A method for extracting effective components of a traditional Chinese medicine composition

Abstract

The extraction method of effective components of traditional Chinese medicine composition for treating knee osteoarthritis based on entropy weight method and response surface method belongs to the technical field of traditional Chinese medicine extraction, establishes the content determination method of index components in the composition, i.e., sinomenine, magnoflorine, paeonol glycoside, paeoniflorin and dioscin, and carries out methodology investigation; the comprehensive score of index component content is calculated by the entropy weight method as an evaluation index, the influence factors are ethanol concentration, solid-liquid ratio and soaking time, the experiment is designed by applying the response surface method, the index weight is determined by the comprehensive weighting theory of evaluation index, the comprehensive score is calculated, the multiple quadratic regression model of comprehensive score and investigation factors is established, the variance analysis is carried out, the optimal process is predicted and verified, and the optimized extraction process parameters of Jiantongkang are determined. The extraction condition optimization research of index components of the composition based on the entropy weight method and the response surface method is strong in pertinence, simple in operation, high in accuracy, scientific and reasonable in process, and is suitable for the content determination of medicinal material effective components.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction technology, specifically involving a combined extraction method based on entropy weight method and response surface methodology for the effective components of a traditional Chinese medicine composition for treating knee osteoarthritis—sinomenine, magnoflorine, paeoniflorin, paeoniflorin and diosgenin. Background Technology

[0002] Knee osteoarthritis, a type of bone and joint disease, is a major cause of leg pain in the elderly. In recent years, its incidence among middle-aged and elderly people has shown an increasing trend, making it one of the most common clinical diseases. Clinical symptoms include knee pain, swelling and clicking of the knee joint, stiffness and coldness in the knee joint, etc. Orthopedic experts at the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine have developed a traditional Chinese medicine formula called Xitongkang (Knee Pain Relief) based on the classic formula Lujiao Wan (Deer Antler Pill) and continuously adjusting the dosage according to clinical feedback. This formula uses five Chinese medicinal herbs: deer antler, Achyranthes bidentata, Sinomenium acutum, Dioscorea nipponica, and Paeonia lactiflora. Deer antler has the effects of warming kidney yang, strengthening tendons and bones, promoting blood circulation and reducing swelling, and is the principal herb. Achyranthes bidentata is neutral in nature, bitter, sweet, and sour in taste, and enters the liver and kidney meridians. Its effects are to tonify the liver and kidneys, strengthen tendons and bones, and is mainly used to treat lower back and knee pain and weakness of tendons and bones. The *Yilue Liushu* states, "Achyranthes bidentata strengthens the liver and kidneys to enhance tendons and bones." Sinomenium acutum is neutral in nature, bitter and pungent in taste, and enters the liver and spleen meridians. Its effects are to dispel wind and dampness, and unblock the meridians, and is mainly used to treat rheumatic pain and joint swelling. Its active ingredient, sinomenine, mainly works by inhibiting the expression and synthesis of tumor suppressor factor (TNF) and interleukin (IL), inhibiting prostaglandin synthesis, and inhibiting cyclooxygenase-2 activity. Achyranthes bidentata and Sinomenium acutum can assist in the treatment of knee osteoarthritis symptoms such as knee weakness, joint swelling, and numbness, serving as adjuvant herbs. Dioscorea nipponica is warm in nature, sweet and bitter in taste, and enters the liver, kidney, and lung meridians. It has the effects of dispelling wind and dampness, relaxing muscles and tendons, promoting blood circulation, and relieving pain, and is mainly used to treat joint numbness and rheumatic arthralgia. Its active ingredient, total saponins from Dioscorea nipponica, inhibits synovial cell proliferation, clears inflammatory cells, reduces tissue damage, and alleviates synovial cell inflammation by downregulating the levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6). Paeonia lactiflora is slightly cold in nature, bitter and sour in taste, and enters the liver and spleen meridians. It has the effects of nourishing blood and astringing yin. Paeonia lactiflora enters the liver to promote blood circulation, thereby achieving the effect of promoting blood circulation. Its active ingredient, total paeoniflorin, can lower the levels of nitric oxide and IL-1β, thereby regulating cartilage metabolism and relieving pain symptoms. Dioscorea nipponica and Paeonia lactiflora, along with the other three herbs, are used as adjuvants to treat joint swelling, pain, numbness, and spasms in knee osteoarthritis. The combination of Paeonia lactiflora and Achyranthes bidentata can prevent excessive nourishment and stagnation while promoting blood circulation and removing blood stasis, thus replenishing new blood and maximizing its physiological functions. The combination of these five herbs can achieve anti-inflammatory and analgesic effects. Pharmacological studies of traditional Chinese medicine have found that they mainly exert their effects by regulating and inhibiting the production of related inflammatory factors in the body.

[0003] Many methods are commonly used in laboratories to optimize extraction processes, including single-factor experiments, orthogonal experiments, and Box-Behnken response surface methodology. Compared with other methods, response surface methodology can achieve continuous-level optimization, has better predictive ability, and data visualization makes experimental results more intuitive, clearly revealing the interactions between various factors. It is widely used in the extraction research of traditional Chinese medicine. Based on this, this invention integrates entropy weight method and Box-Behnken response surface methodology to optimize the liquid-to-material ratio, soaking time, reflux time, number of refluxes, and ethanol concentration in the extraction process based on single-factor experiments, aiming to provide a reference for the production and application of traditional Chinese medicine compositions (such as *Sinomenium acutum*, *Dioscorea nipponica*, and stir-fried *Paeonia lactiflora*). The method of this invention has the characteristics of simple equipment, low cost, and stable extraction process. In addition, the method of this invention has good application value for the extraction processes of various traditional Chinese medicinal materials and can provide a theoretical basis for the production, application, and expansion of traditional Chinese medicine compositions for the treatment of knee osteoarthritis. Summary of the Invention

[0004] The present invention aims to provide an extraction process for a traditional Chinese medicine composition for treating knee osteoarthritis, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for extracting the effective components of a traditional Chinese medicine composition (Sinomenium acutum, Dioscorea nipponica, and stir-fried Paeonia lactiflora) for treating knee osteoarthritis based on entropy weight method and response surface methodology includes the following steps:

[0007] Based on the principle of response surface methodology and preliminary experimental results, this invention employs the Box-Behnken response surface methodology in Design-Expert 13 software to design an experimental scheme. Ethanol concentration, liquid-to-solid ratio, and soaking time were used as factors to extract the traditional Chinese medicine composition. High-performance liquid chromatography (HPLC) was used to determine the contents of sinomenine, magnoflorine, paeoniflorin, paeoniflorin, and diosgenin in the composition, and the methodology was validated. The comprehensive score of the contents of sinomenine, magnoflorine, paeoniflorin, paeoniflorin, and diosgenin was calculated using the entropy weight method as an evaluation index. The experiment was conducted using the Box-Behnken response surface methodology with 17 experimental points across 3 factors and 3 levels, and the results of each index were recorded.

[0008] Calculate the weight coefficients for each indicator: Based on the results of the 17 sets of experiments, the indicators are weighted using a comprehensive evaluation index system. Calculate the weight coefficients w. j j=1, 2, 3, 4, 5, which correspond to the contents of sinomenine w1, magnoflorine w2, paeoniflorin w3, paeoniflorin w4, and diosgenin w5 in the traditional Chinese medicine composition, respectively.

[0009] Calculate the overall score H: based on the obtained weight coefficients w of each indicator. j The comprehensive score H was calculated using the formula H = (Sinomenine content / maximum Sinomenine content) × w1 + (Magnoline content / maximum Magnoline content) × w2 + (Paeoniflorin content / maximum paeoniflorin content) × w3 + (Paeoniflorin content / maximum paeoniflorin content) × w4 + (Diosgenin content / maximum diosgenin content) × w5. Here, maximum Sinomenine content, maximum Magnoline content, maximum paeoniflorin content, maximum paeoniflorin content, and maximum diosgenin content represent the maximum values ​​of Sinomenine content, maximum Magnoline content, maximum paeoniflorin content, maximum diosgenin content, and maximum diosgenin content in the 17 sets of experimental data, respectively.

[0010] Model establishment and analysis of variance: The Design-Expert 13 software was used to perform a multivariate fitting analysis on the obtained comprehensive score H, and the multivariate quadratic regression equation model and analysis of variance results of the comprehensive score H with the three independent variables of ethanol concentration, liquid-to-solid ratio and soaking time were obtained.

[0011] Process prediction and validation: The obtained regression equation model was analyzed using Design-Expert 13 software. The optimal extraction process parameters and the predicted H-value of the traditional Chinese medicine composition were obtained. This process was validated by calculating the actual H-value and comparing it with the predicted H-value, demonstrating the stability and feasibility of the process.

[0012] The specific steps are as follows:

[0013] Step 1: Extraction of sinomenine, magnoflorine, paeoniflorin, paeoniflorin and diosgenin

[0014] Weigh out the Chinese ginseng, smilax china, and stir-fried white peony root and place them in a reaction vessel. Add ethanol to soak them, heat to reflux, filter, add ethanol to the residue again, heat to reflux, filter, combine the two filtrates, concentrate, and obtain the extract.

[0015] Step 2: Determination of the content of sinomenine, magnoflorine, paeoniflorin, paeoniflorin and diosgenin

[0016] Chromatographic conditions for sinomenine, magnoflorine, paeoniflorin, and paeoniflorin: The column was a Welch Ultimate® XB-C18 column, 4.6 mm × 250 mm, 5 μm in diameter; the flow rate was 1.0 mL / min; the column temperature was 20 ℃; the detection wavelength was 203 nm; the injection volume was 3 μL; the gradient elution program was as follows: the initial mobile phase ratio was 0.2% aqueous phosphoric acid-acetonitrile (88:12 v / v), held for 13 min, and then reduced to 0.2% aqueous phosphoric acid-acetonitrile (83:17 v / v) at 35 min.

[0017] Chromatographic conditions for diosgenin: The column was a Welch Ultimate® XB-C18 column, 4.6 mm × 250 mm, 5 μm in diameter; the flow rate was 1.0 mL / min; the column temperature was 30 ℃; the detection wavelength was 203 nm; the injection volume was 5 μL; the isocratic elution program was as follows: mobile phase water-acetonitrile (45:55 v / v), hold for 15 min;

[0018] Preparation of reference solutions: Accurately weigh sinomenine, magnoflorine, paeoniflorin, paeoniflorin and diosgenin, dissolve them in methanol to prepare reference stock solutions, accurately transfer the reference stock solutions and dilute them with methanol to obtain a series of mixed reference solutions of different mass concentrations for later use.

[0019] Preparation of test solution: The extract obtained from the concentration in step 1 was diluted to 50 mL with ethanol, sonicated for 20 min, and 200 μL was taken and diluted with ethanol. The solution was then vortexed, centrifuged, and filtered to obtain the test solution. The contents of sinomenine, magnoflorine, paeoniflorin, paeoniflorin and diosgenin in the extract were determined by high performance liquid chromatography.

[0020] Step 3: Methodological Validation

[0021] Specificity: Inject the test solution and the mixed reference solution into the chromatograph and analyze them according to the chromatographic conditions in step 2 to determine the specificity of the content determination method in step 2;

[0022] Linearity and Range: The mixed reference solution was precisely pipetted and injected for analysis. A standard curve was plotted with peak area (Y) on the ordinate and injection volume (X) on the abscissa.

[0023] Precision: Take the mixed reference solution and inject it continuously under the chromatographic conditions in step 2. Record the peak area of ​​each component to verify the instrument precision.

[0024] Stability: Take the test solution from step 2 and place it at room temperature for 0-24 h, then inject it for analysis according to the chromatographic conditions in step 2, record the peak area, and judge the stability of the test solution based on the RSD of the peak area.

[0025] Repeatability: Prepare the test solution according to the method in step 2, and inject it according to the chromatographic conditions in step 2. Record the peak area, calculate the average content of paeoniflorin lactone, paeoniflorin, magnoflorine, sinomenine and diosgenin peaks according to the external standard method, and verify the repeatability of the content determination method in step 2.

[0026] Recovery rate: Nine portions of the traditional Chinese medicine composition were accurately weighed and the test solution was prepared according to the method in step 2. The mixture was divided into three groups of three portions each. Low, medium, and high concentrations of mixed reference solutions were added to each group. The peak areas of paeoniflorin lactone, paeoniflorin, magnoflorine, sinomenine, and diosgenin were determined according to the chromatographic conditions in step 2. The recovery rate and RSD value of each component were calculated by substituting the values ​​into the accompanying standard curve. The accuracy and reliability of the method described in step 2 were verified based on the RSD value range. The low, medium, and high concentrations refer to the ratio of the added reference standard to the content of each component in the test sample being 0.5:1, 1:1, and 1.5:1, respectively.

[0027] Step 4: Calculate the overall score

[0028] Calculate the weight coefficients for each indicator: Based on the results of the 17 sets of experiments, the indicators are weighted using a comprehensive evaluation index system. Calculate the weight coefficients w. j j = 1, 2, 3, 4, 5, which correspond to the contents of sinomenine w1, magnoflorine w2, paeoniflorin w3, paeoniflorin w4, and diosgenin w5 in the traditional Chinese medicine composition, respectively.

[0029] Calculate the overall score H: based on the obtained weight coefficients w of each indicator. j The comprehensive score H was calculated and recorded using the formula H = (Sinomenine content / maximum Sinomenine content) × w1 + (Magnoline content / maximum magnoflorine content) × w2 + (Paeoniflorin content / maximum paeoniflorin content) × w3 + (Paeoniflorin content / maximum paeoniflorin content) × w4 + (Dioscorea saponin content / maximum dioscorea saponin content) × w5. The maximum contents of Sinomenine, magnoflorine, paeoniflorin, paeoniflorin, and dioscorea saponin represent the maximum values ​​of these contents in the 17 sets of experimental data, respectively.

[0030] Step 5: Response Surface Design

[0031] Single-factor experiments were conducted by sequentially changing the soaking time, ethanol concentration, and solid-liquid ratio, and high-performance liquid chromatography (HPLC) was used to extract the traditional Chinese medicine composition. The range of soaking time, ethanol concentration, and solid-liquid ratio was screened using the content of the traditional Chinese medicine composition as an indicator.

[0032] Based on the principles of response surface methodology and the results of single-factor experiments, a response surface methodology (RSM) experimental design was implemented. A Box-Behnken model was used to design a 3-factor, 3-level RSM experiment with 17 experimental points, including 12 factorial experiments and 5 central experiments. Ethanol concentration, material-to-liquid ratio, and soaking time were used as the factors of investigation. The comprehensive score of the calculated contents of paeoniflorin lactone, paeoniflorin, magnoflorine, sinomenine, and diosgenin was used as the evaluation index. The combined medicinal materials were extracted sequentially according to the experimental design, and the results of each index were recorded.

[0033] Step 6: Response Surface Analysis

[0034] The Design-Expert 13 software was used to perform a multivariate fitting analysis on the obtained comprehensive score H. The multivariate quadratic regression equation model and variance analysis results of the comprehensive score H with the three independent variables of ethanol concentration, material-liquid ratio and soaking time were obtained. Process prediction was also performed to obtain the optimal extraction process parameters of the traditional Chinese medicine composition and the model predicted H value. The process was verified, the actual H value was calculated and compared with the predicted H value to prove the stability and feasibility of the process.

[0035] The beneficial effects of this invention are:

[0036] This invention establishes a method for determining the content of the indicator components sinomenine, magnoflorine, paeoniflorin, paeoniflorin, and diosgenin in a traditional Chinese medicine composition and conducts methodological investigation. The comprehensive score of the indicator component content is calculated using the entropy weight method as the evaluation index. Ethanol concentration, material-to-liquid ratio, and soaking time are considered as influencing factors. Response surface methodology is applied to design experiments, and the weights of each indicator are determined using the comprehensive weighting theory of evaluation indicators. The comprehensive score is calculated, and a multiple quadratic regression model of the comprehensive score and the investigated factors is established. Analysis of variance is performed, and the optimal process is predicted and verified. Finally, the optimized extraction process parameters for Xitongkang (a traditional Chinese medicine) are determined.

[0037] This invention specifically relates to the optimization of extraction conditions for index components in a composition based on entropy weight method and response surface methodology. This method is highly targeted, simple to operate, highly accurate, and scientifically sound, making it particularly suitable for the determination of the content of effective components in medicinal materials. The index components selected for content determination in this invention have pharmacological and efficacy effects consistent with the clinical efficacy of Xitongkang (a traditional Chinese medicine), and are commonly used indicators in process research. Using the comprehensive score of the content of five active ingredients in the traditional Chinese medicine composition as the response value, the optimized process is scientifically sound and highly accurate.

[0038] Response surface methodology (RSM) is suitable for the design and optimization of experiments involving multiple factors and indicators. It can accurately predict function models and reflect the mathematical relationship between each factor and the response value. Compared with orthogonal design, it can more intuitively analyze the impact of each interaction factor on the experimental results. The comprehensive weighting theory of evaluation indicators assigns weights to the indicators, while also objectively and reasonably reflecting the importance of each indicator. Compared with subjective weighting methods, it is more scientific. This invention applies RSM to design experiments, uses comprehensive weighting theory to determine the weights of each indicator, calculates the comprehensive score, and determines the optimal extraction process parameters by analyzing the regression equation model. Compared with existing methods, it is more scientific, rigorous, and simple to implement.

[0039] This invention addresses, to some extent, the deficiencies in the prior art, providing theoretical basis and technical support for the later development of Xikangtong preparations and the research on the medicinal value of related medicinal materials. It also provides a reference for the optimization of preparation extraction processes in traditional Chinese medicine and has a promising application prospect. Attached Figure Description

[0040] Figure 1 The results of the linear relationship investigation of paeoniflorin lactone, paeoniflorin, magnoflorine, sinomenine and diosgenin in the traditional Chinese medicine composition;

[0041] Figure 2 This is a summary plot of response surfaces and contour lines showing the effects of various factors on the comprehensive score of the contents of paeoniflorin lactone, paeoniflorin, magnoflorine, sinomenine, and diosgenin in the traditional Chinese medicine composition. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0043] Example 1

[0044] Chromatographic conditions for sinomenine, magnoflorine, paeoniflorin, and paeoniflorin:

[0045] The chromatographic column was a Welch Ultimate® XB-C18 column, with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm; the flow rate was 1.0 mL / min; the column temperature was 20 ℃; the detection wavelength was 203 nm; the injection volume was 3 μL; and the gradient elution program was as follows: the initial mobile phase ratio was 0.2% aqueous phosphoric acid-acetonitrile (88:12 v / v), held for 13 min, and then reduced to 0.2% aqueous phosphoric acid-acetonitrile (83:17 v / v) at 35 min.

[0046] Chromatographic conditions for diosgenin:

[0047] The chromatographic column was a Welch Ultimate® XB-C18 column with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm; the flow rate was 1.0 mL / min; the column temperature was 30 ℃; the detection wavelength was 203 nm; the injection volume was 5 μL; and the isocratic elution program was as follows: mobile phase water-acetonitrile (45:55 v / v), hold for 15 min.

[0048] Example 2

[0049] 2.1 Establishment of content determination method

[0050] 2.1.1 Preparation of reference solution

[0051] Accurately weigh 2.0 mg of sinomenine, 2.34 mg of magnoflorine, 2.1 mg of paeoniflorin, 2.46 mg of paeoniflorin, and 2.22 mg of diosgenin, and dissolve them separately in methanol to prepare reference stock solutions with a mass concentration of 2.0 mg / mL. Accurately transfer the reference stock solutions and dilute them with methanol to prepare a series of mixed reference solutions with different mass concentrations (as shown in Table 1 below) for later use.

[0052] Table 1. Mixed reference solutions of various mass concentrations

[0053]

[0054] 2.1.2 Preparation of test solution

[0055] Weigh 5 g of the Chinese herbal medicine combination *Sinomenium acutum*, 7.5 g of *Dioscorea nipponica*, and 5 g of stir-fried *Paeonia lactiflora*, place them in a round-bottom flask, add 10 times the amount of 80% ethanol (solid-liquid ratio 1:10 g / mL), soak for 120 min, heat under reflux for 1.5 h, filter, add 10 times the amount of 80% ethanol to the residue again, heat under reflux for 1.5 h, filter, combine the two filtrates, concentrate to obtain the extract, add ethanol to make up to 50 mL, sonicate for 20 min, take 200 μL of the sample after making up to 50 mL, add 80% ethanol to dilute to 1000 μL, vortex, centrifuge, filter, and obtain the test solution.

[0056] 2.2 Methodological Validation

[0057] 2.2.1 Specificity

[0058] The test solution and reference solution were injected into the chromatograph and analyzed under the chromatographic conditions described in Example 1. The results showed that the method has good specificity.

[0059] 2.2.2 Linearity and Range

[0060] A mixed reference solution was injected for analysis. A standard curve was plotted with peak area (Y) on the ordinate and injection volume (X·μg / mL) on the abscissa. Figure 1 The results showed that the regression equations for paeoniflorin, magnoflorine, paeoniflorin, and sinomenine were Y = 2.7074X - 12.107 (R²). 2 = 0.9992), Y = 11.137X-374.93 (R 2 = 0.9993), Y = 4.304X-9.34 (R 2 = 0.9997), Y = 23.153X-706.9 (R 2 = 0.9997), with linear ranges of 50–300 μg / mL, 120–720 μg / mL, 60–720 μg / mL, and 100–600 μg / mL, respectively. The regression equation for diosgenin was Y = 1.5306X - 78.956 (R² = 0.9997). 2 = 0.9993), with a linear range of 100~850 μg / mL.

[0061] 2.2.3 Precision

[0062] The mixed reference solution was injected six times consecutively under the chromatographic conditions described in Example 1, and the peak areas of each component were recorded. The results showed that the RSDs of the peak areas of paeoniflorin lactone, paeoniflorin, magnoflorine, and sinomenine were 0.73%, 0.64%, 0.34%, and 0.43% (n = 6), respectively, while the RSD of the peak area of ​​diosgenin was 1.61% (n = 6), indicating good instrument precision.

[0063] 2.2.4 Stability

[0064] The prepared test solution was placed at room temperature for 0, 2, 4, 8, 12, and 24 hours, and then injected for analysis under the chromatographic conditions described in Example 1. The peak areas were recorded. The results showed that the RSDs of the peak areas of paeoniflorin lactone, paeoniflorin, magnoflorine, and sinomenine were 0.79%, 0.19%, 0.57%, and 0.26% (n = 6), respectively, while the RSD of the peak area of ​​diosgenin was 1.79% (n = 6), indicating that the test solution had good stability within 24 hours at room temperature.

[0065] 2.2.5 Repeatability

[0066] Prepare six parallel solutions of the test sample. Inject and determine the peak areas according to the chromatographic conditions of Example 1. Calculate the content using the external standard method. The results showed that the average contents of paeoniflorin lactone, paeoniflorin, magnoflorine, and sinomenine were 477.7 mg / g, 1560.9 mg / g, 2081.2 mg / g, and 6525.8 mg / g, respectively, with RSDs of 1.28%, 1.63%, 1.97%, and 1.30% (n = 6). The average content of diosgenin was 770.3 mg / g, with an RSD of 1.53% (n = 6), indicating that the method has good repeatability.

[0067] 2.2.6 Recovery rate

[0068] Nine portions of the traditional Chinese medicine composition were accurately weighed and the test solution was prepared according to Example 2. The mixture was divided into three groups of three portions each. Low, medium, and high concentrations of mixed reference standard solutions were added to each group, ensuring that the ratio of the added reference standard to the content of each component in the test sample was approximately 0.5:1, 1:1, and 1.5:1, respectively. The peak areas of paeoniflorin lactone, paeoniflorin, magnoflorine, and sinomenine were determined according to the chromatographic conditions of Example 1. The measured amounts were calculated using the external standard method, and the results were calculated according to the formula... The recovery rate and RSD value of each component were calculated. The average recovery rate of the four analytes ranged from 93.0% to 106.0%, and the RSD value ranged from 0.20% to 1.97%, all of which met the requirement of RSD≤2%, indicating that the method is accurate and reliable. The results are shown in Table 2.

[0069] Table 2. Results of spiking recovery tests for paeoniflorin lactone, paeoniflorin, magnoflorine, and sinomenine (n=9)

[0070]

[0071] Nine portions of the traditional Chinese medicine composition were accurately weighed and the test solution was prepared according to Example 2. The mixture was divided into three groups of three portions each. Low, medium, and high concentrations of mixed reference standard solutions were added to each group, ensuring that the ratio of the added reference standard to the content of each component in the test sample was approximately 0.5:1, 1:1, and 1.5:1, respectively. The peak area of ​​diosgenin was determined according to the chromatographic conditions of Example 1. The measured amount was calculated using the external standard method, and the result was calculated according to the formula... The recoveries and RSDs of each component were calculated. The results showed that the recoveries of diosgenin at low, medium, and high levels were 95.8%, 98.7%, and 101.2%, respectively, and the RSDs for these three levels were 0.58%, 0.75%, and 0.83%, respectively, indicating high accuracy of the method. The results are shown in Table 3.

[0072] Table 3. Results of the recovery test of diosgenin (n=9)

[0073]

[0074] The following examples screen soaking time, material-to-liquid ratio, and ethanol concentration;

[0075] Example 3

[0076] The herbal composition was weighed and placed in a round-bottom flask. Ten times the volume of 80% ethanol was added, and the mixture was soaked for 120 min. The solution was then filtered through gauze, refluxed for 1.5 h, filtered again, and 10 times the volume of 80% ethanol was added to the residue. The mixture was refluxed for 1.5 h, filtered again, and the two filtrates were combined and concentrated. The effects of different soaking times on the extraction of paeoniflorin lactone, paeoniflorin, magnoflorine, and sinomenine were investigated under the chromatographic conditions of Example 1.

[0077] Example 4

[0078] Unlike Example 3, this example involves soaking in 10 times the amount of 80% ethanol for 40 minutes and then filtering with gauze.

[0079] Example 5

[0080] Unlike Example 3, this example involves soaking in 10 times the amount of 80% ethanol for 60 minutes and then filtering with gauze.

[0081] Example 6

[0082] Unlike Example 3, this example involves soaking the food in 10 times the amount of 80% ethanol for 80 minutes and then filtering it with gauze.

[0083] Example 7

[0084] Unlike Example 3, this example involves soaking in 10 times the amount of 80% ethanol for 100 minutes and then filtering with gauze.

[0085] Examples 3-7 illustrate the effects of different soaking times on the extraction of sinomenine, magnoflorine, paeoniflorin, and paeoniflorin. Based on a comprehensive consideration of the extract content results, a soaking time of 60-80 min was selected for Box-Behnken response surface methodology experiments.

[0086] Example 8

[0087] The herbal composition was weighed and placed in a round-bottom flask. Ten times the volume of 80% ethanol was added, and the mixture was soaked for 120 min. The solution was then filtered through gauze, refluxed for 1.5 h, filtered again, and 10 times the volume of 80% ethanol was added to the residue. The mixture was refluxed for 1.5 h, filtered again, and the two filtrates were combined and concentrated. The effects of different ethanol concentrations on the extraction of paeoniflorin lactone, paeoniflorin, magnoflorine, and sinomenine were investigated under the chromatographic conditions of Example 1.

[0088] Example 9

[0089] Unlike Example 8, this time it was soaked in 10 times the amount of 50% ethanol for 120 min.

[0090] Example 10

[0091] Unlike Example 8, this time it was soaked in 10 times the amount of 60% ethanol for 120 min.

[0092] Example 11

[0093] Unlike Example 8, this time it was soaked in 10 times the amount of 70% ethanol for 120 min.

[0094] Example 12

[0095] Unlike Example 8, this time it was soaked in 10 times the amount of 90% ethanol for 120 min.

[0096] Examples 8-12 illustrate the effects of different concentrations of ethanol on the extraction of sinomenine, magnoflorine, paeoniflorin, and paeoniflorin. Based on a comprehensive consideration of the extract content results, ethanol concentrations of 70%–90% were selected for Box-Behnken response surface methodology experiments.

[0097] Example 13

[0098] The herbal composition was weighed and placed in a round-bottom flask. Ten times the volume of 80% ethanol was added, and the mixture was soaked for 120 min. The solution was then filtered through gauze, refluxed for 1.5 h, filtered again, and 10 times the volume of 80% ethanol was added to the residue. The mixture was refluxed for 1.5 h, filtered again, and the two filtrates were combined and concentrated. The effects of different liquid-to-solid ratios of ethanol volume on the extraction of paeoniflorin lactone, paeoniflorin, magnoflorine, and sinomenine were investigated under the chromatographic conditions of Example 1.

[0099] Example 14

[0100] The difference from Example 13 is that the liquid-to-solid ratio is 4:1 mL·g -1 Soak in 80% ethanol for 120 min.

[0101] Example 15

[0102] The difference from Example 13 is that the liquid-to-solid ratio is 6:1 mL·g. -1 Soak in 80% ethanol for 120 min.

[0103] Example 16

[0104] The difference from Example 13 is that the liquid-to-solid ratio is 8:1 mL·g. -1 Soak in 80% ethanol for 120 min.

[0105] Example 17

[0106] The difference from Example 13 is that the liquid-to-solid ratio is 12:1 mL·g. -1 Soak in 80% ethanol for 120 min.

[0107] Example 18

[0108] The difference from Example 13 is that the liquid-to-solid ratio is 14:1 mL·g -1 Soak in 80% ethanol for 120 min.

[0109] Example 19

[0110] The difference from Example 13 is that the liquid-to-solid ratio is 16:1 mL·g. -1 Soak in 80% ethanol for 120 min.

[0111] Examples 13-19 illustrate the effects of different concentrations of ethanol on the extraction of sinomenine, magnoflorine, paeoniflorin, and paeoniflorin. Based on a comprehensive consideration of the extract content results, a liquid-to-solid ratio of 10:1 to 14:1 mL·g was selected. -1 Conduct Box-Behnken response surface experiments.

[0112] Example 20

[0113] The herbal composition was weighed and placed in a round-bottom flask. Ten times the volume of 80% ethanol was added, and the mixture was soaked for 120 min. The solution was then filtered through gauze, heated under reflux for 1.5 h, filtered again, and 10 times the volume of 80% ethanol was added to the residue. The mixture was heated under reflux for 1.5 h, filtered again, and the two filtrates were combined and concentrated. The effect of different soaking times on the extraction of diosgenin was investigated under the chromatographic conditions described in Example 1.

[0114] Example 21

[0115] Unlike Example 20, this example involves soaking in 10 times the amount of 80% ethanol for 40 minutes and then filtering with gauze.

[0116] Example 22

[0117] Unlike Example 20, this example involves soaking in 10 times the amount of 80% ethanol for 60 minutes and then filtering with gauze.

[0118] Example 23

[0119] Unlike Example 20, this example involves soaking in 10 times the amount of 80% ethanol for 80 minutes and then filtering with gauze.

[0120] Example 24

[0121] Unlike Example 20, this one was soaked in 10 times the amount of 80% ethanol for 100 minutes and then filtered through gauze.

[0122] Examples 20-24 illustrate the effect of different soaking times on diosgenin. Based on a comprehensive consideration of the extract content results, a soaking time of 60-80 min was selected for Box-Behnken response surface methodology.

[0123] Example 25

[0124] The herbal composition was weighed and placed in a round-bottom flask. Ten times the volume of 80% ethanol was added, and the mixture was soaked for 120 min. The solution was then filtered through gauze, refluxed for 1.5 h, filtered again, and 10 times the volume of 80% ethanol was added to the residue. The mixture was refluxed for 1.5 h, filtered again, and the two filtrates were combined and concentrated. The effect of different ethanol concentrations on the extraction of diosgenin was investigated under the chromatographic conditions described in Example 1.

[0125] Example 26

[0126] Unlike Example 25, this one was soaked in 10 times the amount of 50% ethanol for 120 min.

[0127] Example 27

[0128] Unlike Example 25, this one was soaked in 10 times the amount of 60% ethanol for 120 min.

[0129] Example 28

[0130] Unlike Example 25, this one was soaked in 10 times the amount of 70% ethanol for 120 min.

[0131] Example 29

[0132] Unlike Example 25, this one was soaked in 10 times the amount of 90% ethanol for 120 min.

[0133] Examples 25-29 illustrate the effect of different concentrations of ethanol on the extraction of diosgenin from diosgenin. Based on a comprehensive consideration of the extract content results, ethanol concentrations of 70%–90% were selected for Box-Behnken response surface methodology experiments.

[0134] Example 30

[0135] The herbal composition was weighed and placed in a round-bottom flask. Ten times the volume of 80% ethanol was added, and the mixture was soaked for 120 min. The mixture was then filtered through gauze, refluxed for 1.5 h, filtered again, and 10 times the volume of 80% ethanol was added to the residue. The mixture was refluxed for 1.5 h, filtered again, and the two filtrates were combined and concentrated. The effect of different liquid-to-solid ratios of ethanol volume on the extraction of diosgenin was investigated under the chromatographic conditions described in Example 1.

[0136] Example 31

[0137] The difference from Example 30 is that the liquid-to-solid ratio is 4:1 mL·g-1 Soak in 80% ethanol for 120 min.

[0138] Example 32

[0139] The difference from Example 30 is that the liquid-to-solid ratio is 6:1 mL·g -1 Soak in 80% ethanol for 120 min.

[0140] Example 33

[0141] The difference from Example 30 is that the liquid-to-solid ratio is 8:1 mL·g -1 Soak in 80% ethanol for 120 min.

[0142] Example 34

[0143] The difference from Example 30 is that the liquid-to-solid ratio is 12:1 mL·g -1 Soak in 80% ethanol for 120 min.

[0144] Example 35

[0145] Unlike Example 30, the liquid-to-solid ratio was 14:1 mL·g -1 Soak in 80% ethanol for 120 min.

[0146] Example 36

[0147] The difference from Example 30 is that the liquid-to-solid ratio is 16:1 mL·g -1 Soak in 80% ethanol for 120 min.

[0148] Examples 30-36 illustrate the effect of different concentrations of ethanol on the extraction of diosgenin from traditional Chinese medicine compositions. Based on a comprehensive consideration of the extract content results, a liquid-to-solid ratio of 10:1 to 14:1 mL·g was selected. -1 Conduct Box-Behnken response surface experiments.

[0149] Example 37

[0150] Comprehensive weighting of evaluation indicators

[0151] (1) G1 Subjective Empowerment

[0152] The G1 method is a fast, subjective evaluation method that does not require consistency testing. First, the order of the rating indicators is determined. Based on the effective components of the traditional Chinese medicine composition, this experiment determined the order of the five evaluation indicators in the composition as follows: paeoniflorin lactone (1) = paeoniflorin (2) = magnoflorine (3) = sinomenine (4) = diosgenin (5). Next, the relative importance between adjacent indicators is determined, and their values ​​are r1 = r2 = r3 = r4 = r5 = 1 (ri To represent the degree of importance, the five indicators are first ranked by importance, and then adjacent indicators are compared. Since the five indicators are equally important in this study, the relative importance r is calculated. i =1), and finally, the weighting coefficients of each indicator are calculated ( w 1j ) calculation. ( m (Number of evaluation indicators).

[0153] (2) Objective assignment of entropy weight method

[0154] The entropy weight method determines the weights of indicators based on the degree of variation in their values. This is an objective weighting method that avoids biases caused by human factors, resulting in higher accuracy, stronger objectivity, and better interpretation of the results. The smaller the degree of variation in an indicator, the less information it reflects, and therefore, its corresponding weight should be lower. First, an original evaluation indicator matrix is ​​established ( x ij Then, based on the principle (positive for effective treatment and negative for worsening of disease), the positive and negative values ​​of each indicator of the traditional Chinese medicine composition are determined, the maximum, minimum and difference values ​​of each indicator are calculated, and then standardized to obtain a matrix. y ij ) Positive indicators are standardized using the first formula, and negative indicators are standardized using the second formula. Then, a probability matrix is ​​constructed ( p ij ) Then, calculate the information entropy of each indicator ( e 2j ) and the coefficient of difference ( g 2j () g 2j =1- e 2j Finally, the weighting coefficients are calculated. w 2j ) , n Indicates the number of experiments. m This indicates the number of evaluation indicators.

[0155] (3) Determining the combined weights

[0156] Let the subjective weights obtained by the G1 method be... w 1j The objective weights obtained by the entropy weight method are: w 2j The combined weight is w j The results are shown in Table 4.

[0157] Table 4 Weight values ​​of extraction process evaluation indicators

[0158]

[0159] (4) Comprehensive evaluation method

[0160] The combined weight values ​​of each index calculated in (3) can be further calculated to obtain the comprehensive score H of each experimental condition index. Then, we conduct intuitive analysis and analysis of variance.

[0161] Example 38

[0162] Box-Behnken response surface methodology

[0163] (1) Experimental design and results of Box-Behnken response surface methodology

[0164] Based on the results of the single-factor experiments, the comprehensive score H of the contents of paeoniflorin lactone, paeoniflorin, magnoflorine, sinomenine, and diosgenin was used as the evaluation index, and ethanol concentration (A), liquid-to-solid ratio (B), and soaking time (C) were used as influencing factors. The three-factor, three-level Box-Behnken response surface methodology was used for the experiment. The comprehensive score of the contents of paeoniflorin lactone, paeoniflorin, magnoflorine, sinomenine, and diosgenin was calculated according to the comprehensive scoring method in Example 37 (4). The factors and levels of the Box-Behnken response surface experimental design are shown in Table 5. The scheme and results are shown in Table 6.

[0165] Table 5. Factors and levels in the Box-Behnken response surface methodology experiment.

[0166]

[0167] Table 6. Experimental Design and Results of Box-Behnken Response Surface Methodology

[0168]

[0169] (2) Model fitting and analysis of variance

[0170] The Design-Expert 13 software was used to perform multiple regression fitting and analysis of variance on the data in Table 4. The results are shown in Table 7. The quadratic multinomial regression equation between factors A, B, and C and the comprehensive score H is: H = 0.9581 - 0.0074A - 0.0040B - 0.0041C - 0.0003AB - 0.0172AC - 0.0135BC - 0.0256A 2 -0.0114B 2 -0.0295C 2 As shown in Table 7, the model... P <0.05, lack of analogyP =0.5746, the model is significant, and the lack of fit is not significant, indicating that the unknown factors have little impact on the experiment, and the model can be used to predict experimental results. The model correlation coefficient R0 2 =0.9726, adjusted R 2 =0.9374, coefficient of variation =0.6946, indicating that the model has a good simulation effect and the method is reliable.

[0171] Table 7. Results of ANOVA for the Regression Model

[0172]

[0173] The F-value indicates the significance of the entire fitted equation. The larger the F-value, the more significant the equation and the better the fit. The P-value is an indicator of the difference between the control group and the experimental group.

[0174] (3) Response surface analysis and prediction

[0175] Box-Behnken response surface analysis was performed on the model equations using Design-Expert 13 software, yielding contour plots and response surface plots. The results are shown below. Figure 2 .Depend on Figure 2 It is evident that the interaction between ethanol concentration and liquid-to-solid ratio, and between liquid-to-solid ratio and soaking time, is stronger, with the contour plots tending towards ellipses and the response surface plots being steeper. This indicates that the interaction between ethanol concentration and liquid-to-solid ratio, and between liquid-to-solid ratio and soaking time, has a more significant impact on the overall score. The response surface plot for liquid-to-solid ratio is steeper than that for ethanol concentration and soaking time, indicating that the liquid-to-solid ratio has a greater impact on the overall extraction process score. Based on the model fitting results, the optimal extraction process for paeoniflorin lactone, paeoniflorin, magnoflorine, sinomenine, and diosgenin is predicted to be an ethanol concentration of 78.507%, a liquid-to-solid ratio of 11.632:1, and a soaking time of 70.153 min. Further adjustments based on actual operation resulted in an optimal extraction process of an ethanol concentration of 78%, a liquid-to-solid ratio of 12:1, and a soaking time of 70 min.

[0176] (4) Validation of extraction processes for paeoniflorin lactone, paeoniflorin, magnoflorine, sinomenine and diosgenin

[0177] The optimal extraction process was repeated three times for validation. The results showed that the average comprehensive score of the three validation experiments was 0.946, and the RSD was 0.42%, indicating that the established extraction process of the traditional Chinese medicine composition was stable and had good repeatability. The deviation between the actual average comprehensive score and the predicted value (0.959) obtained by the Box-Behnken response surface methodology was 1.32%, indicating that the model had good predictive ability. The results are shown in Table 8.

[0178] Table 8. Validation results of extraction processes for paeoniflorin lactone, paeoniflorin, magnoflorine, sinomenine, and diosgenin.

[0179] 。

Claims

1. A method for extracting the effective components of a traditional Chinese medicine composition, characterized in that, The extraction of the traditional Chinese medicine composition includes the following steps: Weigh 5g of Qingfengteng, 7.5g of Chuanshanlong, and 5g of stir-fried Baishao, place them in a reaction vessel, add 78% ethanol at a liquid-to-solid ratio of 12:1 and soak for 70 minutes, heat under reflux for 1.5 hours, filter, add 78% ethanol to the residue again, heat under reflux for 1.5 hours, filter, combine the two filtrates, concentrate, and obtain the extract.

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