Response surface method-optimized extraction process for total chromone aglycones of radix saposhnikoviae and application of total chromone aglycones

Abstract

The invention provides an extraction process for optimizing total chromone aglycones of radix saposhnikoviae through a response surface method and application of the total chromone aglycones. By taking the total content of cimicifugin and 5-O-methylvisammiol in an extracting solution as an index, the extraction process of the total chromone aglycone of the radix saposhnikoviae is optimized. The extraction process is optimized through a center point (CCD) design test by taking the enzyme adding amount, the reaction time and the reaction temperature as single factors and the total extraction rate (%) of the cimicifugin and the 5-O-methylvisammiol as response values, so that the optimal process is that the enzyme adding amount is 89.87%, the reaction temperature is 55.95 DEG C, the reaction time is 59.97 min, and under the optimal extraction condition, the theoretical extraction rate is 0.458%, and the reaction time is 55.95 min. The actual extraction rate is 0.449%; a high performance liquid chromatograph is used for qualitative analysis, and it is speculated that an extracting solution contains compounds such as cimicifugin and 5-O-methylvisamidol; the radix saposhnikoviae total chromone aglycone is prepared by using a D101 macroporous resin chromatographic column, the activity of the radix saposhnikoviae total chromone aglycone is studied, and the result shows that the radix saposhnikoviae total chromone aglycone has a good inhibition effect on NO secretion of mouse macrophages (RAW 264.7) induced by lipopolysaccharide (LPS).

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and particularly relates to a method for extracting total chromone aglycones from siler and application thereof. Background Art

[0002] Saposhnikovia divaricata (Turcz.) Schisck is a commonly used Chinese medicine with a long history of use. It is the dried root of the Umbelliferae plant Saposhnikovia divaricata (Turcz.) Schisck. It tastes spicy and slightly sweet, is warm in nature, and enters the liver, bladder, and spleen meridians. It has the effects of dispelling wind and relieving exterior symptoms, eliminating dampness and relieving pain, and stopping spasms. It was first recorded in Shennong's Herbal Classic and listed as a top-grade Chinese medicine. Today, the production areas of Saposhnikovia divaricata are mainly distributed in Inner Mongolia, Jilin, Heilongjiang, Liaoning and other provinces. Baicheng area in Jilin Province is one of the important authentic production areas of Saposhnikovia divaricata, and the current planting area is large. Modern research shows that Saposhnikovia divaricata contains rich chemical components, including chromones, volatile oils, coumarins and polysaccharides. Among them, chromone components are the main components that play a pharmacological role in Saposhnikovia divaricata, and have multiple pharmacological activities such as antipyretic, analgesic, anti-inflammatory, antioxidant, and antiplatelet coagulation.

[0003] The 2020 edition of the Chinese Pharmacopoeia uses the content of two chromone components, cimicifuga and 5-O-methylvisaminol glycoside, as the quality control standard for Saposhnikovia divaricata, stipulating that it must not be less than 0.24%. Studies have shown that cimicifuga and 5-O-methylvisaminol glycoside are converted into cimicifuga in the body and enter the blood, exerting their efficacy indirectly through cimicifuga. The aglycone cimicifuga is a direct-acting ingredient with obvious anti-inflammatory and analgesic effects. Its activity is 4-12 times that of cimicifuga glycoside, and far exceeds that of 5-O-methylvisaminol glycoside. Therefore, this article extracts and prepares total chromone aglycones from Saposhnikovia divaricata for quality control and activity testing, providing a basis for the research, development and utilization of Saposhnikovia divaricata medicinal materials.

[0004] Literature reports that the extraction methods of chromone components of Saposhnikovia divaricata mainly include ultrasonic assisted extraction, reflux extraction, microwave assisted extraction, enzyme assisted extraction, rapid solvent extraction, flash extraction and cold soaking. Among them, the most common extraction methods are ultrasonic method and reflux method, the solvent is ethanol, and the extraction rate is relatively high. The chromone components of Saposhnikovia divaricata often use orthogonal experimental method or response surface analysis method to optimize the extraction process and determine the optimal extraction conditions. At present, there are many studies on chromone components of Saposhnikovia divaricata, and few studies on chromone aglycones of Saposhnikovia divaricata. There is no relevant report on the optimization of the extraction process of total chromone aglycones of Saposhnikovia divaricata using response surface analysis method and the activity. The present invention adopts high performance liquid phase method to determine, and takes the total content of cimicifuga and 5-O-methylvisaminol in the extract as an index to optimize the extraction process of total chromone aglycones of Saposhnikovia divaricata. And by macroporous resin chromatography, the total chromone aglycones of Saposhnikovia divaricata are separated and the activity is studied. Summary of the invention

[0005] The invention aims to provide a method for optimizing the extraction of total chromone aglycones by using a response surface methodology and its application.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A method for optimizing the extraction of total chromone aglycones from Radix Saposhnikoviae by response surface methodology comprises the following steps:

[0008] (1) Dry and crush the radix fangfeng to obtain radix fangfeng powder, and pass it through a 60-mesh sieve.

[0009] (2) Take the fangfeng powder, add a certain amount of water and enzyme to hydrolyze it at a certain temperature for a certain time, extract it with ethanol ultrasonic for 1 hour, and determine the contents of cimicifuga and 5-O-methylvisaminol in the extract.

[0010] (3) Selecting the three factors of enzyme addition amount, reaction temperature and reaction time to conduct a single factor experiment on step (2) to determine the optimal levels of enzyme addition amount, reaction temperature and reaction time. Based on the single factor optimal point and actual conditions such as yield and cost, two level values ​​were selected as the levels of the response surface. A three-factor response surface experimental design was performed according to the central point design principle. The enzyme addition amount, reaction temperature and reaction time were used as independent variables, and the total content of cimicifuga and 5-O-methylvisaminol was used as the response value. A multivariate quadratic equation for the total chromone aglycone content of Saposhnikovia divaricata was established.

[0011] The multivariate quadratic equation established in step (3) was subjected to response surface analysis, and the total content of cimicifuga and 5-O-methylvisaminol was taken as the maximum value to obtain the optimal extraction conditions.

[0012] 2. The method according to claim 1, characterized in that: in step (3), with the three factors of enzyme addition amount X1, reaction temperature X2 and reaction time X3 as independent variables, and the total content Y of cimicifuga and 5-O-methylvisaminol as the response value, the multivariate quadratic equation established is:

[0013] Y=0.4370+0.0746X1+0.2013X2+0.2344X3-0.0442X1X2-0.0320X1X3-0.1571X2X3-0.0418X1 2 -0.0807X2 2 -0.0665X3 2

[0014] 3. The method according to claim 2, characterized in that during the extraction in step (3), the amount of enzyme added is 50% to 100%, the reaction time is 30 min to 90 min, and the reaction temperature is 40 to 60°C.

[0015] 4. The method according to claim 3, characterized in that the amount of enzyme added in step (3) is 90%, the time is 60 minutes, and the temperature is 56°C.

[0016] 5. The purpose of the present invention is to provide that the total chromone aglycones of Saposhnikovia divaricata obtained by the extraction process have the effect of inhibiting the secretion of NO by RAW 264.7 cells under the condition of lipopolysaccharide (LPS) induction. Description of the drawings:

[0018] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0019] Figure 1 The curve of the effect of enzyme addition on the content of total chromone aglycones in Saposhnikovia divaricata

[0020] Figure 2 The curve of the effect of reaction temperature on the content of total chromone aglycones in Saposhnikovia divaricata

[0021] Figure 3 The curve of the effect of reaction time on the content of total chromone aglycones in Saposhnikovia divaricata

[0022] Figure 4 Response surface diagram and contour diagram of enzyme dosage, reaction temperature and reaction time on total chromone aglycone content

[0023] Figure 5 Effects of total chromone aglycones from Saposhnikovia divaricata on the viability of RAW 264.7 cells

[0024] Figure 6 Effects of total chromone aglycones from Saposhnikovia divaricata on NO secretion in RAW 264.7 cells under normal conditions and LPS-induced conditions DETAILED DESCRIPTION

[0025] Example 1

[0026] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0027] (1) Dry and crush the radix fangfeng to obtain radix fangfeng powder, and pass it through a 60-mesh sieve.

[0028] (2) A certain amount of water and enzyme were added to the radix fangfengii powder for hydrolysis at a certain temperature for a certain time, and then the powder was extracted with ethanol by ultrasonic for 1 h. The contents of cimicifuga and 5-O-methylvisaminol were detected by high performance liquid chromatography.

[0029] (3) Selecting the three factors of enzyme addition amount, reaction temperature and reaction time to conduct a single factor experiment on step (2) to determine the optimal levels of enzyme addition amount, reaction temperature and reaction time. Based on the single factor optimal point and actual conditions such as yield and cost, two level values ​​were selected as the levels of the response surface. A three-factor response surface experimental design was performed according to the star point design principle. The enzyme addition amount, reaction temperature and reaction time were used as independent variables, and the total content of cimicifuga and 5-O-methylvisaminol was used as the response value. A multivariate quadratic equation for the total chromone aglycone content of Saposhnikovia divaricata was established.

[0030] (4) Performing response surface analysis on the multivariate quadratic equation established in step (3), taking the total chromone aglycone content as the maximum value, and obtaining the optimal extraction conditions.

[0031] Determination method of chromone content:

[0032] 1. Chromatographic conditions

[0033] The contents of four chromone components in Saposhnikovia divaricata were determined by HPLC. Chromatographic column: Agilent C 18 250×4.6mm, mobile phase: methanol-water, gradient elution program: 0-5min, 40%-45% methanol; 5-10min, 45%-60% methanol; 10-15min, 60%-80% methanol; 15-20min, 80%-95% methanol; 20-30min, 95%-40% methanol; flow rate 1mL·min -1 , detection wavelength 254nm, column temperature 30℃, injection volume 10μL. Under the above chromatographic conditions, the mixed reference and test samples of cimicifuga glycosides, cimicifuga, 5-O-methylvisaminol glycosides, 5-O-methylvisaminol were well separated, and the theoretical plate numbers calculated based on the chromatographic peaks of each component were ≥5000.

[0034] 2. Draw a standard curve

[0035] Accurately weigh 2 mg of cimicifuga glycosides, cimicifuga, 5-O-methylvisaminol glycosides, and 5-O-methylvisaminol reference substances respectively, dissolve them in methanol and make up to volume to prepare a reference stock solution with a mass concentration of 2 mg / mL. Take an appropriate amount of the reference, dilute it step by step with methanol to make up to volume, and prepare a mixed reference solution of concentrations A to G.

[0036] With cimicifuga glycoside, cimicifuga, 5-O-methylvisaminol glycoside, and 5-O-methylvisaminol as standard substances, according to the linear regression equation of peak area and concentration: cimicifuga glycoside y1 = 10453593x1 + 2214, cimicifuga y2 = 17378553x2 + 4810, 5-O-methylvisaminol glycoside y3 = 10922106x3 + 5540, 5-O-methylvisaminol y4 = 20925730x4 + 5931

[0037] R 2 The concentrations were calculated as 0.9995, 0.9995, 0.9995 and 0.9994 respectively. Chromone content = mass of chromone / mass of fangfeng powder.

[0038] Single factor experiment results

[0039] Effect of enzyme dosage on the extraction rate of total chromone aglycones from Saposhnikovia divaricata

[0040] According to the conditions of (2), while keeping other conditions unchanged, the enzymatic reaction time was 60 min, the reaction temperature was 50 °C, the amount of enzyme added was changed (mass fraction: 5, 10, 20, 50, 80, 100%), and the total extraction rate of cimicifuga and 5-O-methylvisaminol was measured. The results are as follows: Figure 1 As shown, the enzyme addition reached a maximum at 80%.

[0041] Effect of reaction temperature on the extraction rate of total chromone aglycones from Saposhnikovia divaricata

[0042] According to the conditions of (2), while keeping other conditions unchanged, the enzyme addition amount was 80%, the enzymatic reaction time was 60 min, the reaction temperature was changed (20, 30, 40, 50, 60, 70 ° C), and the total extraction rate of cimicifuga and 5-O-methylvisaminol was measured. The results are as follows: Figure 2 As shown, the reaction temperature reaches a maximum at 50°C.

[0043] Effect of reaction time on the extraction rate of total chromone aglycones from Saposhnikovia divaricata

[0044] According to the conditions of (2), while keeping other conditions unchanged, the enzyme addition amount was 100%, the constant temperature air bath shaking reaction temperature was 50°C, and the enzymatic hydrolysis reaction time was changed (10, 20, 30, 60, 90, 120 min). The total extraction rate of cimicifuga and 5-O-methylvisaminol was measured. The results are as follows: Figure 3 As shown, the reaction time reaches a maximum at 1 h.

[0045] Response surface methodology optimization design

[0046] The optimal yield (Y) of the two chromone aglycones extraction was optimized by optimizing the enzyme concentration (X1), reaction time (X2) and reaction temperature (X3) by CCD (Design Expert software, Version 12.0). Based on the single factor experiment, enzyme concentration (X1: 50% to 100%), reaction time (X2: 30 to 90 min) and reaction temperature (X3: 40 to 60 ° C) were selected as the optimized independent variables in this design. The coding levels and actual levels of the three variables are shown in Table 1.

[0047] Table 1 Factor level design of response surface experiment

[0048] The Design-Expert software was used to conduct a three-factor three-level experimental design according to the central point design principle. The experimental design and response value results are shown in Table 2, and the equation variance analysis is shown in Table 3.

[0049] Table 2 Response surface design scheme and response values

[0050] With the amount of enzyme added X1, reaction temperature X2, and reaction time X3 as independent variables, and the total content of cimicifuga and 5-O-methylvisaminol as the response value Y, a multivariate quadratic equation was established:

[0051] Y=0.4370+0.0746X1+0.2013X2+0.2344X3-0.0442X1X2-0.0320X1X3-0.1571X2X3-0.0418X1 2 -0.0807X2 2 -0.0665X3 2

[0052] Table 3 Variance analysis of the regression equation of total chromone aglycone content

[0053] The response surface analysis of the multivariate quadratic equation was performed using the Design-Expert software, and the response surface and contour lines of the regression equation were obtained. Figure 4

[0054] Further analysis of the variance results shown in Table 3 shows that there is a complex interactive relationship between the various factors in the experimental process and the response target value. 2 、X3 2 The first-order factor X1 was significant, X2 and X3 were extremely significant, and the interaction term X2X3 had a significant effect, while the other terms were not significant. By comparing the P values, it was found that the order of influence of the single factor on the model was reaction temperature > reaction time > enzyme addition. According to the determination coefficient R 2=0.9658, it can be seen that the reliability of the model is good and can be used to explain 96.58% of the data. The prediction of the model is reliable and repeatable within the range allowed by the parameters. The results of variance analysis show that the precision of the model 20.490>4, which means that the model formula can be predicted after any combination of enzyme addition, reaction time, and reaction temperature. From the experimental data, the model is suitable for the analysis and prediction of the extraction results of total chromone aglycones of Saposhnikovia divaricata.

[0055] Optimal condition verification experiment

[0056] Through a deeper analysis of the fitted linear equation, the optimal extraction parameters were finally given through software analysis, namely, 89.87% enzyme addition, 55.95℃ reaction temperature, and 59.97min reaction time. Under the optimal extraction conditions, the total extraction rate of cimicifuga and 5-O-methylvisaminol should theoretically be 0.458%.

[0057] In order to verify the accuracy of the model prediction, the optimal extraction parameters were modified: 90% enzyme addition, 56°C reaction temperature, 60 min reaction time, and the experiment was repeated three times using the optimized best extraction conditions. The average total extraction rate of cimicifuga and 5-O-methylvisaminol was 0.449%. It can be seen that the experimental results are basically the same as the predicted values ​​provided by the software, indicating that the model used in the experiment is applicable to the extraction of total chromone aglycones of Saposhnikovia divaricata, and provides a theoretical basis for the future development of Saposhnikovia divaricata.

[0058] Example 2

[0059] 1. Preparation of total chromone aglycones from Saposhnikovia divaricata

[0060] After the Fangfeng medicinal material is dried and crushed, it is passed through a 60-mesh sieve. Take 5g of the medicinal material powder, add 300mL of distilled water and 90% of the enzyme according to the optimal extraction parameters, react at 56°C for 60min, continue to add 300mL of anhydrous ethanol, let it stand overnight, ultrasonically extract for 1h, filter, and obtain the filtrate. Use a rotary evaporator to reduce pressure and concentrate until there is no alcohol taste. After concentration, add 95% ethanol solution to adjust the alcohol concentration to 80%, let it stand for 12h, collect the precipitate and repeat the alcohol precipitation twice, and reduce the pressure and concentrate the supernatant until there is no alcohol taste, add water to make up to 0.5g crude drug / mL as the loading solution.

[0061] 2. Macroporous resin separation of fangfeng extract

[0062] Take the pretreated macroporous resin D101 and wet-load it into a glass chromatographic column. Use distilled water to balance the column until the resin is evenly arranged, there are no bubbles, and the eluent has no alcohol taste. Pipette the above solution onto the column at a flow rate of 1.5mL / min, and let it stand for 3h after dynamic adsorption. First elute with distilled water, and collect the eluent according to the column bed volume (BV) for detection. When the Molish color reaction and ninhydrin color reaction in the eluent are negative, record the volume of the water-washed column. Continue to elute with 50% ethanol, and collect an eluent for every 1BV. The mixed reference solution of Saposhnikovia divaricata (Cimicifuga glycosides, Cimicifuga glycosides, 5-O-methylvisamin alcohol glycosides, 5-O-methylvisamin alcohol), Saposhnikovia divaricata extract (total extract), column liquid, and each column volume of alcohol eluent are respectively applied to the same GF 254 On the thin layer chromatography plate, chloroform: methanol (4:1) lower layer solution was used as the developing solvent, and the color was developed under ultraviolet light. The volume of alcohol eluent was recorded when the alcohol eluent of each column volume did not develop color on the silica gel plate. The 50% ethanol eluent was collected and recovered by a rotary evaporator under reduced pressure until the solvent had no alcohol taste, and then freeze-dried to obtain the total chromone aglycone powder of Saposhnikovia divaricata.

[0063] Example 3

[0064] Effects of total chromone aglycones from Saposhnikovia divaricata on RAW 264.7 cell viability under optimal extraction parameters

[0065] 1. Preparation of drug solution

[0066] The total chromone aglycone extracts of Saposhnikovia divaricata extracted under the optimal conditions and before optimization (sample 1 and sample 2) were dissolved in DMSO respectively, and diluted into 6.25, 12.5, 25, 50, and 100 mg / L solutions by adding complete culture medium, and the final concentration of DMSO did not exceed 0.1%.

[0067] 2. Test methods

[0068] (1) Mouse macrophage RAW 264.7 cells in the logarithmic growth phase were inoculated into a 96-well plate, with 1.5 × 10 4 The cells were plated in 6 replicate wells and cultured in a CO2 incubator at 37°C for 24 h.

[0069] (2) The old culture medium was discarded and replaced with complete culture medium containing different concentrations of total chromone aglycones of Saposhnikovia divaricata. The culture was continued for 24 h, and the cell viability was determined by CCK-8 method. The experiment was repeated three times.

[0070] like Figure 5 As shown in Figure 2, total chromone aglycones from Saposhnikovia divaricata had no killing effect on RAW 264.7 cells.

[0071] Example 4

[0072] Effects of total chromone aglycones from Saposhnikovia divaricata on NO secretion in RAW 264.7 cells under normal conditions and LPS-induced conditions before and after process optimization

[0073] Total chromone aglycones were used to act on lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophages, and the NO level in the culture supernatant was detected by Griess reagent colorimetric method. The ability of the test drug to inhibit NO release was used as an indicator to evaluate the activity of the compound.

[0074] 1. Preparation of drug solution

[0075] The total chromone aglycones were dissolved in DMSO to prepare 5, 25, 50, and 100 mg / L solutions, and the final concentration of DMSO did not exceed 0.1%.

[0076] 2. Test methods

[0077] (1) Mouse macrophage RAW 264.7 cells in the logarithmic growth phase were inoculated into a 96-well plate, with 1.5 × 10 4 The cells were plated in triplicate wells and cultured in a CO2 incubator at 37°C for 24 h.

[0078] (2) Discard the old culture medium and add different concentrations of the test drug to each well, along with 1 μg of LPS.

[0079] (3) After culturing for 48 hours, the supernatant was collected by centrifugation and grouped. The cells were tested by the Griess method and the amount of NO produced in different groups was calculated according to the standard curve prepared in the instruction manual.

[0080] 3. Experimental results

[0081] Effect of total chromone aglycones of Saposhnikovia divaricata on NO secretion induced by LPS in RAW 264.7 mouse macrophages Figure 6 shown.

[0082] The results showed that total aglycones above 25 mg / L had a significant inhibitory effect on LPS-induced NO production in RAW 264.7 mouse macrophages, and the inhibitory effect of the optimized concentration of 25 mg / L was equivalent to that of 100 mg / L before optimization, indicating that the compounds of the present invention have more obvious anti-inflammatory activity than before optimization, and therefore can be used to prepare new anti-inflammatory active drugs.

[0083] Using Saposhnikovia divaricata as raw material, enzyme-assisted ethanol extraction of total chromone aglycones from Saposhnikovia divaricata was used, and the extraction process was optimized using response surface methodology. It was found that total chromone aglycones from Saposhnikovia divaricata were non-toxic to RAW 264.7 cells and had good anti-inflammatory activity, providing technical support for the further development and utilization of Saposhnikovia divaricata.

[0084] The preferred embodiments of the present invention are described. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

Claims

1. A method for optimizing the extraction of total chromone aglycones from Radix Saposhnikoviae by response surface methodology, comprising the following steps: (1) drying and crushing the radix fangfeng to obtain radix fangfeng powder, and passing the powder through a 60-mesh sieve; (2) Take the fangfeng powder, add a certain amount of water and enzyme to hydrolyze at a certain temperature for a certain time, extract with ethanol by ultrasonic for 1 h, and determine the contents of cimicifuga and 5-O-methylvisaminol in the extract; (3) Selecting the three factors of enzyme addition amount, reaction temperature and reaction time to conduct a single factor experiment on step (2) to determine the optimal levels of enzyme addition amount, reaction temperature and reaction time; taking the single factor condition as the independent variable and the content Y of cimicifuga and 5-O-methylvisaminol as the response value, a multivariate quadratic equation is established: Y=4.3970+0.0746X1+0.2013X2+0.2344X3-0.0442X1X2-0.0320X1X3-0.1571X2X3-0.0418X1 2 -0.0807X2 2 -0.0665X3 2 In step (3), the amount of enzyme added was 90%, the reaction temperature was 56° C., and the reaction time was 60 min.

2. According to the method of claim 1, the extract is preliminarily judged to contain compounds such as cimicifuga and 5-O-methylvisaminol by high performance liquid chromatography.

3. Use of the chlorogenic acid aglycone extract of Saposhnikovia divaricata prepared by the method according to claim 1 in anti-inflammatory aspects.

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