution method was isocratic (40% A, 60% B). The peak area and separation of sanguisorba officinalis saponin II in Jiubiying were investigated at detection wavelengths of 195 nm, 203 nm, and 210 nm, respectively.

[0034] The results are shown in Table 4 and Figure 4 .

[0035] Table 4: Investigation of different wavelengths

[0036] The results show that Sanguisorbaside II can be detected at wavelengths of 195 nm, 203 nm, and 210 nm. The peak area of Sanguisorbaside II is smaller at 210 nm, while the response is higher at 203 nm, with a resolution greater than 1.5. At 195 nm, the response is higher, but the resolution is less than 1.5. Based on a resolution greater than 1.5, the detection wavelength for Sanguisorbaside II is 203-210 nm, with 203 nm being preferred.

[0037] Based on the above investigation results, it can be seen that the detection of Sanguisorba officinalis saponin II in Jiubijing is carried out with acetonitrile (A)-0.1% phosphoric acid water (B) as the mobile phase and Diamonsil® C 18 The elution conditions were isocratic (38% A to 40% A) or gradient (35% to 40% A, 0-10 min; 40% to 46% A, 10-45 min). The column temperature was 30°C-35°C, the detection wavelength was 203-210 nm, and the flow rate was 1.0 mL / min. Since the concentration of the test sample determines the peak area of sanguisorbaside II during detection, the preparation and concentration of the test sample were further optimized to determine the sample injection volume.

[0038] Experimental Example 2: Optimization of test sample preparation conditions 2.1 Investigation of different extraction methods The effects of reflux extraction and ultrasonic extraction on the content of sanguisorba officinalis saponin II in Jiubiying medicinal materials were investigated.

[0039] Approximately 0.5 g of jelly powder (passed through a No. 3 sieve) was accurately weighed, 25 mL of methanol was added, and the mixture was weighed. Ultrasonic treatment was performed for 40 min (power 300 W, frequency 45 kHz). The mixture was allowed to cool and the weight was re-weighed. The weight loss was made up with methanol, and the mixture was filtered and tested by injection. A Diamonsil® C18 (250 × 4.6 mm, 5 μm) column was used with acetonitrile (A) and 0.1% phosphoric acid water (B) as the mobile phases. The column temperature was 35°C, and isocratic elution (40% A, 60% B) was used. The detection wavelength was 203 nm.

[0040] Weigh approximately 0.5 g of jelly powder (passed through a No. 3 sieve), accurately weigh it, add 25 mL of methanol, heat and reflux for extraction for 40 min, let it cool, weigh it again, make up the weight loss with methanol, filter it, and inject it for detection.

[0041] Two samples were prepared in parallel for each of the two extraction methods, and each sample was injected twice. The results of the detection of sanguisorbaside II in the samples obtained by different extraction methods are shown in Tables 5 and Figure 5 .

[0042] Table 5: Detection results of Sanguisorba officinalis saponin II using different extraction methods

[0043] The results of the effects of reflux extraction and ultrasonic extraction on the content of Sanguisorba officinalis saponin II showed that the content of Sanguisorba officinalis saponin II measured by reflux extraction and ultrasonic extraction was equivalent. Since the reflux extraction operation was complicated and the ultrasonic method was simple and easy, the ultrasonic extraction method was preferred to prepare the Jiubiying test sample.

[0044] 2.2 Investigation of different extraction solvents Approximately 0.5 g of jubilee powder (passed through a No. 3 sieve) was weighed into five accurately weighed portions. 25 mL of 30% methanol, 50% methanol, 70% methanol, methanol, or ethanol was added, weighed, and ultrasonicated for 40 min (power 300 W, frequency 45 kHz). The mixture was allowed to cool and weighed again. The weight loss was compensated for with the corresponding solvent concentration, filtered, and sampled for detection. A Diamonsil® C18 (250 × 4.6 mm, 5 μm) column was used with acetonitrile (A) and 0.1% phosphoric acid (B) as the mobile phases. The column temperature was 35°C, and isocratic elution (40% A, 60% B) was used. Detection was at 203 nm. Two replicates were prepared for each sample, and the injections were repeated twice.

[0045] The results of the detection of Sanguisorba officinalis saponin II with different extraction solvents are shown in Table 6 and Figure 6 .

[0046] Table 6: Detection results of Sanguisorba officinalis saponin II with different extraction solvents

[0047] The results showed that when the extraction solvent was ethanol, the peak shape of Sanguisorba officinalis saponin II in Jiubiying medicinal materials was poor, while the peak shape of Sanguisorba officinalis saponin II was good when the extraction solvent was methanol. Further investigation of the content of Sanguisorba officinalis saponin II using 30%, 50%, 70%, and 100% methanol as the extraction solvent revealed that when the methanol concentration was ≤50%, Sanguisorba officinalis saponin II was difficult to dissolve, and was not detected or detected at a low level with an RSD greater than 12%. 70% and 100% methanol were able to fully dissolve Sanguisorba officinalis saponin II, and the highest content of Sanguisorba officinalis saponin II was achieved when 100% methanol was used, making 100% methanol the preferred extraction solvent.

[0048] 2.3 Investigation of different extraction times Approximately 0.5 g of jubilee powder (passed through a No. 3 sieve) was accurately weighed, and 25 mL of pure methanol was weighed. The mixture was sonicated for 30, 40, 50, and 60 min (power 300 W, frequency 45 kHz). The mixture was allowed to cool and reweighed. The weight loss was adjusted with methanol. The mixture was filtered and tested by injection. A Diamonsil® C18 (250 × 4.6 mm, 5 μm) column was used with acetonitrile (A) and 0.1% phosphoric acid (B) as the mobile phases. The column temperature was 35°C, and isocratic elution (40% A, 60% B) was used. The detection wavelength was 203 nm. Two replicates were prepared for each sample, and the injections were repeated twice.

[0049] The detection results of Sanguisorba officinalis saponin II at different extraction times are shown in Table 7.

[0050] Table 7: Detection results of Sanguisorba officinalis saponin II at different extraction times

[0051] According to the results, the extraction content of Sanguisorbaside II when the ultrasonic time is 30 min is lower than that when the ultrasonic time is 40-60 min. The extraction content of Sanguisorbaside II when the ultrasonic time is 40 min, 50 min and 60 min is higher and the difference is not large. Considering the extraction time and content comprehensively, the extraction time of 50 min is preferred.

[0052] 2.4 Investigation of different material-liquid ratios (1) Weigh approximately 0.2 g, 0.5 g, 1.0 g, or 2.0 g of scutellaria baicalensis powder (passed through a No. 3 sieve) and accurately weigh the mixture. Add 25 mL of pure methanol, weigh the mixture, sonicate for 50 min (power 300 W, frequency 45 kHz), cool the mixture, weigh the mixture again, make up the weight loss with methanol, and filter the mixture to obtain medicinal material test solutions with concentrations of 8 mg / mL, 20 mg / mL, 40 mg / mL, and 80 mg / mL. The injection volume of each sample was 30 μL. A Diamonsil® C18 (250×4.6 mm, 5 μm) column was used with acetonitrile (A) and 0.1% phosphoric acid water (B) as the mobile phases. The column temperature was 35°C, and the elution was isocratic (40% A, 60% B). The detection wavelength was 203 nm. The peak area of sanguisorbaside II in the samples was recorded. The results are shown in Table 8.

[0053] Table 8: Content of Sanguisorba officinalis saponin II in Jiubiying medicinal materials at different material-liquid ratios

[0054] Due to the low content of Sanguisorba officinalis saponin II in Jiubiying (Shangyu Bijing), the injection volume was fixed at 30 μM. The data in the table show that the Sanguisorba officinalis saponin II peak area is ≥100 mAU when the concentration of Jiubiying (Shangyu Bijing) is above 40 mg / mL. Because detection error is large when the Sanguisorba officinalis saponin II peak area is less than 100 mAU, the sample preparation concentration should be determined based on the injection volume, peak area, and detection stability when detecting Sanguisorba officinalis saponin II in Jiubiying (Shangyu Bijing). Therefore, the injection volume should be optimized.

[0055] (2) Accurately weigh 0.2 g, 0.5 g, and 1.0 g of the medicinal material of Jiubiying, respectively, and place them in a 10 mL volumetric flask. Add methanol to the scale line and sonicate for 50 min (power 300 W, frequency 45 kHz). After cooling, add methanol to make up the weight. Pass through a 0.22 μm organic filter membrane to prepare medicinal material test solutions with concentrations of 20 mg / mL, 50 mg / mL, and 100 mg / mL. Prepare two parallel samples for each concentration. A Diamonsil® C18 (250×4.6 mm, 5 μm) column was used with acetonitrile (A) and 0.1% phosphoric acid water (B) as the mobile phase. The column temperature was 30°C and the elution conditions were 0-10 min, 35%→40% A; 10-45 min, 40%→46% A. 10 μL, 20 μL, and 30 μL were injected, respectively, and the peak area of sanguisorbaside II in the samples was recorded. The results are shown in Table 9.

[0056] Table 9: Content of Sanguisorba officinalis saponin II in Jiubiying medicinal materials at different material-liquid ratios and injection volumes

[0057] Based on the response values for Sanguisorbaside II in the herbal medicine Jiubiying at a 30 μL injection volume, the effects of injection volume and sample preparation concentration on the stability of Sanguisorbaside II content detection were investigated to determine the optimal test sample concentration and injection volume. According to the results in Table 9, the detected Sanguisorbaside II content was similar when the herbal medicine concentration was 100 mg / mL and the injection volume was 10 μL, as compared to when the herbal medicine concentration was 20 mg / mL and the injection volume was 30 μL. However, when the sample concentration was lower and the injection volume was larger, the detection RSD was ≥5%. Therefore, the injection volume was determined to be 10-30 μL, and the herbal medicine test sample preparation concentration was determined to be 20-120 mg / mL, with 100 mg / mL being preferred.

[0058] Experimental Example 3: Preparation of reference solution Accurately weigh 5.02 mg of sanguisorbaside II into a 25 mL volumetric flask, add methanol, dilute to the mark, sonicate for 50 min, filter through a 0.22 μm filter membrane, and the concentration of sanguisorbaside II is 0.2008 mg / mL. Store in a 4 °C refrigerator and dilute 10-fold with methanol to a concentration of 20.08 μg / mL when used.

[0059] Experimental Example 4: Investigation of Isocratic Elution Methodology 4.1 Chromatographic conditions C 18 The chromatographic column was 250 × 4.6 mm, 5 μm; the column temperature was 35°C, the detection wavelength was 203 nm, the injection volume was 20 μL, and the flow rate was 0.8 mL / min. The mobile phase was acetonitrile (A)-0.1% phosphoric acid (B) in water, with a volume ratio of 38:62.

[0060] 4.2 Solution preparation Preparation of reference solution: Accurately weigh 5.03 mg of Sanguisorbaside II into a 50 mL volumetric flask, add methanol, dilute to the mark, sonicate for 50 min, filter through a 0.22 μm filter to a concentration of 0.1006 mg / mL of Sanguisorbaside II, and store in a 4°C refrigerator until use.

[0061] Preparation of test solution: Weigh 0.5 g of Jiubiying medicinal material, place it in a 25 mL volumetric flask, add methanol, dilute to the scale, sonicate for 50 min, let cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to make the final concentration of 20 mg / mL.

[0062] 4.3 System adaptability test The test solution and reference solution prepared under 4.2 were tested on the chromatographic conditions under 4.1, and the retention time, peak width and half-peak width of Sanguisorba officinalis saponin II were recorded. The theoretical plate number and separation degree were calculated according to the provisions of the General Chapter 0512 High Performance Liquid Chromatography of the Chinese Pharmacopoeia. The results are shown in Table 10 and Figure 7 .

[0063] Table 10: Results of the adaptability test of the isocratic elution system of Sanguisorba officinalis saponin II

[0064] According to the results, there is no interfering peak in the blank solution, the peak elution time of the test solution and the reference solution is consistent, the target peak separation is greater than 1.5, and the number of theoretical plates is greater than 2000 (Table 10), indicating that the method has good specificity.

[0065] 4.4 Linear Relationship Dilute the reference solution under 4.2 into a series of standard working solutions with 6 concentrations of 0.1006 mg / mL, 0.0503 mg / mL, 0.02515 mg / mL, 0.02012 mg / mL, 0.012575 mg / mL, and 0.0062875 mg / mL, and perform the test on the instrument. Draw a standard curve with concentration (X) as the horizontal axis and the corresponding peak area (Y) as the vertical axis, and calculate the regression equation and correlation coefficient. Figure 8 .

[0066] The linear regression equation of sanguisorbaside II was y = 9575.7x + 6.3111, indicating that sanguisorbaside II had a good linear relationship with the peak area in the range of 0.0062875-0.1006 mg / mL (R²=0.9998).

[0067] 4.5 Precision Six consecutive injections of the reference solution from 4.2 were made, and the peak areas were recorded and the relative standard deviation (RSD) was calculated. The RSD of the peak area after six consecutive injections was 1.05%, indicating that the instrument had good precision under these chromatographic conditions. The results are shown in Table 11.

[0068] Table 11: Instrument precision test

[0069] 4.6 Stability Weigh 0.5 g of the herb, add 25 mL of methanol, and prepare a 20 mg / mL suibiying test solution according to the method in 2.4.2. Samples were injected and analyzed after 0 h, 2 h, 6 h, 8 h, 18 h, and 24 h. The peak area of sanguisorbaside II was recorded, and the relative standard deviation (RSD) was calculated to determine the stability of sanguisorbaside II in the suibiying extract. The RSD values for the peak areas at 0 h, 2 h, 6 h, 8 h, 18 h, and 24 h were 0.54%, indicating that sanguisorbaside II in the test sample was stable within 24 h. The results are shown in Table 12.

[0070] Table 12: Stability test

[0071] 4.7 Repeatability Weigh 6 aliquots of 0.5 g of Jiubiying from the same batch and accurately add 25 mL of methanol. Prepare the test solution according to the method in 4.2, perform the analysis, and calculate the RSD. The RSD of the peak area after six consecutive injections was 1.29%, indicating good instrument precision. The results are shown in Table 13.

[0072] Table 13: Repeatability test

[0073] 4.8 Sample recovery Three portions of the same batch of Jiubiying medicinal materials were accurately weighed and spiked with a Sanguisorbaside II reference solution at 80%, 100%, and 120% of the known Sanguisorbaside II content, respectively. Test solutions were prepared according to the method in 4.2. After injection and testing, the average recovery and RSD values were calculated. Results: Recovery rates ranged from 92.45% to 99.37%, with an average recovery of 94.66% and an RSD of less than 3%. This method is reliable for determining Sanguisorbaside II content. Specific data are shown in Table 14.

[0074] Table 14: Sample recovery test

[0075] Experimental Example 5: Gradient Elution Methodology 5.1 Chromatographic conditions C 18 The column was 250 × 4.6 mm, 5 μm. The column temperature was 30°C, the detection wavelength was 210 nm, the injection volume was 10 μL, and the flow rate was 1 mL / min. The mobile phase was acetonitrile (A) and 0.1% phosphoric acid in water (B). The column temperature was 30°C, and the elution conditions were: 35% to 40% A (0-10 min); 40% to 46% A (10-45 min).

[0076] 5.2 Solution preparation Preparation of reference solution: Accurately weigh 5.03 mg of Sanguisorbaside II into a 25 mL volumetric flask, add methanol, dilute to the mark, sonicate for 50 min, filter through a 0.22 μm filter to a concentration of 0.2012 mg / mL of Sanguisorbaside II, and store in a 4°C refrigerator until use.

[0077] Preparation of test solution: Weigh 0.5 g of Jiubiying medicinal material, place it in a 25 mL volumetric flask, add methanol, dilute to the scale, sonicate for 50 min, let cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to make the final concentration of 20 mg / mL.

[0078] 5.3 System adaptability test The test solution and reference solution prepared under 5.2 were tested on the chromatographic machine according to the chromatographic conditions under 5.1. The retention time, peak width and half-peak width of Sanguisorba officinalis saponin II in the Jiubiying medicinal material were recorded. The theoretical plate number and separation degree were calculated according to the provisions of General Chapter 0512 High Performance Liquid Chromatography of the "Chinese Pharmacopoeia".

[0079] Chromatogram Figure 9 As shown in Table 15, there is no interfering peak in the blank solution, the elution time of the test solution and the reference solution is consistent, the target peak separation is greater than 1.5, and the number of theoretical plates is greater than 2000. The data are shown in Table 15, indicating that the method has good specificity.

[0080] Table 15: Results of the adaptability test of the gradient elution system of Sanguisorba officinalis saponin II

[0081] 5.4 Linear Relationship Dilute the reference solution under 5.2 into a series of standard working solutions with 6 concentrations of 0.2012 mg / mL, 0.1006 mg / mL, 0.08048 mg / mL, 0.0503 mg / mL, 0.02515 mg / mL, and 0.012575 mg / mL, and perform the test on the instrument. Draw a standard curve with concentration (X) as the horizontal axis and the corresponding peak area (Y) as the vertical axis, and calculate the regression equation and correlation coefficient. See the standard curve for details. Figure 10 The linear regression equation of sanguisorbaside II was y = 3378.3x + 2.7602, indicating that sanguisorbaside II had a good linear relationship with the peak area in the range of 0.012575-0.2012 mg / mL (R²=0.9999).

[0082] 5.5 Precision The reference solution from 5.2 was injected six times, and the peak areas were recorded and the RSD was calculated. The RSD for the peak areas after six injections was 1.479%, indicating good instrument precision under these chromatographic conditions (Table 16).

[0083] Table 16: Instrument precision test

[0084] 5.6 Stability Weigh 0.5 g of the herb, add 25 mL of methanol, and prepare a 20 mg / mL suibiying test solution according to the method in Section 5.2. Samples were injected and analyzed after 0 h, 1 h, 4 h, 16 h, 18 h, and 24 h. The peak area of sanguisorbaside II was recorded, and the relative standard deviation (RSD) was calculated to determine the stability of sanguisorbaside II in the suibiying extract. Results: The RSD values of the peak areas at 0 h, 1 h, 4 h, 24 h, 28 h, and 48 h were 1.737%, indicating that sanguisorbaside II in the test sample was stable within 48 h. Specific data are shown in Table 17.

[0085] Table 17: Stability test

[0086] 5.7 Repeatability Weigh 0.5 g of six samples of Jiubiying herbal medicine from the same batch and accurately add 25 mL of methanol. Prepare the test solution according to the method in Section 5.2, perform the analysis, and calculate the RSD. Results: After six consecutive injections, the RSD of the peak area was 1.29%, indicating good instrument precision. Specific data are shown in Table 18.

[0087] Table 18: Repeatability test

[0088] 5.8 Sample recovery Three portions of the same batch of Jiubiying medicinal materials were accurately weighed and spiked with a Sanguisorbaside II reference solution at 80%, 100%, and 120% of the known Sanguisorbaside II content, respectively. Test solutions were prepared according to the method in 5.2. After injection and testing, the average recovery and RSD values were calculated. Results: Recovery rates ranged from 98.03% to 104.13%, with an average recovery of 99.98% and an RSD of less than 3%. This experimental method is reliable for determining Sanguisorbaside II content. Specific data are shown in Table 19.

[0089] Table 19: Sample recovery test

[0090] Through the above methodological investigation, the detection method of Sanguisorba officinalis saponin II in the medicinal material Jiubiying provided by the present invention has good methodological investigation, whether it is isocratic elution or gradient elution.

[0091] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting Sanguisorba officinalis saponin II in the medicinal material of Jiubiying, comprising the following steps: preparation of a reference solution, preparation of a test solution, liquid chromatography detection and data analysis, wherein: The data analysis includes calculating the content of Sanguisorba officinalis saponin II in the Jiubiying medicinal material; The conditions used for liquid chromatography detection include: the chromatographic column filler is octadecylsilane bonded silica gel; the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, and an isocratic elution of 38% A to 40% A is used, or a gradient elution of 0-10 min, 35%→40% A; 10-45 min, 40%→46% A is used.

2. The detection method according to claim 1, wherein The conditions adopted for the liquid chromatography detection also include: a mobile phase flow rate of 0.80-1.20 mL / min, a column temperature of 30-35° C., and a detection wavelength of 203-210 nm.

3. The detection method according to claim 1, wherein The chromatographic column is selected from Diamonsil® C 18 (250 × 4.6 mm, 5 μm) column or Inno C 18 -AQ (250 × 4.6 mm, 5 μm) column.

4. The detection method according to claim 1, wherein The preparation of the test solution includes the following steps: taking the powder of the medicinal material Jiubing, accurately weighing it, placing it in a container, accurately adding the extraction solvent, sealing it, weighing it, after extraction, cooling it, weighing it again, making up the lost weight with the extraction solvent, shaking it evenly, and filtering it to obtain it.

5. The detection method according to claim 4, characterized in that The concentration of the test solution is 20-120 mg / mL, and the injection volume of the test solution in liquid chromatography detection is 10-30 μL.

6. The detection method according to claim 4, characterized in that The extraction solvent is a methanol aqueous solution, and the concentration of the methanol aqueous solution is greater than or equal to 70% by volume of methanol.

7. The detection method according to claim 4, characterized in that The extraction is carried out by ultrasonic extraction or heating reflux extraction, and the extraction time is 40-60 minutes.

8. The detection method according to claim 1, wherein The preparation of the reference solution comprises the following steps: taking a Sanguisorba officinalis saponin II reference substance, placing it in a container, accurately adding an extraction solvent, sealing the container, weighing the weight, cooling the container after extraction, weighing the weight again, making up the lost weight with the extraction solvent, shaking well, and filtering to obtain the solution.

9. The detection method according to claim 8, characterized in that The solvent is a methanol aqueous solution, and the concentration of the methanol aqueous solution is greater than or equal to 70% by volume of methanol.

10. Use of the detection method according to any one of claims 1 to 9 in quality detection of a sample containing Herba Cibotii, wherein the sample containing Herba Cibotii is selected from Herba Cibotii medicinal materials, Herba Cibotii decoction pieces, Herba Cibotii extracts, Herba Cibotii intermediates or Herba Cibotii preparations.

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