CN112778431A - Response surface method optimized lamiophlomis rotata polysaccharide enzymolysis extraction process - Google Patents

Abstract

The invention extracts the lamiophlomis rotata crude polysaccharide by enzymolysis, takes the enzyme dosage, the temperature, the extraction time and the extraction frequency as influencing factors, takes the polysaccharide extraction rate as an investigation factor, and screens out the optimal influencing factor of the polysaccharide extraction rate by single factor investigation. By utilizing the response surface design, the optimal extraction process is optimized as follows: extracting at 60 deg.C for 30min with 2% enzyme-drug ratio and pH of 8. According to the invention, through single factor investigation and RSM test, the optimal extraction process conditions of the lamiophlomis rotata polysaccharide are determined to be extraction time of 60min, PH of 8 and extraction temperature of 60 ℃; the research result shows that the enzymolysis extraction of the lamiophlomis rotata polysaccharide is an effective, convenient, reliable and feasible method, and the optimal extraction process conditions are that the extraction time is 60min, the PH is 8, the extraction temperature is 60 ℃, and the enzyme-drug ratio is 2%.

Description

Response surface method optimized lamiophlomis rotata polysaccharide enzymolysis extraction process

Technical Field

The invention relates to the technical field of lamiophlomis rotata polysaccharide extraction, in particular to a response surface method optimized lamiophlomis rotata polysaccharide enzymolysis extraction process.

Background

The Tibetan medicine unique flavor is a drug for minority nationality, and is originally collected in Tibetan medical treatises of four medical classics and Jingzhu herbal, and has a history of one thousand to two hundred years from now. It is sweet, slightly bitter and warm in taste, mainly used for setting up bone and dry yellow water. The modern clinical application is used for treating traumatic injury, arthralgia and myalgia, cancellous bone inflammation and other symptoms, has obvious effects of relieving pain and diminishing inflammation, and has reliable curative effect. The research reports at home and abroad that the lamiophlomis rotata has pharmacological activity of oxidation resistance, inflammation resistance and pain relief, but the lamiophlomis rotata pays attention to iridoid in the lamiophlomis rotata, and the biological function of polysaccharide components in the lamiophlomis rotata is not reported. The research provides an early-stage basis for the research and extraction of biological activities such as oxidation resistance, immunoregulation and the like through the characterization of a polysaccharide structure. The natural plant polysaccharide belongs to a very important biological macromolecule in a plant body, is a basic substance for effectively maintaining and ensuring the normal operation of the life activity of an organism, has various pharmacological actions such as anti-tumor, immunoregulation, antivirus and antioxidation, can be used as a natural immunoregulator, and has become one of the research hotspots of scholars at home and abroad.

At present, the methods for extracting the plant polysaccharide comprise a hot water extraction method, an acid-base solution extraction method, a microwave extraction method, an ultrasonic extraction method and other methods. Research reports that the lamiophlomis rotata polysaccharide can be obtained by using a water extraction and alcohol precipitation method, but the extraction rate of the extraction method is low, and the lamiophlomis rotata is a rare traditional Chinese medicine and cannot exert the maximum value. The enzymes, especially pectinase, cellulase and papain, have high activity, are easy to obtain and low in cost, and can be applied to the extraction of plant polysaccharide, so the research uses enzymolysis extraction and optimizes the extraction process.

Disclosure of Invention

The invention aims to provide a response surface method for optimizing a lamiophlomis rotata polysaccharide enzymolysis extraction process so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

the response surface method for optimizing the enzymolysis extraction process of the lamiophlomis rotata polysaccharide comprises the following steps:

the method comprises the following steps: heating and refluxing the lamiophlomis rotata powder twice by 80% ethanol, degreasing, decoloring, removing oligosaccharide and micromolecule impurities, and volatilizing the solvent to obtain a treated sample;

step two: weighing 3g of a pretreatment sample, adding a proper amount of distilled water, soaking for 2h, controlling by single factors of enzyme adding amount, enzymolysis temperature and pH value, then extracting in a water bath, collecting supernatant, concentrating under reduced pressure to be below 20% of the original volume, adding absolute ethyl alcohol until the final concentration of the ethyl alcohol is 80%, carrying out alcohol precipitation for 24h at the temperature of 4 ℃, centrifuging, collecting precipitate, deproteinizing by a Sevag method, dialyzing for 48h by using a dialysis bag with the molecular weight cutoff of 3500Da, and finally freeze-drying to obtain the lamiophlomis rotata crude polysaccharide;

step three: on the basis of the single-factor test in the second step, by utilizing Box-Behnken Design (BBD) center combination Design and taking the PPs extraction yield Y (%) as a response value, the influence of enzyme addition, enzymolysis temperature and pH value on the extraction yield is inspected.

Preferably, the enzyme adding amount is 1.0-3.0%; the PH is 4-8; the enzymolysis temperature is 20-60 ℃.

Preferably, the polysaccharide content is determined by a sulfuric acid-phenol method in the extraction process;

the extraction yield (Y) of the PPs is represented by the formula of Y (%) (M/W) multiplied by 100%, wherein M is the content of polysaccharide, and W represents the net weight of the sample; taking the glucose concentration (C) as an abscissa and the absorbance (Abs) as an ordinate, and linearly fitting to draw a glucose quasi-curve to obtain a regression equation:

Abs＝0.06329C-0.03835(R²＝0.9994)。

preferably, the method for measuring the sugar content in the extraction process comprises the following steps:

weighing 0.01g of glucose standard product dried at 105 ℃ and having constant weight, dissolving the glucose standard product in ultrapure water, and fixing the volume to 100m1 to obtain 0.1mg/m1 solution; precisely sucking 0ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1.0ml to a suction pipe, adding ultrapure water to supplement 2ml, respectively adding 5% phenol solution 1m1, shaking up, quickly adding concentrated sulfuric acid 5ml to a vertical liquid level, and shaking up; cooling in cold water to room temperature, and developing; measuring the absorbance at the 490mm wavelength by using an ultraviolet spectrophotometer by taking a blank as a comparison;

drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance as an ordinate to obtain a regression equation y of 0.0063x-0.0077 (R)²0.9992); x is the concentration of a glucose standard substance, unit mg/ml, and y is an absorbance value;

calculating polysaccharide yield:

in formula (1), y: the yield (%) of the polysaccharide extracted by ultrasonic waves; c: the polysaccharide concentration (mg/mL) was calculated from the standard; v: volume-fixed volume (mL); n: dilution times; m: measuring the mass (mg) of the crude polysaccharide; m: crude polysaccharide mass (g); w: the mass (g) of the lamiophlomis rotata powder for extraction.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, through single factor investigation and RSM test, the optimal extraction process conditions of the lamiophlomis rotata polysaccharide are determined to be extraction time of 60min, PH of 8 and extraction temperature of 60 ℃; the research result shows that the enzymolysis extraction of the lamiophlomis rotata polysaccharide is an effective, convenient, reliable and feasible method, and the optimal extraction process conditions are that the extraction time is 60min, the PH is 8, the extraction temperature is 60 ℃, and the enzyme-drug ratio is 2%.

Drawings

FIG. 1 is a schematic diagram showing the effect of extraction temperature on the yield of Lamiophlomis rotate (Benth.) kudo polysaccharide;

FIG. 2 is a schematic diagram showing the effect of extraction time on the yield of Lamiophlomis rotate (Benth.) kudo polysaccharide;

FIG. 3 is a schematic diagram showing the effect of the amount of enzyme used in the present invention on the yield of Lamiophlomis rotate (Benth.) kudo polysaccharide;

FIG. 4 is a graph showing the effect of pH on the yield of Lamiophlomis rotate (Lamiophlomis rotate) polysaccharide according to the present invention;

FIG. 5 is an infrared absorption spectrum of Lamiophlomis rotate (Benth.) kudo polysaccharide of the present invention;

FIG. 6 is the ultraviolet absorption chart of Lamiophlomis rotate (Benth.) kudo polysaccharide of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The response surface method for optimizing the enzymolysis extraction process of the lamiophlomis rotata polysaccharide comprises the following steps:

the method comprises the following steps: heating and refluxing the lamiophlomis rotata powder twice by 80% ethanol, degreasing, decoloring, removing oligosaccharide and micromolecule impurities, and volatilizing the solvent to obtain a treated sample;

step two: weighing 3g of a pretreatment sample, adding a proper amount of distilled water, soaking for 2h, controlling by single factors of enzyme adding amount, enzymolysis temperature and pH value, then extracting in a water bath, collecting supernatant, concentrating under reduced pressure to be below 20% of the original volume, adding absolute ethyl alcohol until the final concentration of the ethyl alcohol is 80%, carrying out alcohol precipitation for 24h at the temperature of 4 ℃, centrifuging, collecting precipitate, deproteinizing by a Sevag method, dialyzing for 48h by using a dialysis bag with the molecular weight cutoff of 3500Da, and finally freeze-drying to obtain the lamiophlomis rotata crude polysaccharide;

step three: on the basis of the single-factor test in the second step, by utilizing Box-Behnken Design (BBD) center combination Design and taking the PPs extraction yield Y (%) as a response value, the influence of enzyme addition, enzymolysis temperature and pH value on the extraction yield is inspected.

The enzyme dosage in this example is 1.0-3.0%; the PH is 4-8; the enzymolysis temperature is 20-60 ℃.

In the extraction process of the embodiment, the content of polysaccharide is determined by a sulfuric acid-phenol method;

the extraction yield (Y) of the PPs is represented by the formula of Y (%) (M/W) multiplied by 100%, wherein M is the content of polysaccharide, and W represents the net weight of the sample; taking the glucose concentration (C) as an abscissa and the absorbance (Abs) as an ordinate, and linearly fitting to draw a glucose quasi-curve to obtain a regression equation:

Abs＝0.06329C-0.03835(R²＝0.9994)。

the method for measuring the sugar content in the extraction process of the embodiment comprises the following steps:

weighing 0.01g of glucose standard product dried at 105 ℃ and having constant weight, dissolving the glucose standard product in ultrapure water, and fixing the volume to 100m1 to obtain 0.1mg/m1 solution; precisely sucking 0ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1.0ml to a suction pipe, adding ultrapure water to supplement 2ml, respectively adding 5% phenol solution 1m1, shaking up, quickly adding concentrated sulfuric acid 5ml to a vertical liquid level, and shaking up; cooling in cold water to room temperature, and developing; measuring the absorbance at the 490mm wavelength by using an ultraviolet spectrophotometer by taking a blank as a comparison;

drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance as an ordinate to obtain a regression equation y of 0.0063x-0.0077 (R)²0.9992); x is the concentration of a glucose standard substance, unit mg/ml, and y is an absorbance value;

calculating polysaccharide yield:

in formula (1), y: the yield (%) of the polysaccharide extracted by ultrasonic waves; c: the polysaccharide concentration (mg/mL) was calculated from the standard; v: volume-fixed volume (mL); n: dilution times; m: measuring the mass (mg) of the crude polysaccharide; m: crude polysaccharide mass (g); w: the mass (g) of the lamiophlomis rotata powder for extraction.

On the basis of a single-factor test, a Box-Behnken Design (BBD) center combination Design is utilized, PPs extraction yield Y (%) is taken as a response value, and the influence of three independent variables, namely enzyme addition amount (A), enzymolysis temperature (B) and pH value (C) (each factor takes 3 levels) on the extraction yield is examined. The experimental design and the PPs yield are shown in table 1, and all experiments were performed 3 times; and performing data analysis by using Design Expert (version 8.0) software; analysis of variance was performed using ANOVA. It is statistically significant when P is less than 0.05.

TABLE 1 response surface level Experimental design

Response surface optimization experimental design

On the basis of a single-factor experimental result, a Box-Benhnken central combination design is adopted to optimize the ultrasonic-assisted extraction process of the lamiophlomis rotata polysaccharide, and the experimental factors and the level of response surface optimization are shown in a table 2. And carrying out a verification experiment according to the response surface optimization design experiment result, and comparing the predicted polysaccharide yield with the actual polysaccharide yield.

TABLE 2 analysis of variance of fitted quadratic polynomial model

Effect of Single factor on polysaccharide yield

As shown in figure 1, under the conditions that the pH value is 6, the addition amount of the complex enzyme is 3 percent, and the fixed enzymolysis time is 90min, the extraction enzymolysis temperature is 30-50 ℃ and the polysaccharide yield is in a positive effect relationship, and then the enzymolysis temperature is further increased, so that the polysaccharide yield is reduced. In the process of raising the temperature from 30 ℃ to 50 ℃, the polysaccharide extraction yield is steadily raised, which indicates that the temperature can improve the mass transfer efficiency of the polysaccharide, and along with the improvement of the extraction temperature, the solubility and the diffusion coefficient of the polysaccharide in the solvent are increased, the viscosity of the extraction solvent is reduced, and the processes are favorable for the extraction of the polysaccharide. However, too high a temperature may also destroy the structure of the polysaccharide, causing it to degrade during extraction and reducing the extraction yield. Therefore, it is preferable to select an extraction temperature of about 50 ℃.

As shown in figure 2, when the pH value is 6, the addition amount of the complex enzyme is 3%, the enzymolysis temperature is 50 ℃, the extraction enzymolysis time is 60-90 min, the positive effect relationship is formed between the extraction enzymolysis temperature and the polysaccharide yield, and then the polysaccharide yield is reduced by further increasing the extraction time. In the process that the extraction time is 60-90 min, the polysaccharide extraction yield is steadily increased, which indicates that the extraction time can improve the polysaccharide extraction yield, so that the extraction time of about 90min is more suitable.

As shown in figure 3, under the conditions of pH value of 6 and fixed enzymolysis time of 90min, the extraction temperature is 30-50 ℃, the fixed enzymolysis temperature is 60 ℃, the positive effect relationship is formed between the compound enzyme addition amount of 3% and the polysaccharide yield, and then the polysaccharide yield is reduced when the enzyme addition amount is further increased. The results show that the 3 kinds of biological enzymes have good enzymolysis function on plant cell tissues and can improve the solubility of polysaccharide in water, and the polysaccharide yield begins to decrease after the total amount is higher than 3 percent, which is probably caused by the degradation of polysaccharide by excessive cellulase, protease and pectinase. Therefore, the amount of enzyme to be added should be selected to be about 3%.

As shown in FIG. 4, the influence of pH during the enzymolysis process on the extraction yield of polysaccharide is complicated under other fixed experimental conditions. When the pH value is 5-7, the polysaccharide yield and the pH value have a positive effect, and when the pH value is 7, the polysaccharide yield is reduced, possibly under the over-alkali condition, the spatial structure of the enzyme is damaged, the combination with a substrate is influenced, and therefore the extraction yield is reduced. When the pH value is increased from 5 to 7, the polysaccharide yield is greatly increased and then is reduced again, which indicates that the activity of the enzyme is reduced by the overhigh pH value in the extraction process. Therefore, the pH value should be 5-7 for enzymolysis extraction.

Table 3 response surface experimental design and results

Regression analysis is carried out on the response surface experiment result of the yield of the lamiophlomis rotata polysaccharide, and the result is shown in a table (2). Regression fitting is carried out on the three factors to obtain a regression equation:

the extraction rate is +4.53+0.14 a + 0.12B + 0.26C-0.29 a B-0.18 a C + 0.82B C +1.22 a²+0.22*B²+0.81*C²

As can be seen from the analysis of variance,correction factor R²＝0.9796,R²The P value of the model is less than 0.0001, and is extremely remarkable; the mismatching term P value is more than 0.05 and is not significant. This shows that the degree of fitting of the model is good, and that the degree of fitting of the quadratic model of the experiment is high, and the variation relationship between the factors and the response value can be correctly reflected. The analysis of variance of the model can obtain that the extraction temperature (A), the extraction time (B), the PH (C), the extraction temperature and time interaction item (AB), the extraction temperature and PH interaction item (AC), the extraction time and PH interaction item (BC) and the secondary item of each factor have obvious influence on the response value. The influence significance of each factor on the response value is ranked as A > B > C.

As can be seen from the response surface and the contour lines, the response surface among the factors is steeper, which indicates that the interaction among the factors is stronger. According to the obtained model, the optimal technological conditions are predicted to be extraction temperature of 60 ℃, extraction time of 30min and PH 8, and the polysaccharide extraction rate can reach 6.34573% theoretically under the conditions.

Analysis of infrared spectra results

As shown in FIG. 5, in 3600 to 3000^-1And (3) a broad peak with strong absorption: 3420.16 and 3389.46cm^-1And is-OH stretching vibration peak. At 2325cm^-1The vibration is in the range of 1400-1200 cm^－1There are C-H variable angle vibrations of 1399.81 cm each^-1Absorption peak. At 1200-1000 cm^-1The absorption peak is caused by ether bond (C-O-C) of pyranose ring and-OH stretching vibration, and is 1168.68cm^-1All the 4 groups of absorption peaks are characteristic absorption peaks of saccharides, which indicates that all the extracts obtained by the experimental method are polysaccharides. In addition, 1654.66 cm^-1A strong absorption peak is respectively positioned at the two parts, namely the absorption peak of the amide I band; 1558.26 cm^-1Is the variable angle vibration of N-H, is the absorption peak of the amide II band, and proves that a small amount of protein (such as glycoprotein) combined with the sugar exists in the polysaccharide. 592.95cm^－1Is the O-H out-of-plane bending vibration absorption peak.

Ultraviolet absorption spectrum result analysis

As shown in FIG. 6, the UV absorption spectrum analysis of Lamiophlomis rotata polysaccharide shows that Lamiophlomis rotata polysaccharide absorbs slightly at 280 nm.

Verification experiment

By predicting the extreme point of the model by Design-Expert8.0.6, the result shows that the optimal extraction conditions are as follows: the extraction temperature is 60 deg.C, the extraction time is 30min, and the pH is 8, under the optimum extraction condition, the polysaccharide yield is 6.34573%. Designing and verifying experiments according to actual operation, wherein the extraction conditions are as follows: the extraction temperature is 60 ℃, the extraction time is 30min, the PH is 8, 3 times of parallel experiments are carried out according to the extraction conditions, the experiment result shows that the polysaccharide yield under the secondary conditions is 5.93 +/-0.02%, and the verification experiment result is close to the predicted value, which shows that the fitting degree of the model is good, and the process for extracting the lamiophlomis rotata polysaccharide under the extraction conditions is optimized reasonably and effectively.

The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (4)

1. The response surface method optimizes the lamiophlomis rotata polysaccharide enzymolysis extraction process, which is characterized by comprising the following steps of:

the method comprises the following steps: heating and refluxing the lamiophlomis rotata powder twice by 80% ethanol, degreasing, decoloring, removing oligosaccharide and micromolecule impurities, and volatilizing the solvent to obtain a treated sample;

step two: weighing 3g of a pretreatment sample, adding a proper amount of distilled water, soaking for 2h, controlling by single factors of enzyme adding amount, enzymolysis temperature and pH value, then extracting in a water bath, collecting supernatant, concentrating under reduced pressure to be below 20% of the original volume, adding absolute ethyl alcohol until the final concentration of the ethyl alcohol is 80%, carrying out alcohol precipitation for 24h at the temperature of 4 ℃, centrifuging, collecting precipitate, deproteinizing by a Sevag method, dialyzing for 48h by using a dialysis bag with the molecular weight cutoff of 3500Da, and finally freeze-drying to obtain the lamiophlomis rotata crude polysaccharide;

step three: on the basis of the single-factor test in the second step, by utilizing Box-Behnken Design (BBD) center combination Design and taking the PPs extraction yield Y (%) as a response value, the influence of enzyme addition, enzymolysis temperature and pH value on the extraction yield is inspected.

2. The response surface method optimized lamiophlomis rotata polysaccharide enzymolysis extraction process according to claim 1, wherein the enzyme adding amount is 1.0-3.0%; the PH is 4-8; the enzymolysis temperature is 20-60 ℃.

3. The response surface method optimized enzymatic hydrolysis extraction process of lamiophlomis rotata polysaccharide according to claim 1, wherein a sulfuric acid-phenol method is adopted in the extraction process to determine the polysaccharide content;

the extraction yield (Y) of the PPs is represented by the formula of Y (%) (M/W) multiplied by 100%, wherein M is the content of polysaccharide, and W represents the net weight of the sample; taking the glucose concentration (C) as an abscissa and the absorbance (Abs) as an ordinate, and linearly fitting to draw a glucose quasi-curve to obtain a regression equation:

Abs＝0.06329C-0.03835(R²＝0.9994)。

4. the response surface method optimized enzymatic extraction process of lamiophlomis rotata polysaccharide according to claim 1, wherein the sugar content in the extraction process is determined by the following method:

weighing 0.01g of glucose standard product dried at 105 ℃ and having constant weight, dissolving the glucose standard product in ultrapure water, and fixing the volume to 100m1 to obtain 0.1mg/m1 solution; precisely sucking 0ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1.0ml to a suction pipe, adding ultrapure water to supplement 2ml, respectively adding 5% phenol solution 1m1, shaking up, quickly adding concentrated sulfuric acid 5ml to a vertical liquid level, and shaking up; cooling in cold water to room temperature, and developing; measuring the absorbance at the 490mm wavelength by using an ultraviolet spectrophotometer by taking a blank as a comparison;

drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance as an ordinate to obtain a regression equation y of 0.0063x-0.0077 (R)²0.9992); x is the concentration of a glucose standard substance, unit mg/ml, and y is an absorbance value;

calculating polysaccharide yield:

in formula (1), y: the yield (%) of the polysaccharide extracted by ultrasonic waves; c: the polysaccharide concentration (mg/mL) was calculated from the standard; v: volume-fixed volume (mL); n: dilution times; m: measuring the mass (mg) of the crude polysaccharide; m: crude polysaccharide mass (g); w: the mass (g) of the lamiophlomis rotata powder for extraction.

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