CN110638858A - Method for optimizing ultrasonic extraction of multi-index components in gentiana lineata by response surface method - Google Patents
Method for optimizing ultrasonic extraction of multi-index components in gentiana lineata by response surface method Download PDFInfo
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Abstract
The invention discloses a method for optimizing and extracting multi-index components in gentiana lineata by a response surface method, which comprises the following steps: A. pulverizing whole plant of Gentiana scabra Bunge; B. putting the powder into a conical flask, and adding water, absolute ethyl alcohol, methanol and 50-75% methanol according to the solid-to-liquid ratio of 20-60: 1; C. ultrasonic extracting at room temperature for 10-50min to obtain extractive solution; D. centrifuging the extractive solution at 3000r/s for 20min to obtain supernatant containing multiple index components. The optimal extraction solvent for extracting the multi-index components in the gentiana straminea maxim is methanol, the concentration of the solution is 60%, the extraction time is 40min, the volume of the solvent is 15mL, and the solid-to-liquid ratio is 30: 1. The method realizes the purpose of extracting multiple index components of the gentiana straminea at one time, has the advantages of high precision and efficiency, can be used for comprehensively evaluating the quality of the gentiana straminea, and provides a basis for comprehensive utilization and development of the gentiana straminea.
Description
Technical Field
The invention belongs to the technical field of plant component extraction, and particularly relates to a method for optimizing ultrasonic extraction of multi-index components in gentiana lineata by a response surface method.
Background
Gentiana belongs to Gentianales Gentianaceae, more than 400 Gentianaceae are available worldwide, and are widely distributed in the global world and mainly produced in the northern temperate zone and tropical mountain areas. I have 22 genera and about more than 500, and the total number of the genera is 12 and 70 for medical use. Gentian is an annual or perennial herb. The plants of the genus gentian of the family gentianaceae are the most diverse, and gentian plants can be used for various purposes such as ornamental and medical purposes, and are mostly traditional Tibetan medicines. The Tibetan medicine 'Banjian' is a general name of various medicinal plants of gentian, is a representative common large Tibetan medicinal material, has the effects of treating virus diseases, various pyretic symptoms and laryngitis fever, and has definite curative effect and wide application.
Gentiana farreri Balf. f. also known as Gentiana farreri Gentiana, "Banglan Yunbao" and Gentiana scabra, are distributed in Sichuan, Tibet, Gansu and Qinghai areas of continental China, and grow in the area with elevation of 2, 410 m to 4, 600 m, with small leaves and light blue flowers, and mostly grow in shrubs, alpine meadows and swamp beaches. The Bingzhong is derived from various medicinal plants of gentiana of Gentianaceae, is a representative common large Tibetan medicinal material, and has the effects of treating virus diseases, various pyretic symptoms and laryngitis. At present, more than 10 Tibetan medicine enterprises have 14 Tibetan medicine compound preparations with national medicine standard word sizes, namely ten-ingredient gentiana granule (capsule), three-ingredient gentiana tablet pill and fifteen-ingredient gentiana pill (powder); the gentian flower is sold in medicinal material markets of Qinghai, Sichuan, Tibet, Gansu and the like and local specialty door markets, and is one of the most common Tibetan medicinal materials in China. If the efficacy is different according to the classification of 'between charts', the 'between charts' nature is rather cool, and the situation that the basic varieties of 'between charts' are complex and are mutually substituted and used is seen in the current situation of use.
Researches on gentiana straminea show that the main components of the whole plant include secoiridoid glycosides, flavonoids, triterpenes and the like.
The response surface design is an effective statistical and optimization method, and compared with the traditional uniform design and orthogonal design, the response surface method has the advantages of high test precision, few test times, good mathematical model predictability and the like, and can reflect the relationship between each factor and a response value.
Disclosure of Invention
The invention aims to provide a method for optimizing and extracting multi-index components in gentiana lineata by a response surface method, which optimizes the extraction process of the multi-index components of gentiana lineata and provides a research basis for analysis of the multi-index components in gentiana lineata medicinal materials.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for optimizing and ultrasonically extracting multiple index components in gentiana lineata by a response surface method comprises the following steps:
A. pulverizing whole plant of Gentiana striolata into powder, and sieving with 60 mesh sieve;
B. putting the powder into a conical flask, and adding water, absolute ethyl alcohol, methanol and 50-75% methanol according to the solid-to-liquid ratio of 20-60: 1;
C. ultrasonic extracting at room temperature for 10-50min to obtain extractive solution;
D. centrifuging the extractive solution at 3000r/s for 20min to obtain supernatant containing multiple index components.
The optimal extraction solvent for extracting the multi-index components in the gentiana straminea by utilizing the response surface method is methanol, the solution concentration is 60%, the extraction time is 40min, the solvent volume is 15mL, and the solid-to-liquid ratio is 30: 1.
The index components include loganin, swertiamarin, 6' -O-beta-D-glucosyl gentiamarin, isoorientin, isovitexin, ursolic acid and oleanolic acid.
A method for optimizing and ultrasonically extracting multiple index components in gentiana straminea by using a response surface method comprises the following steps:
(1) single factor screening: designing a single-factor test according to three single factors of solvent concentration, solvent volume and extraction time;
(2) designing a response surface method: taking the ultraviolet absorption value of index components in gentiana straminea as a response value, and performing a secondary response surface regression analysis experiment on the basis of single factor screening in the step (1) by using Design-Expert software to obtain a simulation equation and a determination coefficient as follows:
the simulated equation for loganine is:
y＝4.76-0.55*A-0.069*B+0.062*C+0.0218AB-0.049*AC+6.864*BC-0.29*A2+0.13*B2+0.16*C2determining the coefficient R2＝0.997；
The simulation equation of the swertiamarin is as follows:
y＝4.82-0.3*A-0.04*B+0.051*C+4.65AB-0.031*AC+8.837*BC-0.091*A2+0.041*B2+0.077*C2determining the coefficient R2＝0.9565；
The simulation equation for gentiopicroside is:
y＝4.65+0.27*A-0.048*B+0.022*C-0.012*AB-0.022*AC+0.029*BC+0.44*A2+0.093*B2+0.16*C2determining the coefficient R2＝0.9806；
The simulation equation of the swertiamarin is as follows:
y＝4.78-0.52*A-0.038*B+0.043*C+5.793AB-0.029*AC+0.011*BC-0.34*A2+0.15*B2+0.13*C2determining the coefficient R2＝0.9961；
The simulation equation of isoorientin is as follows:
y＝7.73+0.16*A-0.056*B-0.14*C+0.16AB-0.22*AC+0.08*BC+0.11*A2-0.22*B2+0.13*C2determining the coefficient R2＝0.9245；
The simulation equation of isovitexin is:
y＝7.8+0.09*A-0.067*B-0.045*C+0.15AB-0.087*AC+0.064*BC+0.069*A2-0.14*B2+0.067*C2determining the coefficient R2＝0.9008；
The simulation equation of 6' -0-beta-D-glucosyl gentiopicroside is as follows:
y＝4.79-0.25*A-0.066*B+0.048*C-0.046AB-3.709*AC-6.38*BC-0.11*A2+0.11*B2+0.078*C2determining the coefficient R2＝0.8966；
The simulation equation for ursolic acid is:
y＝4.67+0.27*A-8.13*B+0.043*C-0.028AB-0.027*AC-9.527*BC+0.18*A2-0.12*B2-0.13*C2determining the coefficient R2＝0.9721；
The simulation equation for oleanolic acid is:
y＝4.67+0.27*A-8.13*B+0.043*C-0.028AB-0.027*AC-9.527*BC+0.18*A2-0.12*B2-0.13*C2determining the coefficient R2＝0.9721；
In the formula, y is the ultraviolet absorption value of each component, a variable parameter A is the concentration of the solvent, a variable parameter B is the extraction time, and a variable parameter C is the volume of the solvent; the calculation determined that the optimal solvent concentration is 60%, the extraction time is 40min, and the solvent volume is 15 mL.
The extraction and separation of chemical components of Chinese medicine is the basis for researching the chemical components of Chinese medicine. This process should generally be performed under the follow-up of biological activity or pharmacological indicators. The extraction and separation method should be selected according to the main physicochemical properties of the extracted components and considering the principles and characteristics of various extraction and separation techniques, so that the required components can be fully extracted and separated.
The design is carried out on the factors influencing the components of the gentiana straminea by taking several types of chemical components as main lines, so that the components are completely extracted, the cost is low, the toxicity is low, the efficiency is high, and the operation is convenient.
(1) Selection of solvent type
The solvent extraction method is to select proper solvent and method for extraction according to the solubility of the extracted components. The action principle is that the solvent penetrates the cell membrane of the medicinal powder to dissolve the solute, so that the concentration difference of the solute inside and outside the cell is formed, and the solute is seeped out of the cell membrane, thereby achieving the purpose of extraction. Therefore, according to the characteristics of 9 components in the gentiana straminea, methanol is used as a solvent, and all components can be extracted to the maximum extent.
(2) Selection of solvent concentration and selection of feed-liquid ratio
The selection of the solvent can be divided into three categories according to polarity, namely lipophilic organic solvents, hydrophilic organic solvents and water. According to the principle of similar compatibility, the required chemical components are extracted to the maximum extent, and the boiling point of the solvent is moderate, easy to recover, low in toxicity and safe. Methanol is the most commonly used solvent because it can be mixed with water at any ratio, and most lipophilic organic solvents, and has strong infiltration capability into medicinal material cells, and can dissolve most of Gentiana lineolata components according to the material-to-liquid ratio, so that the hydrophilic organic solvent methanol is selected to be 60%, and the material-to-liquid ratio is 30:1, which is the best.
(3) Selection of extraction mode
The extraction method is to select proper solvent and method, and make the material dissolve by physical action. The principle of ultrasonic extraction mainly comprises mechanical action, thermal action and cavitation, and the frequency and the speed of the molecular motion of a substance and the penetrating power of a solvent are increased by utilizing various actions such as strong cavitation effect, mechanical vibration, disturbance effect, high acceleration, emulsification, diffusion, crushing, stirring and the like generated by ultrasonic radiation, so that the target component is accelerated to enter the solvent. The ultrasonic extraction can greatly improve the extraction efficiency, save the solvent and avoid the influence of high temperature on the extraction components. According to the characteristics and the extraction efficiency of the components of the gentiana straminea, an ultrasonic extraction process is adopted, the extraction time is shortened, and the extraction temperature is reduced because the effective components can be accelerated to enter a solvent by strong vibration, high acceleration, strong cavitation effect, stirring action and the like generated by ultrasonic waves, so that the extraction rate is improved, the extraction time is shortened, the influence of high temperature on the extracted components is avoided, and the method is an advanced extraction means. Therefore, the best extraction method in the screening process is ultrasonic extraction. The power was 50kHz and the temperature was 20 ℃.
(4) Selection of extraction time
In the aspect of selection of extraction time, the influence of ultrasonic time, temperature and factors on the ultrasonic extraction is inspected by adopting a single-factor alternation test method. Finally, the optimal time is 40 min. Thus, the time is short, the energy is saved, and the extraction yield is highest.
Compared with the prior art, the invention has the beneficial effects that:
the method selects proper parameter levels through single-factor screening, inspects the solvent concentration, the solvent volume and the extraction time of the ultrasonic extraction process of the gentiana lineata by using a three-factor three-level response surface analysis method, constructs a secondary polynomial simulation equation of nine components of loganin, swertiamarin, gentiamarin, swertiamarin, isoorientin, isovitexin, 6' -O-beta-D-glucosyl gentiamarin, ursolic acid and oleanolic acid in the gentiana lineata, draws a three-dimensional curved surface diagram, and finds the optimal combination of factors and the optimal value of the response value in the whole area according to the obtained model: on the basis of 0.5g of gentiana lineata, the method is carried out according to optimized extraction conditions, and results show that the total extraction effect of 9 components in gentiana lineata is optimal under the conditions of 60% of solvent concentration, 15mL of methanol dosage (liquid-material ratio of 30:1) and 40min of extraction time, under the process conditions, the extraction effect of the chemical components of the gentiana lineata is more comprehensive and sufficient.
Drawings
FIG. 1 is a schematic diagram showing the effect of solvent concentration on extraction efficiency;
FIG. 2 is a schematic representation of the effect of solvent volume on extraction performance;
FIG. 3 is a schematic diagram showing the effect of extraction time on the extraction effect;
FIG. 4 is a graph of the response of solvent concentration and solvent volume to logenin extraction;
FIG. 5 is a graph of the response of solvent concentration and extraction time to logenin extraction;
FIG. 6 is a response surface plot of the effect of solvent volume and extraction time on logenin extraction;
FIG. 7 is a graph of response surface of solvent concentration and solvent volume to effect swertiamarin extraction;
FIG. 8 is a graph of response surface of solvent concentration and extraction time on the effect of swertiamarin extraction;
FIG. 9 is a response surface plot of the effect of solvent volume and extraction time on swertiamarin extraction;
FIG. 10 is a response surface plot of the effect of solvent concentration and solvent volume on gentiopicroside extraction;
FIG. 11 is a response surface plot of the effect of solvent concentration and extraction time on gentiopicroside extraction;
FIG. 12 is a response surface plot of the effect of solvent volume and extraction time on gentiopicroside extraction;
FIG. 13 is a response surface plot of the effect of solvent concentration and solvent volume on swertiamarin extraction;
FIG. 14 is a response surface graph of the effect of solvent concentration and extraction time on swertiamarin extraction;
FIG. 15 is a response surface plot of solvent volume and extraction time effects on swertiamarin extraction;
fig. 16 is a response surface diagram of the influence of solvent concentration and solvent volume on isoorientin extraction;
FIG. 17 is a response surface diagram of the effect of solvent concentration and extraction time on isoorientin extraction;
FIG. 18 is a response surface diagram of the effect of solvent volume and extraction time on isoorientin extraction;
FIG. 19 is a graph of the response of solvent concentration and solvent volume to the effect of isovitexin extraction;
FIG. 20 is a graph showing the response of solvent concentration and extraction time to the effect of isovitexin extraction;
FIG. 21 is a graph of the response of solvent volume and extraction time to the effect of isovitexin extraction;
FIG. 22 is a response surface plot of the effect of solvent concentration and solvent volume on the extraction of 6-0- β -D-glucosyl gentiopicroside;
FIG. 23 is a response surface plot of the effect of solvent concentration and extraction time on the extraction of 6-0- β -D-glucosyl gentiopicroside;
FIG. 24 is a response surface plot of the effect of solvent volume and extraction time on the extraction of 6-0- β -D-glucosyl gentiopicroside;
FIG. 25 is a graph of the response of solvent concentration and solvent volume to ursolic acid extraction;
FIG. 26 is a graph of the response of solvent concentration and extraction time on ursolic acid extraction;
FIG. 27 is a graph of the response of solvent volume and extraction time to ursolic acid extraction;
FIG. 28 is a graph of the response of solvent concentration and solvent volume to oleanolic acid extraction;
FIG. 29 is a graph of the response of solvent concentration and extraction time on oleanolic acid extraction;
FIG. 30 is a graph of the response of solvent volume and extraction time on the effect of oleanolic acid extraction.
Detailed Description
The invention is further described with reference to the following figures and detailed description.
A method for optimizing and ultrasonically extracting multiple index components in gentiana lineata by a response surface method comprises the following steps:
A. pulverizing whole plant of Gentiana striolata into powder, and sieving with 60 mesh sieve;
B. putting the powder into a conical flask, and adding water, absolute ethyl alcohol, methanol, 50% methanol or 75% methanol at a solid-to-liquid ratio of 20-60: 1;
C. ultrasonic extracting at room temperature for 10-50min to obtain extractive solution;
D. centrifuging the extractive solution at 3000r/s for 20min to obtain supernatant containing index components.
The preferred embodiment is as follows:
a method for optimizing and extracting multi-index components in gentiana lineata by a response surface method is characterized by comprising the following steps:
A. pulverizing whole plant of Gentiana striolata into powder, and sieving with 60 mesh sieve;
B. putting the powder into a conical flask, and adding a methanol solution with the mass concentration of 60% according to the solid-to-liquid ratio of 30: 1;
C. ultrasonic extracting at room temperature for 40min to obtain extractive solution;
D. centrifuging the extractive solution at 3000r/s for 20min to obtain supernatant containing index components.
First, preparation work
1. Instrumentation and equipment
Ultraviolet visible spectrophotometer (Shimadzu UV-2600); a cuvette (quartz, Shih glass instrument factory, Yixing city); a universal high-speed pulverizer (model FW200, beijing kewei Yongxing instruments ltd); centrifuge (BECKMAN Avanti J-26XP, Beckman, USA); electronic balances (one in ten thousand, mettler-toledo instruments (shanghai) ltd); DHG-9123A electric heat air blast constant temperature drying cabinet (Shanghai Qixin scientific instruments Co., Ltd.); a KQ-250TDB type high-frequency numerical control ultrasonic cleaner (kunlun mountain ultrasonic instruments ltd); pipette (sky wave TB9EX, 20 ℃, 5 mL); volumetric flask (50 mL); erlenmeyer flask (100 mL).
2. Reagent
Absolute ethanol (analytically pure, Tianjin, Daoyantan chemical reagent plant), methanol (analytically pure, Tianjin, Daoyantan chemical reagent plant), and Drech distilled water.
3. Material
The sample used was identified as a dry aerial part of Gentiana striolata falrei balf. The samples used in the single-factor experiment and the response surface optimization experiment are all the whole grass of Gentiana scabra Bunge which is produced by cooperation of Gansu and Ganan.
4. Selection of ultraviolet wavelength of gentiana straminea
The maximum absorption wavelength of 9 components of loganin, swertiamarin, 6-O-beta-D-glucosyl gentiamarin, isoorientin, isovitexin, ursolic acid and oleanolic acid is shown in Table 1.
TABLE 1 maximum absorption wavelength of 9 ingredients of Gentiana filifolia
Table 1The maximum absorption wavelength of 9ingredients ingentiana
Second, design and embodiment of experiment
1. Single factor screening
Weighing a certain amount of gentiana straminea maxim, carrying out ultrasonic extraction under different solvent concentrations, volumes and extraction time, centrifuging, taking supernate, carrying out full-wavelength scanning on an ultraviolet spectrophotometer, reading absorption values under the maximum absorption wavelengths of nine components of loganin, swertiamarin, 6' -O-beta-D-glucosyl gentiamarin, gentiopicroside, isoorientin, isovitexin, ursolic acid and oleanolic acid, and respectively inspecting the influence of different solvent concentrations (absolute ethyl alcohol, water, methanol, 50% methanol and 75% methanol), solvent volumes (10mL, 15mL, 20mL, 25mL and 30mL) and extraction time (10min, 20min, 30min, 40min and 50min) on the extraction efficiency of gentiana straminea maxim by taking the ultraviolet absorption values measured by the ultraviolet spectrophotometer as evaluation indexes.
Example 1: effect of solvent concentration on extraction Effect
Weighing about 0.5g of gentiana straminea whole grass into a conical flask, respectively adding water, absolute ethyl alcohol, methanol, 50% methanol and 20mL of 75% methanol (liquid-material ratio is 40:1), performing ultrasonic extraction at normal temperature for 30min, centrifuging at 3000r/s for 20min, taking supernate, measuring under an ultraviolet spectrophotometer, taking three parallels for each group of conditions, taking average values, and obtaining the measurement results shown in table 2 and figure 1, wherein the optimal condition is 75% methanol.
TABLE 2 Effect of solvent concentration on extraction
Example 2: influence of solvent volume (liquid-to-material ratio) on extraction efficiency
Weighing about 0.5g of gentiana straminea whole grass into a conical flask, respectively adding 10mL, 15mL, 20mL, 25mL and 30mL of 75% methanol, performing ultrasonic extraction for 30min at normal temperature, centrifuging at 3000r/s for 20min, taking supernate, measuring under an ultraviolet spectrophotometer, performing three parallels under each condition, taking an average value, and obtaining a measurement result shown in table 3 and figure 2, wherein the optimal condition is 10 mL.
TABLE 3 influence of solvent volume (liquid to feed ratio) on the extraction efficiency
Example 3: influence of extraction time on extraction effect
Weighing about 0.5g of gentiana straminea whole grass into a conical flask, adding 10mL of 75% methanol (liquid-material ratio is 20:1), performing ultrasonic extraction at normal temperature for 10min, 20min, 30min, 40min and 50min respectively, centrifuging at 3000r/s for 20min, taking supernate, measuring under an ultraviolet spectrophotometer, performing three parallels under each condition, taking an average value, and obtaining a measurement result shown in table 4 and figure 3, wherein the optimal condition is 30 min.
TABLE 4 Effect of extraction time on extraction Effect
As can be seen from examples 1-3, the single factor optimal process is: the concentration of the solvent is as follows: 75% methanol, solvent volume: 10mL, extraction time: and (3) 30 min.
2. Response surface method optimized ultrasonic extraction process
(1) Response surface method optimization design
On the basis of a single-factor test, the level values of 3 factors including solvent concentration, solvent volume and extraction time are determined, Design-expert8.0.6.1 software is used for carrying out Box-Behnken response surface Design and data analysis, and specific experimental factors and all level values are shown in a table 5.
TABLE 5 response surface Experimental factors and levels
Table 5 Factors and levels of response surface test
Designing 3 factors and 3 levels by adopting Design-Expert8.0.6.1 software according to a Box-Behnken combined Design principle to obtain 17 experiments, wherein the center point repeats the experiments for 5 times; the specific experimental design and results are shown in table 6, using the uv absorption value as the response value.
Table 6 response surface experimental conditions and results
Table 6 Conditions and resultsof response surface test
(2) Quadratic response surface regression model analysis
Performing secondary response surface regression analysis on the test data by adopting Design-Expert software, thus solving the correlation equations of the primary effect, the secondary effect and the interaction effect of the influencing factors, and obtaining the simulation equation and the determination coefficient R of the ultraviolet absorption values and the dependent variables of the 9 components through analysis2See table 7:
TABLE 7 simulation equations and coefficient of determination
Table 7 simulated variance and coefficient of decision
As can be seen from Table 7, equestrian acid R20.997 of swertiamarin R20.9565 Gentianoside R20.9806 swertiamarin R20.9961, isoorientin R20.9245 isovitexin R20.9008, 6' -0-beta-D-glucosyl gentiopicroside R20.8966 Ursolic acid R20.9721 Oleanolic acid R20.9721, the regression model is better in fitting, the experimental value is closer to the predicted value, and the reliability of the model can be checked through analysis of variance and correlation coefficients;
TABLE 8 analysis of variance of the marchanotide quadratic response surface regression model
Table 7Loganin acid two response surface regression model analysis of variance
Note: the difference is very obvious when the P is less than 0.001; has a P <0.01, high significance; p <0.05 is significant
TABLE 9 analysis of variance of regression model of swertiamarin secondary response surface
Table 9 swertiamain two response surface regression model analysis of variance
Note: the difference is very obvious when the P is less than 0.001; has a P <0.01, high significance; p <0.05 is significant
TABLE 10 gentiopicroside secondary response surface regression model analysis of variance
Table 10gentiopicrosidetwo response surface regression model analysis of variance
Note: the difference is very obvious when the P is less than 0.001; has a P <0.01, high significance; p <0.05 is significant
TABLE 11 analysis of variance of regression model of swertiamarin secondary response surface
Table 11 swertiamain two response surface regression model analysis of variance
Note: the difference is very obvious when the P is less than 0.001; has a P <0.01, high significance; p <0.05 is significant
TABLE 12 analysis of variance of isoorientin secondary response surface regression model
Table 12Isoorientin two response surface regression model analysis of variance
Note: the difference is very obvious when the P is less than 0.001; has a P <0.01, high significance; p <0.05 is significant
TABLE 13 analysis of variance of regression model of secondary response surface of isovitexin
Table 13 isovitexin two response surface regression model analysis of variance
Note: the difference is very obvious when the P is less than 0.001; has a P <0.01, high significance; p <0.05 is significant
TABLE 146-0-beta-D-glucosyl gentiopicroside secondary response surface regression model variance analysis Table 146' -O-beta-D-glucosylgenietipido response surface regression model analysis of variance
Note: the difference is very obvious when the P is less than 0.001; has a P <0.01, high significance; p <0.05 is significant
TABLE 15 Ursolic acid Secondary response surface regression model analysis of variance
Table 15 ursolic acid two response surface regression model analysis of variance
Note: the difference is very obvious when the P is less than 0.001; has a P <0.01, high significance; p <0.05 is significant
TABLE 16 Oleanolic acid Secondary response surface regression model analysis of variance
Table 16 oleanolic acid two response surface regression model analysis of variance
Note: the difference is very obvious when the P is less than 0.001; has a P <0.01, high significance; p <0.05 is significant
According to the response surface analysis of variance table, in the model, loganin P is less than 0.0001, swertiamarin P is 0.0006, gentiamarin P is less than 0.0001, swertiamarin P is less than 0.0001, isoorientin P is 0.0062, isovitexin P is 0.0087, 6-0-beta-D-glucosyl gentiamarin P is 0.0099, ursolic acid P is 0.0001, oleanolic acid P is 0.0001, and all the results are less than 0.001, which shows that the quadratic equation model is very significant. The results of the experiments show that the quadratic regression equation obtained by the experiment is highly significant, the fitting degree of the equation to the experiment is good, and the error is small, so that the regression equation can be used for replacing the true point of the experiment to analyze and predict the experimental result. The mean square value of the pure error of the model is 0.004, 0.022, 0.018, 0.0035, 0.018, 0.02, 6-0-beta-D-glucosyl gentiopicroside 0.073, 0.011 ursolic acid and 0.011 oleanolic acid, and the values are all small, so that the model is effective and can be used for predicting the process conditions of the extraction process. According to the check value F, the influence of each factor on the dependent variable is as follows: solvent concentration (A) > extraction time (C) > solvent volume (B).
(3) Response surface map analysis
The response surface and contour line graph group obtained by performing ternary quadratic regression fitting analysis on the test data by using Design-Expert8.0.6.1 software can visually reflect the influence results of various factors and interaction thereof on the extraction rates of 9 components. The shape of the contour line can reflect the strength of interaction between two factors, the ellipse represents that the interaction between two factors is strong, and the circle represents that the interaction is weak. The density of the contour lines and the gradient of the response surface can reflect the influence of various factors on the change of the extraction rate, wherein the gradient of the response surface is steeper, the contour lines are denser, the influence is larger, and the gradient of the response surface is gentler, and the influence is smaller.
Fig. 4, 7, 10, 13, 16, 19, 22, 25 and 28 show the influence of the interaction of the solvent concentration and the solvent volume on the extraction rates of loganin, swertiamarin, gentiamarin, swertiamarin, isoorientin, isovitexin, 6-0- β -D-glucosylgentiamarin, ursolic acid and oleanolic acid, respectively, when the extraction time is at the central level, wherein the interaction of the solvent concentration and the solvent volume on the extraction rates of loganin, swertiamarin, harpagoside, 6-0- β -D-glucosylgentiamarin, ursolic acid and oleanolic acid is weak as can be seen from fig. 4, 7, 13, 22, 25 and 28, and the interaction of the solvent concentration and the solvent volume on the extraction rates of gentiamarin, isoorientin and isovitexin is strong as can be seen from fig. 10, 16 and 19. FIGS. 4, 7, 13, 22, 25 and 28 show that the change in solvent volume has relatively little effect on the change in extraction rates of loganin, swertiamarin, 6-0-beta-D-glucosylgentiamarin, ursolic acid and oleanolic acid, as shown by the relatively gentle slope of the response surface and the relatively sparse contour; the change of the solvent concentration has a large influence on the change of the extraction rate, which is represented by a steep gradient and dense contour line of the response curved surface, and fig. 10, 16 and 19 show that the change of the solvent volume has a relatively large influence on the change of the extraction rate of gentiopicroside, isoorientin and isovitexin, which is represented by a steep gradient and dense contour line of the response curved surface, and the change of the solvent concentration has a small influence on the change of the extraction rate, which is represented by a gentle gradient and sparse contour line of the response curved surface;
figures 5, 8, 11, 14, 17, 20, 23, 26 and 29 respectively show that when the solvent volume is at a central level, influence of interaction of solvent concentration and extraction time on extraction rate of loganin, swertiamarin, gentiamarin, swertiamarin, isoorientin, isovitexin, 6-0-beta-D-glucosyl gentiamarin, ursolic acid and oleanolic acid, wherein, as can be seen from fig. 5, 8, 14, 23, 26 and 29, the interaction of the solvent concentration and the extraction time on the extraction rates of loganin, swertiamarin, 6-0-beta-D-glucosyl gentiopicrin, ursolic acid and oleanolic acid is weak, and as can be seen from fig. 11, 17 and 20, the interaction of the solvent concentration and the extraction time on the extraction rates of gentiamarin, isoorientin and isovitexin is strong. FIGS. 5, 8, 14, 17, 20, 23, 26 and 29 respectively show that the extraction time variation has relatively small influence on the variation of the extraction rates of loganic acid, swertiamarin, isoorientin, isovitexin, 6-0-beta-D-glucosylgentiopicroside, ursolic acid and oleanolic acid, and shows that the gradient of the response curved surface is relatively mild and the contour line is sparse, the influence of the solvent concentration variation on the extraction rate variation is large, and shows that the gradient of the response curved surface is relatively steep and the contour line is dense, FIG. 11 shows that the extraction time variation has relatively large influence on the variation of the gentiopicroside extraction rate, shows that the gradient of the response curved surface is relatively steep and the contour line is dense, the influence of the solvent concentration variation on the extraction rate variation is small, and shows that the gradient of the response curved surface is relatively mild and the contour line is sparse;
FIGS. 6, 9, 12, 15, 18, 21, 24, 27 and 30 show the effect of the interaction of the extraction time and the solvent volume on the extraction rate of loganin, swertiamarin, gentiamarin, swertiamarin, isoorientin, isovitexin, 6-0-beta-D-glucosylgentiamarin, ursolic acid and oleanolic acid, respectively, when the solvent concentration is at the central level, wherein the interaction of the extraction time and the solvent volume on the extraction rate of loganin, swertiamarin, gentiamarin, isoorientin, isovitexin, ursolic acid and oleanolic acid is strong as can be seen from 6, 9, 12, 18, 21, 27 and 30; from FIGS. 15 and 24, it is understood that the interaction of the extraction time and the solvent volume with the extraction rates of swertiamarin and 6-0-. beta. -D-glucosyl gentiopicroside is weak. Fig. 6, 9, 15, 18, 21, 24, 27 and 30 show that the change of extraction time has relatively small influence on the change of extraction rates of loganic acid, swertiamarin, gentiopicrin, swertiamarin, isoorientin, isovitexin, ursolic acid and oleanolic acid, and shows that the gradient of the response curved surface is relatively mild, the contour line of the response curved surface is sparse, the change of the volume of the solvent has relatively large influence on the change of the extraction rate, and shows that the gradient of the response curved surface is relatively steep and the contour line is dense, and fig. 12 shows that the change of the extraction time has relatively large influence on the change of the extraction rate of the gentiopicrin, and shows that the gradient of the response curved surface is relatively steep and the contour line is dense, the change of the concentration of the solvent has relatively small influence on the change of the extraction rate, and shows that the gradient of the.
(4) Optimal condition screening
Box-Behnken response surface experimental Design is carried out by using Design-Expert software, and the finally obtained optimal extraction conditions according to the optimized extraction conditions are shown in Table 17.
TABLE 17 optimal extraction conditions obtained by the response surface method
Table 17 optimal extraction conditions of response surface method
As can be seen from the above, on the basis of the content of the gentiana lineata gallet being 0.5g, the results according to the optimized extraction conditions show that the total extraction effect of 9 components in the gentiana lineata is the best under the conditions of solvent concentration of 60%, the dosage of methanol of 15mL (liquid-material ratio; 30:1) and extraction time of 40 min.
(5) Verification experiment
In order to verify the reliability of the result obtained by the response surface method, the process condition of model optimization is verified, and the optimal condition of the response surface method optimization is as follows: solvent concentration 60%, solvent volume: 14.14mL, extraction time: and (4) 40 min. Considering the feasibility and convenience of the experiment, the technological parameters of 60 percent of solvent concentration, volume of the solvent: 15mL, extraction time: experiments were performed 3 times at 40min, the average was taken, and the experimental results and the predicted results are shown in table 18.
Table 18 verifies the experimental measurements and predictions
Table 18 validation test measured and predicted values
Claims (4)
1. A method for optimizing and extracting multi-index components in gentiana lineata by a response surface method is characterized by comprising the following steps:
A. pulverizing whole plant of Gentiana striolata into powder, and sieving with 60 mesh sieve;
B. putting the powder into a conical flask, and adding water, absolute ethyl alcohol, methanol and 50-75% methanol according to the solid-to-liquid ratio of 20-60: 1;
C. ultrasonic extracting at room temperature for 10-50min to obtain extractive solution;
D. centrifuging the extractive solution at 3000r/s for 20min to obtain supernatant containing multiple index components.
2. The method for optimizing the ultrasonic extraction of the multi-index components in the gentiana straminea according to the response surface method of claim 1, wherein the optimal extraction solvent for extracting the multi-index components in the gentiana straminea obtained by optimization of the response surface method is methanol, the solution concentration is 60%, the extraction time is 40min, the solvent volume is 15mL, and the solid-to-liquid ratio is 30: 1.
3. The method for optimized ultrasonic extraction of multi-index components in gentiana lineolata according to claim 1 or 2, wherein the index components include loganin, swertiamarin, 6' -O-beta-D-glucosyl gentiamarin, isoorientin, isovitexin, ursolic acid and oleanolic acid.
4. The method for optimizing the ultrasonic extraction of the multi-index components in the gentiana lineata according to any one of claims 1 to 3, wherein the response surface method comprises the following steps:
(1) single factor screening: designing a single-factor test according to three single factors of solvent concentration, solvent volume and extraction time;
(2) designing a response surface method: and (2) taking the ultraviolet absorption values of the multi-index components in the gentiana straminea as response values, and performing a secondary response surface regression analysis experiment on the basis of single factor screening in the step (1) by using Design-Expert software to obtain a simulation equation and a determination coefficient as follows:
the simulated equation for loganine is:
y＝4.76-0.55*A-0.069*B+0.062*C+0.0218AB-0.049*AC+6.864*BC-0.29*A2+0.13*
B2+0.16*C2determining the coefficient R2＝0.997；
The simulation equation of the swertiamarin is as follows:
y＝4.82-0.3*A-0.04*B+0.051*C+4.65AB-0.031*AC+8.837*BC-0.091*A2+0.041*B2+0.077*C2determining the coefficient R2＝0.9565；
The simulation equation for gentiopicroside is:
y＝4.65+0.27*A-0.048*B+0.022*C-0.012*AB-0.022*AC+0.029*BC+0.44*A2+0.093*B2+0.16*C2determining the coefficient R2＝0.9806；
The simulation equation of the swertiamarin is as follows:
y＝4.78-0.52*A-0.038*B+0.043*C+5.793AB-0.029*AC+0.011*BC-0.34*A2+0.15*B2+0.13*C2determining the coefficient R2＝0.9961；
The simulation equation of isoorientin is as follows:
y＝7.73+0.16*A-0.056*B-0.14*C+0.16AB-0.22*AC+0.08*BC+0.11*A2-0.22*B2+0.13*C2determining the coefficient R2＝0.9245；
The simulation equation of isovitexin is:
y＝7.8+0.09*A-0.067*B-0.045*C+0.15AB-0.087*AC+0.064*BC+0.069*A2-0.14*B2+0.067*C2determining the coefficient R2＝0.9008；
The simulation equation of 6' -0-beta-D-glucosyl gentiopicroside is as follows:
y＝4.79-0.25*A-0.066*B+0.048*C-0.046AB-3.709*AC-6.38*BC-0.11*A2+0.11*B2+0.078*C2determining the coefficient R2＝0.8966；
The simulation equation for ursolic acid is:
y＝4.67+0.27*A-8.13*B+0.043*C-0.028AB-0.027*AC-9.527*BC+0.18*A2-0.12*B2-0.13*C2determining the coefficient R2＝0.9721；
The simulation equation for oleanolic acid is:
y＝4.67+0.27*A-8.13*B+0.043*C-0.028AB-0.027*AC-9.527*BC+0.18*A2-0.12*B2-0.13*C2determining the coefficient R2＝0.9721；
In the formula, y is the ultraviolet absorption value of each component, a variable parameter A is the concentration of the solvent, a variable parameter B is the extraction time, and a variable parameter C is the volume of the solvent; the calculation determined that the optimal solvent concentration is 60%, the extraction time is 40min, and the solvent volume is 15 mL.
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