Optimization of the extraction process of three active ingredients from Dictamni cortex by a hierarchical analysis combined with response surface methodology

Abstract

The invention discloses a hierarchical analysis combined with response surface method to optimize the extraction process of three effective components of Dictamni cortex, adopts hot reflux method to extract Dictamni cortex with alcohol, performs hierarchical analysis on the basis of single factor method, determines weight coefficient, and optimizes extraction conditions by combining multi-index comprehensive scoring with response surface method. Result The optimal extraction process conditions are extraction temperature 80±2°C, solid-liquid ratio 1:15±0.06 (g / mL), extraction time 1.5±0.05h, ethanol concentration 70±0.03%, extraction times 1, and the extraction rate of the extract under the optimal extraction conditions is 17.18±0.86%, and the content determination results of dacricine, halodendron and chalazine in Dictamni cortex are: 0.25±0.03 mg / g, 0.83±0.05 mg / g, and 1.13±0.02 mg / g. This patent provides a theoretical basis for the future resource development and utilization of Dictamni cortex Chinese medicine and its industrial production.

Description

Technical Field

[0001] The invention relates to the field of extraction of effective components of medicinal materials, and specifically to an extraction process for optimizing three effective components of Dictamni cortex by combining hierarchical analysis with response surface methodology. Background Art

[0002] Dictamni Dictamnus dasycarpus Turcz ) is the dried root bark of Dictamnus dasycarpa of the Rutaceae family, mainly distributed in Northeast China, Shaanxi, Gansu, Sichuan, Jiangsu, Hebei, Guizhou and other places in my country. It is a traditional Chinese herbal medicine in my country, bitter in taste, cold in nature, and has the effects of dispelling wind and detoxification, clearing heat and drying dampness. Modern clinical studies have found that Dictamnus dasycarpa has anti-inflammatory, antibacterial, anticancer, and insecticidal biological activities, among which diosmin, kaempferol, and phlegmone are the main active ingredients of Dictamnus dasycarpa. Kaempferol is a new type of liver-protecting drug, and also has anti-inflammatory and antibacterial effects; phlegmone is a high-content limonoid compound that can produce antifeedant phenomena on insect larvae, and also has the effect of increasing the activity of anti-tumor drugs that inhibit microtubules; diosmin also has multiple biological activities, it can be anti-inflammatory, antibacterial, antifungal, vasodilation, and has a dilation effect on tracheal smooth muscle.

[0003] Zhang Hongjing reported [Zhang Hongjing. Study on the extraction, separation and activity evaluation of chemical components in Dictamni Cortex [D]. Jilin Agricultural University, 2011.] The best process for extracting distilled water from Dictamni Cortex was to add 15:1 (mL / g) 80% ethanol to the crude powder of the medicinal material, extract for 3 times, each time for 1 hour, and the distilled water content was 0.1789 mg / g. Liang Hong et al. [Liang Hong, Wei Baohan, Huang Guangwei, et al. Study on the extraction process of Dictamni Cortex by orthogonal test method [J]. Guangxi Light Industry, 2009, 25(05):16-17] The best extraction conditions using the percolation method were to break the medicinal material into 1-2 mm, add 7 times 60% ethanol, and the percolation rate was 4-5 mL / min, and the distilled water content was 0.1722 (mg / g). Qu Wenhua reported [Qu Wenhua, Zhang Li, Li Qingyuan. Optimization of extraction process of tarrone from Dictamni Cortex by orthogonal test method [J]. Health Reading, 2011, 000(004): 357-357.] Taking the tarrone content as an indicator, the optimal extraction process was obtained, which was to use 8 times 95% ethanol for 3 extractions, each for 90 minutes. The total amount of tarrone was 0.72625 mg / g.

[0004] Through the above review, it is found that in the current extraction process of Dictamni cortex, the content of dactylin, kaempferol or berberine is often used as the evaluation index for orthogonal extraction experiments, and no related research has been reported on the weighted score content of the three active ingredients as the evaluation response value. This patent uses hierarchical analysis plus response surface to evaluate the extraction process of the three active ingredients, providing a theoretical basis for the future resource development and utilization of Dictamni cortex Chinese medicine and its industrial production. Summary of the invention

[0005] At present, in the field of Dictamni cortex extraction technology, the process time is long, the ethanol concentration is high, the number of extractions is large, and no relevant research has been reported on the experiment with the weighted score content of the three active ingredients as the investigation response value. Based on the above problems, this patent uses a hot reflux method to extract the active ingredients in Dictamni cortex, takes the content of the three active ingredients in Dictamni cortex as the investigation index, and optimizes the Dictamni cortex extraction process by combining the analytic hierarchy process with the response surface method. A method as claimed in claim 1 that uses a hierarchical analysis method combined with the response surface method to optimize the extraction process of the three active ingredients in Dictamni cortex, characterized in that it is carried out according to the following steps:

[0006] 1. Single Factor Experiment

[0007] The raw medicinal materials of Dictamni were crushed, and the influencing factors were solid-liquid ratio, extraction time, and ethanol concentration for single factor preliminary screening. Five levels were selected under each factor, and other conditions were fixed for experiments. Each group was paralleled three times to examine the influence of different factors on the evaluation indexes of the content of three active ingredients. Finally, the single factor optimized extraction conditions were obtained as follows: extraction temperature 80±2°C, solid-liquid ratio 1:15±0.06 (g / mL), extraction time 1.5±0.05h, ethanol concentration 70±0.03%, and extraction times 1.

[0008] Hierarchical Analysis

[0009] Dictamnin, ashtone and phragmites are the three main components of Dictamnin cortex. The multi-index hierarchical analysis method was used to optimize the Dictamnin cortex cutting process, among which the random consistency ratio was CR=0.0462, and CR<0.1. The comprehensive score Y=(phragmites content / maximum phragmites content×0.66+ashtone content / maximum ashtone content×0.28+dictamnin content / maximum dictamnin content×0.06)×100% was determined by the weight coefficient.

[0010] Response Surface

[0011] This patent is based on the experimental results of a single factor, and uses the solid-liquid ratio, ethanol concentration and extraction time as independent variables to design a response surface experiment, and combines the weight coefficients obtained by hierarchical analysis to comprehensively score the experimental results. The optimal extraction process conditions are extraction temperature 80±2°C, solid-liquid ratio 1:15±0.06 (g / mL), extraction time 1.5±0.05h, ethanol concentration 70±0.03%, and extraction times 1. The results of the determination of damipine, kaempferol and phlorothalone are: 0.25±0.03 mg / g, 0.83±0.05 mg / g, and 1.13±0.02 mg / g.

[0012] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0013] (1) The extraction process was optimized by single factor combined with response surface and hierarchical analysis. The comprehensive score of hierarchical analysis was Y = (flavonoids content / maximum flavonoids content × 0.66 + ash content / maximum ash content × 0.28 + dactylin content / maximum dactylin content × 0.06) × 100%.

[0014] (2) The optimal extraction process conditions were as follows: extraction temperature 80±2°C, solid-liquid ratio 1:15±0.06 (g / mL), extraction time 1.5±0.05h, ethanol concentration 70±0.03%, and extraction times 1.

[0015] (3) This patent adopts single factor, hierarchical analysis plus response surface multiple verification of the extraction process of three active ingredients. Compared with the research in the preface literature, this patent has a shorter process time, a lower ethanol concentration, and a shorter extraction number, which provides a theoretical basis for the future resource development and utilization of Dictamni Cortex as a traditional Chinese medicine, and its industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the specific implementation of the present invention, the drawings required for use in the specific implementation or the description of the prior art will be briefly introduced below.

[0017] Figure 1 , 2 3 is the standard curve of dioscorea alkaloids, eugenol and chloroquine; Figure 4 is the effect of the solid-liquid ratio on the total content of the three active ingredients; Figure 5 is the effect of extraction time on the total content of the three active ingredients; Figure 6 is the effect of ethanol concentration on the total content of the three active ingredients; Figure 7 is the effect of extraction times on the total content of the three active ingredients, Figure 8 It is a response surface software that draws a three-dimensional graph of the interaction of three influencing factors. DETAILED DESCRIPTION

[0018] The present invention is described in detail below in conjunction with diagrams and specific implementation operations. The purpose of providing these implementation modes or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0019] Implementation Plan 1 Extraction and Content Determination of Dictyophore

[0020] Implementation Plan 1: Drawing of Standard Curve

[0021] Take 1 mg of distilled water, ash, and flavonoids standard products, and prepare 0.004, 0.006, 0.008, 0.01, and 0.012 mg / mL distilled water standard solutions, 0.016, 0.020, 0.024, 0.028, and 0.032 mg / mL ash standard solutions, and 0.03, 0.04, 0.05, 0.06, and 0.07 mg / mL flavonoids standard solutions, respectively. Use UV to measure the absorbance A of distilled water, ash, and flavonoids standard solutions of different concentrations at wavelengths of 236 nm, 218 nm, and 212 nm, respectively. Draw the standard curves of the three standards with concentration as the horizontal axis and absorbance as the vertical axis. Measure each group three times, calculate the average value, and calculate its content according to the formula.

[0022] Formula (1-1)

[0023] Formula (1-2)

[0024] Formula (1-3)

[0025] Formula (1-4)

[0026] Where: C: concentration of active ingredient (mg / mL); V: volume (mL); m0: mass of Dictamni cortex medicinal material (g); m: mass of Dictamni cortex after extraction (g).

[0027] The standard curve is as follows Figure 1-3 As shown, the standard curve equation of damipine is y = 1.92x + 3.2589 (4-12 μg / mL), R 2 = 0.9992, standard curve equation of octyl ketone y=0.57x+2.6477 (16-32μg / mL), R 2 =0.9995, standard curve equation of pyruvate ketone y=0.406x+2.6446 (30-70μg / mL), R 2 = 0.9991, and the three curves show good linear relationship in their respective ranges.

[0028] Implementation Plan 2: Single Factor Experiment on Dictamni

[0029] Four factors were investigated: solid-liquid ratio, extraction time, ethanol concentration, and extraction times. Five levels were selected under each factor, and then the experiment was carried out with the other three factors fixed to compare the contents of the three active ingredients. The contents of the three active ingredients were used as reference factors to screen out the optimal level of the single factor, and the experiment was repeated three times.

[0030] (1) Effect of material-liquid ratio on the content of three active ingredients

[0031] Accurately weigh 3 g of Dictamni cortex, fix the extraction temperature at 80°C, the extraction time at 1.5 h, the ethanol concentration at 80%, the extraction number at 1 time, the solid-liquid ratio at 1:5 g / mL, 1:10 g / mL, 1:15 g / mL, 1:20 g / mL, 1:25 g / mL, and each group was run in parallel for three groups to investigate the effects of different solid-liquid ratios on the contents of the three active ingredients.

[0032] Depend on Figure 4 It can be seen that a turning point occurs when the solid-liquid ratio is 1:15 g / mL, and the contents of daunorubicin, quercetin and berberine are the highest. After that, as the solid-liquid ratio continues to increase, the extraction amounts of the three index components tend to be flat, indicating that the content no longer increases, so 1:15 g / mL was selected for the next experiment.

[0033] (2) Effect of extraction time on the contents of three active ingredients

[0034] 3 g of Dictamni cortex was accurately weighed, the extraction temperature was fixed at 80°C, the solid-liquid ratio was 1:15 g / mL, the ethanol concentration was 80%, the number of extractions was 1, and the extraction times were 0.5 h, 1 h, 1.5 h, 2 h and 2.5 h, respectively. The effects of different extraction times on the contents of the three active ingredients were investigated.

[0035] The effects of extraction time on the content of index components in Dictamni are as follows Figure 5 As shown. Figure 5 It can be seen that as the extraction time increases, the contents of dactyline, quercetin and berberine gradually increase, but a turning point appears at 1.5h, when the three active ingredients all reach their maximum values, and then the contents decrease. It can be seen that the optimal condition is an extraction time of 1.5h.

[0036] (3) Effect of ethanol concentration on the contents of three active ingredients

[0037] 3 g of Dictamni cortex was accurately weighed, the extraction temperature was fixed at 80°C, the solid-liquid ratio was 1:15 g / mL, the extraction time was 1.5 h, the extraction number was 1, and the ethanol concentrations were 50%, 60%, 70%, 80%, and 90%, respectively. The effects of different extraction temperatures on the contents of the three active ingredients were investigated.

[0038] Depend on Figure 6It can be seen that using the above-mentioned optimal liquid-to-solid ratio and extraction time, extracting once and changing the ethanol concentration, the change in the content of berberine is the most obvious. Among them, the curves of the first two points rise relatively slowly. When the ethanol is 70%, the content of berberine is significantly higher than the other four points. Although the content of tarterone is higher than the other four points, the difference between its content and the first two points is small, followed by distilled water. It can be concluded that although the content of tarterone and distilled water is slightly different, under 70% ethanol extraction, the three active ingredients are higher than the other four points. Therefore, the optimal condition is an ethanol concentration of 70%.

[0039] (4) Effect of extraction times on the contents of three active ingredients

[0040] 3 g of Dictamni cortex was accurately weighed, the extraction temperature was fixed at 80°C, the solid-liquid ratio was 1:15 g / mL, the ethanol concentration was 80%, the extraction time was 1.5 h, and the number of extractions was 1, 2, 3, 4, and 5, respectively, to investigate the effects of the number of extractions on the contents of the three active ingredients.

[0041] Depend on Figure 7 It can be seen that when the other single factors are fixed and the number of extractions is subjected to 5 different levels, the content of the three active ingredients is the highest when the number of extractions is 1. As the number of extractions increases, the contents of dactyline, quercetin and berberine decrease. Among them, the content difference in the last four extractions is relatively small. This shows that it is more reasonable to extract once.

[0042] Depend on Figure 4-7 It can be seen that the optimal single factor conditions for Dictamni extraction are: extraction temperature 80±2°C, solid-liquid ratio 1:15±0.06 g / mL, extraction time 1.5±0.05h, ethanol concentration 70±0.03%, and extraction times 1.

[0043] Implementation Plan 3 Establishment of the comprehensive weights of the three active ingredients in Dictamni Cortex by chromatography analysis

[0044] The analytic hierarchy process was used to establish comprehensive weights for the three components of dactylin, kaempferol, and phlorothenin in Dactylin Cortex. Based on the above single-factor experimental results and the extraction amounts of the three indicators, the priority order of the three active ingredients was preliminarily determined to be phlorothenin>kaempferol>dactylin, and a priority judgment matrix for target pairwise comparison was constructed to assign relative scores to each indicator. The results are shown in Table 1-2.

[0045] (1) Scoring criteria for each indicator component

[0046] Table 1 Scoring criteria for each level of evaluation indicators

[0047]

[0048] Table 2 Priority judgment matrix for comparison of target component pairs

[0049] Evaluation indicators Houttuynia cordata Catalone Dioscorea alba Houttuynia cordata 1 3 9 Catalone 1 / 3 1 6 Dioscorea alba 1 / 9 1 / 6 1

[0050] According to the scoring results in Table 1-2, the weight coefficients of the three active ingredients, berberine, ash ketone and distilled alcohol, calculated by the hierarchical analysis method are 0.66, 0.28 and 0.06 respectively, and the consistency ratio factor CR=0.0462<0.10, indicating that the priority judgment matrix of the target pairwise comparison is consistent, and the required weight coefficients are valid. The comprehensive score Y=(berberine content / maximum berberine content×0.66+ash ketone content / maximum ash ketone content×0.28+distilled alcohol content / maximum distilled alcohol content×0.06)×100 is determined.

[0051] Implementation Plan 4: Response Surface Optimization of Dictamni

[0052] On the basis of the single factor experiment, the most significant factors were the solid-liquid ratio, extraction time and ethanol concentration, and 3 levels were taken for response surface test to optimize the extraction process of the three active ingredients of Dictamni. The response surface factors and results are shown in Table 3-4:

[0053] Table 3. Response surface experiment factor level table

[0054]

[0055] Table 4 Response surface experimental scheme and results of Dictamni extraction

[0056]

[0057] The statistical analysis software Design-Expert V was used to calculate and analyze the extraction process of Dictamni Cortex. According to the multivariate regression fitting, the solid-liquid ratio (A), ethanol concentration (B), and extraction time (C) were taken as the main influencing variables. The quadratic regression model equation of the Dictamni Cortex extraction process response surface was obtained through analysis:

[0058] Y=+1.00+0.21A-0.042B+0.062C-0.058AB-0.014AC+0.053BC-0.14A 2 -0.21B 2 -0.070C 2 .

[0059] Table 5 Results of variance analysis

[0060]

[0061] Note: *Significant difference ( P <0.05); **The difference is highly significant ( P <0.01);***The difference is extremely significant ( P <0.001)

[0062] According to the software calculation in Table 5, the F value in the experimental results is 9.65. P The value is less than 0.01, indicating that the experimental effect is extremely different. Through software analysis, it can be seen that the extraction model shows a significant linear relationship and the relative difference is very obvious. The coefficient of determination R of the model 2 =0.9254, lack of fit =0.2786>0.05, which is not significant. The experimental data show that the model is reliable and has high accuracy. Among the three indicators, A (liquid-to-solid ratio) is an extremely significant term (P<0.001), B (ethanol concentration) and C (extraction time) are not significant terms; A 2 The results of variance analysis showed that the precision of the model was 8.830>4, which means that the model formula can predict any combination of the three influencing factors, and it is reliable and reasonable. Therefore, the model is suitable for the analysis and prediction of Dictamni extraction.

[0063] According to the response surface analysis results, each data response value is composed of the interaction between different extraction influencing factors in the experiment. Figure 8 As shown in the figure, when the contour line diagram of the 2D figure is closer to an ellipse, it indicates that the extraction condition has a greater influence on the extracting conditions of dioscorea alkaloids, truncatone and phlorothalone in Dictamni cortex, while the 3D figure is more curved, indicating that the factor has a greater influence on the extraction of the three active ingredients in Dictamni cortex, and vice versa. It can be seen from the experimental response surface that the optimal extraction conditions are extraction temperature 80±2°C, solid-liquid ratio 1:15±0.06 (g / mL), extraction time 1.5±0.05h, ethanol concentration 70±0.03%, and extraction times 1. The total extraction rate of the extract is 17.18±0.86%. The results of dioscorea alkaloids, truncatone and phlorothalone are: 0.25±0.03 mg / g, 0.83±0.05 mg / g, and 1.13±0.02 mg / g.

[0064] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and the inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A hierarchical analysis combined with response surface methodology to optimize the extraction process of three active ingredients from Dictamni cortex. Features Taking extraction temperature, solid-liquid ratio, extraction time and number of extractions as investigation factors, the comprehensive weight of the content of active ingredients divacantine, quercetin and phlorothalone in Dictamni Cortex was used as the assessment index. The extraction process was optimized based on single factor combined with chromatography analysis and response surface methodology, and the optimal conditions were extraction temperature 80±2℃, solid-liquid ratio 1:15±0.06 (g / mL), extraction time 1.5±0.05h, extraction number 1 and ethanol concentration 70±0.03%; the extraction rate of the extract was 17.18±0.86%, and the determination results of divacantine, quercetin and phlorothalone in Dictamni Cortex were: 0.25±0.03mg / g, 0.83±0.05mg / g, and 1.13%±0.02mg / g.

2. The hierarchical analysis combined with response surface methodology as claimed in claim 1 optimizes the extraction process of the three active ingredients of Dictamni cortex. Features The analytic hierarchy process was used to establish the comprehensive weights of the three component indicators of diffusine, ash ketone and flavonoids in Dactylic Adenophora cortex, and the comprehensive score Y = (flavonoids content / maximum flavonoids content × 0.66 + ash ketone content / maximum ash ketone content × 0.28 + diffusine content / maximum diffusine content × 0.06) × 100.

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