Method for determining content of effective saponin II in Ulmus pumila var. japonica by one-step method
By combining one-step alkaline ethanol solution reflux hydrolysis with high performance liquid chromatography, the problem of loss of target components for effective detection of elm saponin II in Sanguisorba officinalis was solved, and efficient and accurate efficacy evaluation was achieved.
Patent Information
- Application Number
- CN202610430120.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-02
- Publication Date
- 2026-07-10
AI Technical Summary
Existing methods for detecting effective elm saponin II in Sanguisorba officinalis involve stepwise extraction and hydrolysis processes that lead to the loss of the target component, making it difficult to achieve accurate and comprehensive efficacy evaluation.
A one-step extraction method was adopted, which involved reflux hydrolysis with alkaline ethanol solution to release free sanguisorbin II. The content was determined by high performance liquid chromatography, and the extraction process parameters were optimized to improve extraction efficiency and separation effect.
This method enables highly sensitive and accurate determination of the effective content of elm saponin II in Sanguisorba officinalis, simplifies the operation process, improves the comprehensiveness and accuracy of efficacy evaluation, and reduces testing costs.
Smart Images
Figure CN122361649A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of quality control and analysis of traditional Chinese medicine, and particularly relates to a one-step method for determining the content of effective elm saponin II in Sanguisorba officinalis. Background Technology
[0002] Burnt Sanguisorba officinalis ( Sanguisorba officinalis L.) is the dried root of *Sanguisorba officinalis* or *Sanguisorba longifolia*, both belonging to the Rosaceae family. It has the effects of cooling the blood and stopping bleeding, detoxifying and astringing sores. It is mainly used to treat hematochezia, hemorrhoidal bleeding, bloody dysentery, metrorrhagia, burns, carbuncles, and boils. The Chinese Pharmacopoeia includes methods and indicators for determining the content of tannins and gallic acid in *Sanguisorba officinalis*, but there are no methods or indicators for determining the content of saponins. Modern pharmacological studies have shown that saponins from *Sanguisorba officinalis* have good white blood cell-boosting, anti-inflammatory, antibacterial, antioxidant, anti-allergic, and anti-tumor activities, with saponin I and saponin II being the main active ingredients.
[0003] The content of original sanguisorbin II in Sanguisorba officinalis is relatively low compared to sanguisorbin I, making it susceptible to interference from other components during detection, thus requiring high sensitivity. Most existing reports only cover the determination of total saponins or sanguisorbin I in Sanguisorba officinalis. In fact, sanguisorbin II has effects such as increasing red blood cell count, vasoconstriction, hemostasis, antifungal activity, and antitumor activity, making its drug research prospects very broad.
[0004] Studies have reported that sanguisorbin I is the main component of total saponins from Sanguisorba officinalis, but sanguisorbin II is the main component after hydrolysis with sodium hydroxide. This indicates that the usable and exploitable effective sanguisorbin II in Sanguisorba officinalis includes not only the original sanguisorbin II fraction but also the sanguisorbin II fraction formed by hydrolysis. However, current efficacy evaluations of Sanguisorba officinalis focus only on the original sanguisorbin II fraction, neglecting the sanguisorbin II fraction formed by hydrolysis, which is clearly detrimental to the overall efficacy evaluation of Sanguisorba officinalis. Therefore, establishing a method for determining the effective sanguisorbin II fraction in Sanguisorba officinalis is of great significance for the in-depth development and comprehensive efficacy evaluation of Sanguisorba officinalis.
[0005] However, according to the current extraction and hydrolysis process, it is necessary to extract total saponins from Sanguisorba officinalis, then obtain the hydrolyzable saponin II fraction through alkaline hydrolysis, and finally determine the effective saponin II in Sanguisorba officinalis. This stepwise extraction and hydrolysis method is prone to loss of target components during the process, which is not conducive to the accurate evaluation of effective saponin II in Sanguisorba officinalis.
[0006] In conclusion, how to determine the effective content of elm saponin II in Sanguisorba officinalis using a one-step method to comprehensively and accurately evaluate the efficacy of Sanguisorba officinalis is an urgent problem to be solved. Summary of the Invention
[0007] The purpose of this invention is to provide a one-step method for determining the content of effective elmint saponin II in Sanguisorba officinalis. For the first time, an alkaline ethanol solution is used for reflux hydrolysis extraction during the preparation of the test solution. A certain concentration of NaOH provides an alkaline environment, effectively hydrolyzing the ester-bonded saponin precursors in Sanguisorba officinalis and releasing free elmint saponin II. The ethanol concentration balances the solubility of both polar and non-polar components, maximizing the dissolution of the target analyte. This method is more sensitive and accurate in determining the content of effective elmint saponin II in Sanguisorba officinalis, is simple to operate, has high extraction efficiency, good chromatographic separation, high precision and accuracy, and strong stability and durability, thus enhancing the overall quality control of Sanguisorba officinalis.
[0008] The technical solution of the present invention is as follows: A one-step method for determining the content of effective elm saponin II in Sanguisorba officinalis includes the following steps: (1) Preparation of reference solution: Take the reference standard of Sanguisorba officinalis saponin II and add solvent to prepare the reference solution.
[0009] (2) Preparation of test solution: The test solution was prepared by hydrolyzing and extracting the medicinal material of Sanguisorba officinalis with alkaline ethanol solution.
[0010] In the alkaline ethanol solution, the mass concentration of the alkali is 0.3-0.6%, and the volume concentration of the ethanol solution is 60-80%.
[0011] (3) The content of the reference solution and the test solution was determined by high performance liquid chromatography (HPLC). The detection conditions for HPLC included: The detection wavelength is 200-210 nm, the column temperature is 30-38 ℃, the injection volume is 10-30 μL, acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution is used as mobile phase B for gradient elution. The volume concentration of phosphoric acid aqueous solution is 0.1-0.5%.
[0012] Preferably, in step (1), the concentration of the reference solution is 0.15-0.20 mg / mL, and the solvent is methanol. More preferably, the concentration of the reference solution is 0.17-0.19 mg / mL.
[0013] Preferably, in step (2), the medicinal material of Sanguisorba officinalis is pulverized and passed through a No. 3 sieve to obtain medicinal powder, and then subjected to hydrolysis extraction.
[0014] Preferably, in step (2), the alkali is any one or more of sodium hydroxide, potassium hydroxide, and ammonia water. More preferably, the alkali is sodium hydroxide.
[0015] Preferably, in step (2), the ratio of the medicinal material of Sanguisorba officinalis to the alkaline ethanol solution is 4-20 g / L, and the hydrolysis extraction method is heating and reflux at a temperature of 50-85℃ for 20-60 min.
[0016] Preferably, in step (2), after hydrolysis and extraction of Sanguisorba officinalis, the sample is filtered, the pH is adjusted to 6-7 with hydrochloric acid, and then the volume is adjusted with methanol and filtered to obtain the test solution.
[0017] Preferably, in step (3), the gradient elution conditions are as follows: From 0 to 6 minutes, the volume percentage changes of mobile phase A and mobile phase B were 15%-15% and 85%-85%, respectively. Over 6-15 minutes, the volume percentage changes of mobile phase A and mobile phase B were 15%-30% and 85%-70%, respectively. Over 15-24 minutes, the volume percentage changes of mobile phase A and mobile phase B were 30%-55% and 70%-45%, respectively. Over 24-35 minutes, the volume percentage changes of mobile phase A and mobile phase B were 55%-35% and 45%-65%, respectively. Over 35-50 minutes, the volume percentage changes of mobile phase A and mobile phase B were 35%-15% and 65%-85%, respectively.
[0018] Preferably, in step (3), the detection conditions for high-performance liquid chromatography include: The detection wavelength was 203 nm, the column temperature was 35 °C, the injection volume was 20 μL, acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B for gradient elution.
[0019] Preferably, in step (3), the detection conditions of high performance liquid chromatography also include: using octadecylsilane-bonded silica gel as the packing material, with a column size of 250 mm × 4.6 mm and a particle size of 5 μm.
[0020] Preferably, the one-step method for determining the effective sanguisorbin II content in Sanguisorba officinalis further includes: firstly, investigating the hydrolysis extraction process of Sanguisorba officinalis using a single-factor method, selecting the optimal hydrolysis extraction process using the content of sanguisorbin II as an indicator, and then preparing the test solution using the selected optimal hydrolysis extraction process.
[0021] The selected optimal hydrolysis extraction process includes: taking Sanguisorba officinalis powder, adding alkaline ethanol solution at a material-to-liquid ratio of 4-10 g / L, wherein the alkaline ethanol solution contains 0.4-0.6% NaOH and the volume concentration of the ethanol solution is 60-70%, and then heating and refluxing at 70-80℃ for 30-40 min, and the hydrolysis extraction is performed once.
[0022] A method for preparing sanguisorbin II includes the following steps: 1) Hydrolysis extraction: Take the Sanguisorba officinalis herb and crush it into 1-10 mm pieces. Add the above-mentioned alkaline ethanol solution (containing 0.4-0.6% NaOH, with a volume concentration of 60-70%) and perform hydrolysis extraction 2-4 times, with each extraction lasting 1-2 hours. Combine the extracts, adjust the pH of the extract to neutral using hydrochloric acid, and then concentrate it to an ethanol concentration of 30-40%. Let it stand overnight to obtain the concentrated solution. 2) Primary precipitate collection: Filter the concentrate and collect the filtered primary precipitate; 3) Heating and refluxing with anhydrous ethanol: Add the collected initial precipitate to anhydrous ethanol and heat and reflux. After reflux treatment, cool the resulting mixture, add water to adjust the ethanol concentration in the mixture to 30-40%, and let it stand overnight. 4) Secondary precipitate collection: After the mixture has been left to stand overnight, the precipitate has fully formed. Filter the mixture and collect the filtered secondary precipitate. 5) Water heating and reflux: The collected secondary precipitate is added to water and heated and refluxed. The mixture obtained after reflux treatment is filtered to obtain crude Sanguisorbin II.
[0023] Preferably, in step 1), the amount of alkaline ethanol solution added is 6-15 times the amount of the Sanguisorba officinalis medicinal material.
[0024] Preferably, in step 3), the amount of anhydrous ethanol added is 10-30 times the mass of the initial precipitation, the reflux temperature is 50-85℃, and the time is 0.5-1.5h.
[0025] Preferably, in step 5), the amount of water added is 6-15 times the mass of the secondary sedimentation, the reflux temperature is 95-100℃, and the time is 0.5-1.5h.
[0026] This invention also provides the application of a one-step method for determining the effective content of elm saponin II in Sanguisorba officinalis in the quality control of Sanguisorba officinalis.
[0027] The beneficial effects of this invention are: 1. High hydrolysis extraction efficiency of Sanguisorba officinalis saponin II: Compared to the stepwise method of extracting total saponins from Sanguisorba officinalis and then obtaining the hydrolyzable Sanguisorba officinalis saponin II through alkaline hydrolysis, the extraction and hydrolysis rates of this method inevitably lead to the loss of the target component, Sanguisorba officinalis saponin II. This application adopts a one-step method to determine the content of effective Sanguisorba officinalis saponin II in Sanguisorba officinalis. In preparing the test solution, an alkaline ethanol solution (preferably 70% ethanol + 0.4% NaOH) is used for simultaneous reflux hydrolysis extraction. NaOH provides an alkaline environment, effectively hydrolyzing the ester-bonded saponin precursors in Sanguisorba officinalis and releasing free Sanguisorba officinalis saponin II. 70% ethanol balances the solubility of both polar and non-polar components, maximizing the dissolution of the target compound with minimal loss. Furthermore, by optimizing the hydrolysis extraction process parameters of Sanguisorba officinalis (extraction solvent, extraction method, alkali concentration, solid-liquid ratio, extraction time, and extraction times), the content of effective Sanguisorba officinalis saponin II is effectively improved.
[0028] 2. More comprehensive evaluation of the efficacy of Sanguisorba officinalis: Sanguisorba officinalis saponin II has high medicinal value, but the content of original Sanguisorba officinalis saponin II in Sanguisorba officinalis is low (often only a few parts per thousand). Evaluating only the content of original Sanguisorba officinalis saponin II in Sanguisorba officinalis is not only detrimental to its development and utilization, but also leads to the irrational use of the medicinal material. This application determines the content of effective Sanguisorba officinalis saponin II in Sanguisorba officinalis (the original Sanguisorba officinalis saponin II portion + the Sanguisorba officinalis saponin II portion that can be formed by hydrolysis), enabling a more accurate and comprehensive assessment of the efficacy value of Sanguisorba officinalis and providing a screening basis for the development of Sanguisorba officinalis saponin II sources.
[0029] 3. Excellent separation effect: By optimizing the hydrolysis extraction process and gradient elution procedure of Sanguisorba officinalis, this invention effectively removes impurities around the target peak, achieving good separation of Sanguisorba officinalis saponin II from other components in the complex matrix, thus ensuring the accuracy of quantification.
[0030] 4. Robust and reliable method: After comprehensive and rigorous methodological verification, it has been proven that the method of this invention for the determination of the content of sanguisorbin II has high precision, good accuracy, strong stability and good durability, and meets the requirements of the Chinese Pharmacopoeia for content determination methods.
[0031] 5. Simple and practical operation: It combines high efficiency and practicality, is easy to promote, and allows for direct pulverization and hydrolysis extraction of medicinal materials. There is no need for complicated pretreatment processes and long standing times. It uses conventional HPLC equipment, and the whole process is clear, greatly shortening the detection cycle, reducing costs and solvent pollution. It is easy to operate, which helps in the quality control of Sanguisorba officinalis and provides a good foundation for the development and application of Sanguisorba officinalis saponin II.
[0032] 6. The method of this invention can be used to hydrolyze and extract Sanguisorba officinalis medicinal material, and after two precipitation processes, Sanguisorba officinalis saponin II product with a purity of 92.76% can be obtained, which is beneficial to the development and application of Sanguisorba officinalis saponin II. Attached Figure Description
[0033] Figure 1 Comparison of the liquid chromatograms of Sanguisorba officinalis saponin I reference standard, Sanguisorba officinalis saponin II reference standard, water extract, and alkaline ethanol extract in Example 1 of the present invention; Figure 2 Comparison of the liquid chromatograms of Sanguisorba officinalis saponin I reference standard, Sanguisorba officinalis saponin II reference standard and 70% ethanol extract in Example 1 of the present invention; Figure 3 This is the liquid chromatogram of the reference standard Sanguisorba officinalis saponin II in Example 2 of the present invention; Figure 4 The liquid chromatograms of the test samples obtained in Example 2 of this invention using different extraction solvents (70% ethanol aqueous solution, alkaline aqueous solution, alkaline 40% ethanol aqueous solution, alkaline 70% ethanol aqueous solution, alkaline anhydrous ethanol, and alkaline 70% methanol aqueous solution); Figure 5 The above are liquid chromatograms of the test samples obtained by different extraction methods (ultrasonic extraction, reflux extraction, and reflux extraction with 70% ethanol aqueous solution followed by alkaline hydrolysis) in Example 2 of the present invention. Figure 6 The above are liquid chromatograms of the test samples obtained at different alkali concentrations (0.2%, 0.4%, 0.6%, 0.8%) in Example 2 of the present invention. Figure 7 The liquid chromatograms of the test samples obtained in Example 2 of the present invention with different material-liquid ratios (0.2 g / 50 mL, 0.5 g / 50 mL, 1.0 g / 50 mL, 2.0 g / 50 mL); Figure 8 The liquid chromatograms of the test samples obtained at different extraction times (10 min, 30 min, 60 min, 80 min) in Example 2 of the present invention are shown. Figure 9 The above are liquid chromatograms of the test samples obtained from different extraction times (first extraction, second extraction) in Example 2 of the present invention; Figure 10 The results of the specificity investigation in Example 3 of this invention (blank mobile phase); Figure 11 The results of the specificity examination in Example 3 of this invention (blank methanol); Figure 12 The results of the specificity test in Example 3 of this invention (70% ethanol aqueous solution); Figure 13This is the result of the specificity test in Example 3 of the present invention (Sanguisorba officinalis saponin II reference standard); Figure 14 The results of the specificity investigation in Example 3 of this invention (Sanguisorba officinalis medicinal material test sample); Figure 15 This is the standard curve of sanguisorbin II established in Example 3 of the present invention. Detailed Implementation
[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0035] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.
[0036] In this application, the concentrations of ethanol aqueous solution, methanol aqueous solution, and phosphoric acid aqueous solution are all volume percentages, and the concentrations of alkali are all mass percentages.
[0037] Example 1: Verification of the hydrolyzable saponin II moiety Take 50 g of Sanguisorba officinalis root, crush it into small pieces (1-10 mm), add 6 times the amount of water and decoct for 30 min (to remove water-soluble impurities), filter, and obtain an aqueous extract. Extract the residue twice with 10 times the amount of 70% ethanol aqueous solution and 70% ethanol aqueous solution containing 0.2% NaOH (pH 11-13), 1 h each time. Filter, combine the filtrates, and obtain a 70% ethanol extract and an alkaline 70% ethanol extract, respectively.
[0038] High-performance liquid chromatography (HPLC) was used to determine the contents of sanguisorbin I and sanguisorbin II in the reference standards, aqueous extract, 70% ethanol extract, and alkaline 70% ethanol extract. The results of the two components are shown in Table 1. A comparison of the chromatograms of sanguisorbin I and sanguisorbin II with the aqueous extract and alkaline 70% ethanol extract is shown in Table 1. Figure 1 A comparison of the chromatograms of sanguisorbin I reference standard, sanguisorbin II reference standard, and 70% ethanol extract is shown in [the original text]. Figure 2 .
[0039] Table 1
[0040] From Table 1 and Figure 1 , Figure 2It is known that initial water extraction removes some water-soluble impurities, but also yields a small amount of sanguisorbin I. Subsequent extractions with 70% ethanol and alkaline ethanol show that the 70% ethanol extraction primarily yields sanguisorbin I, indicating that sanguisorbin I is the main component of the total saponins extracted from Sanguisorba officinalis using ethanol. Alkaline ethanol extraction, on the other hand, yields a higher content of sanguisorbin II, which is the main component after alkaline hydrolysis of Sanguisorba officinalis.
[0041] Example 2: Determination of the content of effective elm saponin II in Sanguisorba officinalis. 1. High-performance liquid chromatography detection conditions A Shimadzu LC-20AT high-performance liquid chromatograph and Cosmosil C were used. 18 The chromatographic column (250 mm × 4.6 mm, particle size 5 μm) was used with gradient elution of acetonitrile (phase A) - 0.1% phosphoric acid aqueous solution (phase B). The gradient elution conditions are shown in Table 2. The detection wavelength was 203 nm, the column temperature was 35 °C, and the injection volume was 20 μL.
[0042] Table 2
[0043] 2. Preparation of reference solution The preparation method of the reference solution is as follows: Accurately weigh the sanguisorbin II reference standard, dissolve it in methanol to obtain a reference solution with a concentration of 0.184 mg / mL, and determine the sanguisorbin II reference standard under the aforementioned chromatographic conditions. The obtained chromatogram is shown below. Figure 3 As shown, the characteristic peak of sanguisorbin II can be clearly observed.
[0044] 3. Optimization of process parameters for hydrolysis extraction of sanguisorbin from Sanguisorba officinalis. (1) Comparison of different extraction solvents Accurately weigh 0.5 g of *Sanguisorba officinalis* powder (passed through a No. 3 sieve). Add 50 mL each of 70% ethanol aqueous solution and different extraction solvents containing 0.2% NaOH (alkaline aqueous solution, alkaline 40% ethanol aqueous solution, alkaline 70% ethanol aqueous solution, alkaline anhydrous ethanol, and alkaline 70% methanol aqueous solution). Extract by reflux at 80℃ for 30 min. After cooling, filter, wash the filter with a small amount of methanol, and combine the extracts into a 100 mL volumetric flask. Adjust the pH to 6-7 with dilute hydrochloric acid, and dilute to the mark with methanol. Filter, and inject 20 μL of each solvent according to the chromatographic conditions of this example. The results are shown in Table 3 and... Figure 4 The results indicate that the content of sanguisorbin II obtained by extraction with alkaline 70% ethanol aqueous solution is relatively high. Therefore, alkaline 70% ethanol aqueous solution was selected as the extraction solvent for the test sample.
[0045] Table 3 Comparison results of different extraction solvents (n=3)
[0046] (2) Comparison of different hydrolysis extraction methods ① Ultrasonic extraction or reflux extraction Weigh 0.5 g of Sanguisorba officinalis powder (passed through a No. 3 sieve), accurately weigh it, add 50 mL of 70% ethanol aqueous solution containing 0.2% NaOH, extract for 30 min using different methods (ultrasonic treatment, reflux at 80℃), cool, filter, wash the filter with a small amount of methanol, combine the extracts into a 100 mL volumetric flask, adjust the pH to 6-7 with dilute hydrochloric acid, dilute to the mark with methanol, filter, inject 20 μL of each sample, and determine according to the chromatographic conditions of this example.
[0047] ② Extract by reflux with 70% ethanol aqueous solution, then add alkali hydrolysis. Accurately weigh 0.5 g of *Sanguisorba officinalis* powder (passed through a No. 3 sieve), add 50 mL of 70% ethanol aqueous solution, and extract by reflux at 80℃ for 30 min. After cooling, filter. Add 0.2% NaOH to the filtrate and continue reflux extraction for 30 min. After cooling, filter again, wash the filter with a small amount of methanol, combine the extracts in a 100 mL volumetric flask, adjust the pH to 6-7 with dilute hydrochloric acid, and dilute to the mark with methanol. Filter, inject 20 μL of the extract, and determine the chromatographic conditions according to this example. The results are shown in Table 3 and... Figure 3 The results indicate that the content of sanguisorbin II obtained by extraction with alkaline 70% ethanol aqueous solution is relatively high. Therefore, alkaline 70% ethanol aqueous solution was selected as the extraction solvent for the test sample.
[0048] The results are shown in Table 4 and Figure 5 The results indicate that reflux extraction is superior to ultrasonic extraction; therefore, reflux extraction was chosen as the extraction method for the test sample. Furthermore, simultaneous extraction and hydrolysis with an alkaline 70% ethanol aqueous solution yielded a higher content of Sanguisorba officinalis saponin II than the stepwise operation of reflux extraction followed by alkaline hydrolysis with 70% ethanol. Therefore, direct reflux extraction of Sanguisorba officinalis with an alkaline 70% ethanol aqueous solution was chosen.
[0049] Table 4 Comparison results of different extraction methods (n=3)
[0050] (3) Comparison of different alkali concentrations Accurately weigh 0.5 g of *Sanguisorba officinalis* powder (passed through a No. 3 sieve). Add 50 mL of 70% ethanol aqueous solution with different alkali concentrations (0.2% NaOH, 0.4% NaOH, 0.6% NaOH, and 0.8% NaOH), respectively. Extract by reflux at 80℃ for 30 min. After cooling, filter, wash the filter with a small amount of methanol, and combine the extracts into 100 mL volumetric flasks. Adjust the pH to 6-7 with dilute hydrochloric acid, and dilute to the mark with methanol. Filter, and inject 20 μL of each extract according to the chromatographic conditions of this example. The results are shown in Table 5. Figure 6 The results showed that there was no significant difference in the content of Sanguisorba officinalis saponin II after the alkali concentration reached 0.4%, so a 0.4% alkali concentration was selected for the subsequent preparation of the test sample solution.
[0051] Table 5. Comparison results of different alkali concentrations (n=3)
[0052] (4) Comparison of different feed-liquid ratios Accurately weigh 0.2 g, 0.5 g, 1.0 g, and 2.0 g of *Sanguisorba officinalis* powder (passed through a No. 3 sieve), respectively. Add 50 mL of 70% ethanol aqueous solution containing 0.4% NaOH. Extract by reflux at 80℃ for 30 min. After cooling, filter, wash the filter with a small amount of methanol, and combine the extracts into 100 mL volumetric flasks. Adjust the pH to 6-7 with dilute hydrochloric acid, and dilute to the mark with methanol. Filter, and inject 20 μL of each extract. Analyze according to the chromatographic conditions of this example. The results are shown in Table 6 and... Figure 7 This indicates that there was no significant difference in the content of Sanguisorba officinalis saponin II when the material-to-liquid ratio was 0.5-1.0 g / 50 mL. Given that 0.5 g of sample is appropriate and easy to handle, 0.5 g / 50 mL was chosen as the subsequent material-to-liquid ratio.
[0053] Table 6 Comparison results of different feed-to-liquid ratios (n=3)
[0054] (5) Comparison of different extraction times Accurately weigh 0.5 g of *Sanguisorba officinalis* powder (passed through a No. 3 sieve), add 50 mL of 70% ethanol aqueous solution containing 0.4% NaOH, and extract by reflux at 80℃ for 10 min, 30 min, 60 min, and 80 min respectively. After cooling, filter, wash the filter with a small amount of methanol, combine the extracts into a 100 mL volumetric flask, adjust the pH to 6-7 with dilute hydrochloric acid, and dilute to the mark with methanol. Filter, and inject 20 μL of each extract according to the chromatographic conditions of this example. The results are shown in Table 7 and... Figure 8 The results indicate that the content of sanguisorbin II did not increase significantly after reflux extraction for 30 min, therefore, reflux extraction for 30 min was selected as the extraction time for the test sample.
[0055] Table 7 Comparison results of different extraction times (n=3)
[0056] (6) Comparison of different extraction times Accurately weigh 0.5 g of *Sanguisorba officinalis* powder (passed through a No. 3 sieve), add 50 mL of 70% ethanol aqueous solution containing 0.4% NaOH, and extract by reflux at 80℃ for 30 min. After cooling, filter, wash the filter with a small amount of methanol, combine the extracts in a 100 mL volumetric flask, adjust the pH to 6-7 with dilute hydrochloric acid, and dilute to the mark with methanol. Filter to obtain the solution obtained from the first extraction. Extract the residue again using the same method to obtain the solution obtained from the second extraction. Inject 20 μL of each extraction solution and determine the chromatographic conditions according to this example. The results are shown in Table 8 and... Figure 9 This indicates that one extraction is generally sufficient to extract all samples. Considering energy conservation, cost reduction, and ease of operation, one extraction cycle was chosen.
[0057] Table 8. Comparison results of different extraction times (n=3)
[0058] 4. Determination of the optimal hydrolysis process for Sanguisorba officinalis medicinal material Based on the above single-factor investigation, the optimal hydrolysis process for Sanguisorba officinalis was determined as follows: Weigh 0.5 g of Sanguisorba officinalis powder (passed through a No. 3 sieve), accurately weigh it, add 50 mL of 70% ethanol aqueous solution containing 0.4% NaOH, and heat and reflux at 80℃ for 30 min to extract the product.
[0059] 5. Preparation of test solution Based on the optimal hydrolysis process of *Sanguisorba officinalis* root material determined above, the method for determining the content of effective elmint saponin II in *Sanguisorba officinalis* root material was confirmed. The preparation method of the test sample was as follows: 0.5 g of *Sanguisorba officinalis* root material powder (passed through a No. 3 sieve) was accurately weighed, and 50 mL of 70% ethanol aqueous solution containing 0.4% NaOH was added. The mixture was heated under reflux at 80℃ for 30 min, cooled, filtered, and the filter was washed with a small amount of methanol. The extract was combined with the chromatographic data in a 100 mL volumetric flask, the pH was adjusted to 6-7 with dilute hydrochloric acid, and the volume was brought to the mark with methanol. The sample was then filtered to obtain the final product. The test sample was then analyzed according to the chromatographic conditions of this embodiment.
[0060] Example 3 Methodological Investigation 1. Specificity assessment The test solution of Sanguisorba officinalis was prepared according to the method confirmed in Example 2. A blank mobile phase, blank methanol, 70% ethanol aqueous solution, and Sanguisorba officinalis saponin II reference solution (prepared in Example 2) were simultaneously injected for high-performance liquid chromatography (HPLC) detection. Results are shown below. Figures 10-14This indicates that the method has strong specificity and is free from negative interference.
[0061] 2. Establishment of the standard curve Different concentrations (0.021362 mg / mL, 0.042764 mg / mL, 0.106900 mg / mL, 0.213820 mg / mL, 0.534500 mg / mL, and 1.069000 mg / mL) of sanguisorbin II reference solution were prepared, and 20 μL of each solution was injected. The chromatographic conditions of Example 2 were followed. A standard curve was plotted with the injection volume (μg) as the ordinate and the peak area as the abscissa. The results are shown in [Figure 2]. Figure 15 The peak area showed a good linear relationship between the injection amount of sanguisorbin II and the peak area in the range of 0.427-21.38 μg.
[0062] 3. Precision test The test solution of Sanguisorba officinalis was prepared according to the method confirmed in Example 2, and the prepared test solution was injected 6 times repeatedly under the chromatographic conditions of Example 2, with 20 μL injected each time to examine the precision. The results are shown in Table 9. The peak area RSD did not exceed 0.5%, indicating that the instrument precision was good.
[0063] Table 9 Precision test results
[0064] 4. Repeatability testing Six samples of Sanguisorba officinalis medicinal material were prepared according to the method confirmed in Example 2, and were injected sequentially under the chromatographic conditions of Example 2, with 20 μL injected each time to examine repeatability. The results are shown in Table 10. The RSD of Sanguisorba officinalis saponin II content did not exceed 2.0%, indicating good sample repeatability.
[0065] Table 10 Results of Repeatability Testing
[0066] 5. Stability test The test solution of Sanguisorba officinalis was prepared according to the method confirmed in Example 2, and 20 μL of the sample was injected at 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h according to the chromatographic conditions of Example 2 to investigate its stability. The results are shown in Table 11. The RSD of Sanguisorba officinalis saponin II content did not exceed 1.0%, indicating that the sample had good stability within 48 h.
[0067] Table 11 Stability test results
[0068] 6. Spiking recovery test Accurately weigh a sample of Sanguisorba officinalis with known content, add an appropriate amount of Sanguisorba officinalis saponin II reference solution, prepare a test solution of Sanguisorba officinalis according to the method confirmed in Example 2, and inject the sample for detection under the chromatographic conditions of Example 2, and calculate the recovery rate. The results are shown in Table 12, indicating that the sample recovery rate is good.
[0069] Table 12 Recovery test results (n=9)
[0070] 7. Durability Test The test solution of Sanguisorba officinalis was prepared according to the method confirmed in Example 2, and 20 μL was injected sequentially under the chromatographic conditions of Example 2 for detection. The effects of different flow rates (0.8 mL / min, 0.95 mL / min, 1.0 mL / min), different column temperatures (30℃, 35℃, 40℃), and different concentrations of phosphoric acid aqueous solution (0.05% phosphoric acid aqueous solution, 0.10% phosphoric acid aqueous solution, 0.15% phosphoric acid aqueous solution) were investigated. The results are shown in Table 13, indicating that the method has good robustness.
[0071] Table 13 Durability test results (n=3)
[0072] 8. Detection of Sanguisorba officinalis saponin II content in Sanguisorba officinalis from different origins Following the method confirmed in Example 2, 0.5 g of *Sanguisorba officinalis* medicinal materials from different origins were weighed, and test solutions of *Sanguisorba officinalis* medicinal materials were prepared. The solutions were then injected sequentially under the chromatographic conditions of Example 2, with 20 μL injected each time. The results are shown in Table 14. The content of effective elucsaponin II in *Sanguisorba officinalis* medicinal materials from different origins varied.
[0073] Table 14. Test results of Sanguisorba officinalis medicinal materials from different producing areas
[0074] Example 4 Preparation of Sanguisorba officinalis saponin II 1. Basic preparation process 1) Hydrolysis extraction: Take the medicinal material of Sanguisorba officinalis (produced in Hunan Province, batch number 202504015) and crush it into 1-10 mm pieces. Add 10 times the amount of 70% ethanol aqueous solution containing 0.4% NaOH and perform hydrolysis extraction 3 times in a row, with each extraction time being 1.5 hours. Combine the extracts, adjust the pH value of the extract to neutral with hydrochloric acid, and then concentrate it to an ethanol concentration of 30%. Let it stand overnight to obtain the concentrated solution. 2) Primary precipitate collection: Filter the concentrate and collect the filtered primary precipitate; 3) Heating and refluxing with anhydrous ethanol: Add the collected initial precipitate to 10 times the amount of anhydrous ethanol and reflux at 80°C for 1 hour. After reflux, cool the resulting mixture, add water to adjust the ethanol concentration in the mixture to 30%, and let it stand overnight. 4) Secondary precipitate collection: After the mixture has been left to stand overnight, the precipitate has fully formed. Filter the mixture and collect the filtered secondary precipitate. 5) Water heating and reflux: Add the collected secondary precipitate to 10 times the amount of water and reflux at 100℃ for 1 hour (to remove salt). Filter the mixture obtained after reflux treatment to obtain crude Sanguisorbin II (pale yellow powder).
[0075] 2. Effect of different extraction times on the transfer rate of Sanguisorba officinalis saponin II The *Sanguisorba officinalis* herb was extracted for the first time using the method described above, yielding the extract obtained from the first extraction. The residue 1 was kept for later use, and the remaining steps were followed to obtain the crude *Sanguisorba officinalis* saponin II obtained from the first extraction. The residue 1 was then extracted a second time, yielding the extract obtained from the second extraction. The residue 2 was kept for later use, and the remaining steps were followed to obtain the crude *Sanguisorba officinalis* saponin II obtained from the second extraction. The residue 2 was then extracted a third time, yielding the extract obtained from the third extraction, and the remaining steps were followed to obtain the crude *Sanguisorba officinalis* saponin II obtained from the third extraction. Different amounts of crude *Sanguisorba officinalis* saponin II were weighed, and the content of *Sanguisorba officinalis* saponin II was determined according to the chromatographic conditions of Example 2. The transfer rate of *Sanguisorba officinalis* saponin II was calculated, and the results are shown in Table 15. It can be seen that after extracting the *Sanguisorba officinalis* herb fragments three times, most of the *Sanguisorba officinalis* saponin II can be transferred.
[0076] The transfer rate of Sanguisorba officinalis saponin II (%) = 100% × (mass of crude Sanguisorba officinalis saponin II × content of Sanguisorba officinalis saponin II in crude Sanguisorba officinalis saponin II) / (mass of Sanguisorba officinalis saponin II medicinal material × 3.6%). Wherein, 3.6% is the effective content of Sanguisorba officinalis saponin II in the Sanguisorba officinalis medicinal material from Hunan province, batch number 202504015, as determined in Example 3.
[0077] Table 15 Transfer rate of sanguisorbin II obtained from different extraction times
[0078] 3. Determination of Sanguisorba officinalis saponin II content in precipitates from different processing stages The content of sanguisorbin II in the secondary precipitate and crude sanguisorbin II obtained in the preparation process was determined respectively. The results are shown in Table 16. It can be seen that the present invention can gradually improve the purity of sanguisorbin II product, and finally achieve a sanguisorbin II content of 92.76%.
[0079] Table 16 Content of Sanguisorba officinalis saponin II in different precipitates
[0080] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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 one-step method for determining the content of effective elm saponin II in Sanguisorba officinalis, characterized in that: The test solution was prepared by hydrolyzing and extracting the medicinal material of Sanguisorba officinalis using an alkaline ethanol solution. In the alkaline ethanol solution, the mass concentration of the alkali is 0.3-0.6%, the volume concentration of the ethanol solution is 60-80%, and the alkali is any one or more of sodium hydroxide, potassium hydroxide, and ammonia.
2. The method according to claim 1, characterized in that: The ratio of the medicinal material *Sanguisorba officinalis* to the alkaline ethanol solution is 4-20 g / L. The hydrolysis extraction method is reflux heating at a temperature of 50-85℃ for 20-60 min.
3. The method according to claim 1, characterized in that: It also includes first using single-factor analysis to investigate the hydrolysis extraction process of Sanguisorba officinalis, using the content of Sanguisorba officinalis saponin II as an indicator to screen out the best hydrolysis extraction process, and then using the screened best hydrolysis extraction process to prepare the test solution. The selected optimal hydrolysis extraction process includes: taking Sanguisorba officinalis powder, adding alkaline ethanol solution at a material-to-liquid ratio of 4-10 g / L, wherein the alkaline ethanol solution contains 0.4-0.6% NaOH and the volume concentration of the ethanol solution is 60-70%, and then heating and refluxing at 70-80℃ for 30-40 min, and the hydrolysis extraction is performed once.
4. The method according to claim 1, characterized in that: The content was determined using high-performance liquid chromatography (HPLC). The HPLC detection conditions included: The detection wavelength is 200-210 nm, the column temperature is 30-38 ℃, the injection volume is 10-30 μL, acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution is used as mobile phase B for gradient elution. The volume concentration of phosphoric acid aqueous solution is 0.1-0.5%.
5. The method according to claim 1, characterized in that: It also includes the preparation of a reference solution: take Sanguisorba officinalis saponin II reference standard, add methanol to prepare a reference solution, the concentration of the reference solution being 0.15-0.20 mg / mL.
6. The method according to claim 1, characterized in that: After hydrolysis and extraction of Sanguisorba officinalis, the sample solution is filtered, the pH is adjusted to 6-7 with hydrochloric acid, and then diluted to volume with methanol and filtered again to obtain the test solution.
7. The method according to claim 4, characterized in that: The gradient elution conditions are as follows: From 0 to 6 minutes, the volume percentage changes of mobile phase A and mobile phase B were 15%-15% and 85%-85%, respectively. Over 6-15 minutes, the volume percentage changes of mobile phase A and mobile phase B were 15%-30% and 85%-70%, respectively. Over 15-24 minutes, the volume percentage changes of mobile phase A and mobile phase B were 30%-55% and 70%-45%, respectively. Over 24-35 minutes, the volume percentage changes of mobile phase A and mobile phase B were 55%-35% and 45%-65%, respectively. Over 35-50 minutes, the volume percentage changes of mobile phase A and mobile phase B were 35%-15% and 65%-85%, respectively.
8. A method for preparing sanguisorbin II according to claim 1, characterized in that: Includes the following steps: 1) Hydrolysis extraction: Take the medicinal material of Sanguisorba officinalis and crush it into 1-10 mm pieces. Add the alkaline ethanol solution described in claim 1 and perform hydrolysis extraction 2-4 times, with each extraction time being 1-2 hours. Combine the extracts, adjust the pH of the extract to neutral with hydrochloric acid, and then concentrate it to an ethanol concentration of 30-40%. Let it stand overnight to obtain the concentrated solution. 2) Primary precipitate collection: Filter the concentrate and collect the filtered primary precipitate; 3) Heating and refluxing with anhydrous ethanol: Add the collected initial precipitate to anhydrous ethanol and heat and reflux. After reflux treatment, cool the resulting mixture, add water to adjust the ethanol concentration in the mixture to 30-40%, and let it stand overnight. 4) Secondary sediment collection: After the mixture has been left to stand overnight, it is filtered and the filtered secondary sediment is collected; 5) Water heating and reflux: The collected secondary precipitate is added to water and heated and refluxed. The mixture obtained after reflux treatment is filtered to obtain sanguisorbin II.
9. The method according to claim 8, characterized in that: In step 1), the amount of alkaline ethanol solution added is 6-15 times the weight of the Sanguisorba officinalis medicinal material; In step 3), the amount of anhydrous ethanol added is 10-30 times the mass of the initial precipitation, the reflux temperature is 50-85℃, and the time is 0.5-1.5h. In step 5), the amount of water added is 6-15 times the mass of the secondary sedimentation, the reflux temperature is 95-100℃, and the time is 0.5-1.5h.
10. The application of the method according to claim 1 in the quality control of Sanguisorba officinalis medicinal material.