阅读说明：本技术 一种响应面法优化提取洋紫荆花青素的方法 (Method for optimizing and extracting cercis negundo anthocyanin by response surface method ) 是由 田长恩 刘芳莉 于 2021-09-14 设计创作，主要内容包括：本发明公开了一种响应面法优化提取洋紫荆花青素的方法。通过本发明的响应面法优化提取洋紫荆花青素的方法获得洋紫荆花青素最优提取工艺,提取到的花青素抗氧化活性好,1mL浓度为1OD的洋紫荆花青素提取液抗氧化活性相当于2.733mg的L-抗坏血酸。(The invention discloses a method for optimally extracting cercis chinensis anthocyanin by a response surface method. The optimal extraction process of the cercis chinensis anthocyanin is obtained by the method for optimally extracting the cercis chinensis anthocyanin by the response surface method, the extracted anthocyanin is good in antioxidant activity, and 1mL of the cercis chinensis anthocyanin extracting solution with the concentration of 1OD is equivalent to 2.733mg of L-ascorbic acid in antioxidant activity.)

1. A method for optimizing and extracting cercis chinensis anthocyanin by using a response surface method is characterized by comprising the following steps:

(1) carrying out single-factor test of the extraction of the cercis chinensis anthocyanin by using the cercis chinensis as a raw material, wherein the single-factor test comprises the selection of petal types, the selection of extractant types, the selection of the pH value of a leaching solution, the selection of leaching temperature, the selection of leaching time and the selection of a liquid-material ratio;

(2) taking the leaching temperature, leaching time and liquid-material ratio as influencing factors, taking the absorbance of the anthocyanin extracted from the cercis negundo as a response value, establishing a mathematical model by applying a center combination (Box-Behnken) test, carrying out response surface analysis, and screening out the factors which obviously influence the extraction of the cercis negundo anthocyanin;

(3) and analyzing the contour map, the response surface 3D map and the mathematical model of the interaction of the influencing factors, and determining the optimal extraction process for extracting the cercis chinensis anthocyanin.

2. The method for optimizing and extracting the cercis chinensis anthocyanin by the response surface method according to claim 1, is characterized in that,

the method also comprises the following steps before the step (1): naturally drying fresh cercis negundo petals, and then measuring the drying rate; scanning distilled water extracting solution of dry flowers of the cercis negundo at the wavelength of 400-600 nm to obtain the optimal detection wavelength of the cercis negundo anthocyanin; determining the correlation between the concentration of cercis chinensis anthocyanin and the antioxidant activity of the cercis chinensis anthocyanin;

the method also comprises the following steps before the step (2): the influence of the color of the anthocyanin extract on the result of the absorbance measurement is researched.

3. The method for optimizing and extracting the cercis chinensis anthocyanin by the response surface method according to claim 1, is characterized in that,

the petal type selection in the step (1) is specifically as follows: controlling the reaction conditions of other factors to be consistent by taking the types of the petals as variables, and respectively measuring the absorbance of the cercis negundo petal anthocyanin extracting solution in different states at the optimal detection wavelength of the cercis negundo anthocyanin;

the selection of the types of the extracting agents in the step (1) is specifically as follows: the variety of the extracting agent is taken as a variable, the reaction conditions of other factors are controlled to be consistent, and the absorbance of the cercis chinensis anthocyanin extracting solution extracted by different extracting agents is respectively measured under the optimal detection wavelength of the cercis chinensis anthocyanin;

the selection of the pH value of the leaching liquor in the step (1) is specifically as follows: controlling the reaction conditions of other factors to be consistent by taking the pH value of the leaching liquor as a variable, and respectively measuring the absorbance of the cercis chinensis anthocyanin extracting solution under different pH values under the optimal detection wavelength of the cercis chinensis anthocyanin;

the selection of the leaching time in the step (1) is specifically as follows: taking the leaching time as a variable, controlling other factors to enable reaction conditions of other factors to be consistent, and respectively measuring the absorbance of the cercis chinensis anthocyanin under different leaching times under the optimal detection wavelength of the cercis chinensis anthocyanin;

the selection of the liquid-material ratio in the step (1) is specifically as follows: taking the liquid-material ratio as a variable, controlling the reaction conditions of other factors to be consistent, and respectively measuring the absorbance of the cercis chinensis anthocyanin extracting solution under different liquid-material ratios under the optimal detection wavelength of the cercis chinensis anthocyanin;

the leaching temperature in the step (1) is specifically selected as follows: and (3) taking the leaching temperature as a variable, controlling the reaction conditions of other factors to be consistent, and respectively measuring the absorbance of the cercis chinensis anthocyanin extracting solution at different leaching temperatures under the optimal detection wavelength of the cercis chinensis anthocyanin.

4. The method for optimizing and extracting cercis chinensis anthocyanin by using response surface method as claimed in claim 1, wherein the step (2) of applying center combination test to establish mathematical model specifically comprises the following steps: performing a horizontal test of influence factors by using a Box-Behnken composite model, and performing regression analysis on data obtained by the test by using the absorbance of the anthocyanin extracted from the cercis negundo as a response value and using Design Expert software to obtain a mathematical model between the absorbance of the cercis negundo anthocyanin extracting solution and the influence factors.

5. The method for optimizing and extracting cercis negundo anthocyanin according to claim 4, wherein the mathematical model is: 7.86456+0.046173 XA-0.159535 XB-0.483250 XC-0.00112 XAB +0.004925 XAC +0.000462 XBC-0.000246 XA²+0.001432×B²+0.10419×C²(ii) a Wherein A is leaching time, B is leaching temperature, and C is liquid-material ratio.

6. The method for optimizing and extracting the cercis chinensis anthocyanin by the response surface method according to claim 1, wherein the antioxidant activity of the cercis chinensis anthocyanin extracting solution extracted by the optimum extraction process of the cercis chinensis anthocyanin is determined by an ABTS free radical scavenging experiment.

7. The method for optimizing and extracting cercis negundo anthocyanin according to claim 6, wherein the determination of antioxidant activity is specifically as follows: carrying out ABTS free radical scavenging experiment on the cercis chinensis anthocyanin extracting solution extracted by the optimal extraction process, and calculating the antioxidant activity of the cercis chinensis anthocyanin extracting solution; and meanwhile, an ABTS free radical scavenging experiment is used for making an antioxidant activity standard curve of the existing antioxidant activity product, the cercis chinensis anthocyanin extracting solution is compared with the antioxidant activity of the cercis chinensis anthocyanin extracting solution, and the mass number of the existing antioxidant activity product corresponding to the antioxidant activity of the extracting solution obtained by the optimal extraction process is calculated.

8. A process for extracting cercis chinensis anthocyanidin, which is characterized in that the cercis chinensis anthocyanidin is obtained by optimizing a method for extracting the cercis chinensis anthocyanidin by using a response surface method according to any one of claims 1 to 7.

9. The acacia cerivar anthocyanin extraction process of claim 8, wherein the acacia cerivar anthocyanin extraction process: taking natural flowers of natural air-dried cercis negundo as raw materials, pulverizing, extracting with water with pH close to 7 as extractant at 70 deg.C for 5min, with liquid-material ratio of 1:30 (g/mL).

Technical Field

The invention relates to the technical field of anthocyanin extraction, in particular to a method for optimally extracting cercis negundo anthocyanin by a response surface method.

Background

The anthocyanin has strong oxidation resistance, strong scavenging capacity to free radicals and high bioavailability, and the bioavailability to a human body can reach 100%. In recent years, anthocyanin has been found to have physiological activities such as reducing DNA damage, reducing DNA fragmentation and intracellular reactive oxygen species, alleviating juvenile depression symptoms, targeting testis and the like.

The cercis ocellatus is used as an ornamental plant in southern areas of China, is wide in planting and long in flowering phase, has claret-colored petals, is presumed to be rich in anthocyanin, is used as a raw material to extract the anthocyanin, is low in cost and has great development potential; meanwhile, the research on the oxidation resistance of the anthocyanin can provide reference for subsequent development and utilization, promote the production of more green, environment-friendly and natural skin care products, health care products and the like by utilizing the cercis negundo anthocyanin, and improve the life quality of people.

In the extraction process optimization experiment, the response surface method has higher reliability than the orthogonal method. The prediction model obtained by the response surface optimization method is continuous, and each level of the experiment can be continuously analyzed in the optimization process of the experimental conditions; whereas orthogonal experiments can only analyze isolated experimental points. The response surface optimization method considers experimental random errors, and meanwhile, the complex unknown function relation is fitted in a small area by using a simple first-order or second-order polynomial model, so that the calculation is simple and convenient.

CN110526892A discloses a method for extracting anthocyanin from indigo fruit, which comprises the steps of leaching, ultrafiltration, resin adsorption elution and crystallization to obtain the product. The method takes water as solvent to extract anthocyanin product from indigo honeysuckle, and has the advantages of high anthocyanin content, high yield, mild extraction process conditions, little pollution and suitability for practical production. However, the method has the disadvantages of high cost, high resource consumption and complex process.

CN110437197A discloses a method for extracting anthocyanin from sphenanthera flower, which comprises the steps of degreasing, leaching, ultrafiltration, concentration, alcohol precipitation, resin adsorption, gradient desorption, concentration and drying to obtain the anthocyanin product. The method can simultaneously extract anthocyanin products with various contents from the sphenanthera, has high yield, and the whole extraction process has mild conditions and is environment-friendly. However, the method uses ethanol and various organic acids, and has the disadvantages of high cost, large resource consumption and complex process.

CN109020941A discloses a method for extracting anthocyanins from purple sweet potatoes, which comprises the steps of ultrasonic wave and microwave synergistic extraction, centrifugation, concentration and vacuum freeze drying to obtain anthocyanins. The method has the advantages of high anthocyanin extraction rate, high purity and popularization and application value. However, this method is relatively complicated and expensive, and requires relatively high equipment.

CN107474031A discloses a response surface method optimized lotus seedpod procyanidin extraction method, which optimizes 3 factors influencing procyanidin extraction by using the response surface method to the concentration, extraction temperature and extraction time of a glycerol solution, and obtains an optimal lotus seedpod procyanidin extraction process using glycerol as an extractant. The method is simple to operate, the used glycerin is nontoxic, and the glycerin extract can be directly added into certain foods, cosmetics and other products, so that the method is beneficial to industrial application and has better economic and social benefits. But the cost is relatively high by using the glycerol as the extractant; and the influence of the liquid-material ratio on the extraction rate is not considered.

In summary, organic solvents such as ethanol are often used for extracting anthocyanin, and the dissolved organic solvents are not only anthocyanin, so that the content of impurities in the extract is high, further separation and purification are required, and the production cost is increased. Organic solvent extraction is more costly to produce and environmentally damaging than water extraction, and the solvents used must also meet edible standards if the extracted anthocyanins are to be used in the cosmetic or food industry. Although the water extraction method is more environment-friendly and economical than the organic solvent extraction method, the extraction efficiency of the method still needs to be improved at present, including shortening the extraction period, improving the utilization rate of raw materials and the like.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a method for optimally extracting the cercis negundo anthocyanin by a response surface method.

The purpose of the invention is realized by the following technical scheme: a method for optimizing and extracting cercis negundo anthocyanin by a response surface method comprises the following steps:

(1) carrying out single-factor test of the extraction of the cercis chinensis anthocyanin by using the cercis chinensis as a raw material, wherein the single-factor test comprises the selection of petal types, the selection of extractant types, the selection of the pH value of a leaching solution, the selection of leaching temperature, the selection of leaching time and the selection of a liquid-material ratio;

(2) taking the leaching temperature, leaching time and liquid-material ratio as influencing factors, taking the absorbance of the anthocyanin extracted from the cercis negundo as a response value, establishing a mathematical model by applying a center combination (Box-Behnken) test, carrying out response surface analysis, and screening out the factors which obviously influence the extraction of the cercis negundo anthocyanin;

(3) and analyzing the contour map, the response surface 3D map and the mathematical model of the interaction of the influencing factors, and determining the optimal extraction process for extracting the cercis chinensis anthocyanin.

Preferably, the method further comprises the following steps before the step (1): naturally drying fresh cercis negundo petals, and then measuring the drying rate; scanning distilled water extracting solution of dry flowers of the cercis negundo at the wavelength of 400-600 nm to obtain the optimal detection wavelength of the cercis negundo anthocyanin; and determining the correlation between the concentration of the cercis chinensis anthocyanin and the antioxidant activity of the cercis chinensis anthocyanin.

Preferably, the petal type in step (1) is selected specifically as follows: the variety of the petals is taken as a variable, the reaction conditions of other factors are controlled to be consistent, and the absorbance of the cercis negundo petal anthocyanin extracting solution in different states is respectively measured under the optimal detection wavelength of the cercis negundo anthocyanin.

Preferably, the types of the extracting agents in the step (1) are specifically selected as follows: and (3) taking the types of the extracting agents as variables, controlling the reaction conditions of other factors to be consistent, and respectively measuring the absorbance of the cercis chinensis anthocyanin extracting solution extracted by different extracting agents under the optimal detection wavelength of the cercis chinensis anthocyanin.

Preferably, the selection of the pH value of the leach liquor in step (1) is specifically: and (3) taking the pH value of the leaching liquor as a variable, controlling the reaction conditions of other factors to be consistent, and respectively measuring the absorbance of the cercis chinensis anthocyanin extracting solution under different pH values under the optimal detection wavelength of the cercis chinensis anthocyanin.

Preferably, the leaching time in the step (1) is selected from the following steps: and taking the leaching time as a variable, controlling other factors to enable the reaction conditions of other factors to be consistent, and respectively measuring the absorbance of the cercis chinensis anthocyanin under different leaching times under the optimal detection wavelength of the cercis chinensis anthocyanin.

Preferably, the selection of the liquid-material ratio in the step (1) is specifically as follows: and (3) controlling the reaction conditions of other factors to be consistent by taking the liquid-material ratio as a variable, and respectively measuring the absorbance of the cercis chinensis anthocyanin extracting solution under different liquid-material ratios under the optimal detection wavelength of the cercis chinensis anthocyanin.

Preferably, the leaching temperature in the step (1) is selected from the following specific steps: and (3) taking the leaching temperature as a variable, controlling the reaction conditions of other factors to be consistent, and respectively measuring the absorbance of the cercis chinensis anthocyanin extracting solution at different leaching temperatures under the optimal detection wavelength of the cercis chinensis anthocyanin.

Preferably, the method further comprises the following steps before the step (2): the influence of the color of the anthocyanin extract on the result of the absorbance measurement is researched.

The step (2) of establishing a mathematical model by using the central combination test specifically comprises the following steps: performing a horizontal test of influence factors by using a Box-Behnken composite model, and performing regression analysis on data obtained by the test by using the absorbance of the anthocyanin extracted from the cercis negundo as a response value and using Design Expert software to obtain a mathematical model between the absorbance of the cercis negundo anthocyanin extracting solution and the influence factors.

The mathematical model is as follows: 7.86456+0.046173 XA-0.159535 XB-0.483250 XC-0.00112 XAB +0.004925 XAC +0.000462 XBC-0.000246 XA²+0.001432×B²+0.10419×C²(ii) a Wherein A is leaching time, B is leaching temperature, and C is liquid-material ratio.

The anti-oxidation activity of the cercis chinensis anthocyanin extracting solution extracted by the optimal cercis chinensis anthocyanin extracting process is determined by an ABTS free radical scavenging experiment.

Preferably, the antioxidant activity is determined in particular as: carrying out ABTS free radical scavenging experiment on the cercis chinensis anthocyanin extracting solution extracted by the optimal extraction process, and calculating the antioxidant activity of the cercis chinensis anthocyanin extracting solution; and meanwhile, an ABTS free radical scavenging experiment is used for making an antioxidant activity standard curve of the existing antioxidant activity product, the cercis chinensis anthocyanin extracting solution is compared with the antioxidant activity of the cercis chinensis anthocyanin extracting solution, and the mass number of the existing antioxidant activity product corresponding to the antioxidant activity of the extracting solution obtained by the optimal extraction process is calculated.

A folium Cercis chinensis anthocyanin extraction process is obtained by the above response surface method optimized extraction method.

The extraction process of the cercis chinensis anthocyanin comprises the following steps: taking natural flowers of natural air-dried cercis negundo as raw materials, pulverizing, extracting with water with pH close to 7 as extractant at 70 deg.C for 5min, with liquid-material ratio of 1:30 (g/mL).

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

the optimal extraction process of the cercis chinensis anthocyanin can be obtained by adopting the response surface method optimization method, the cercis chinensis anthocyanin extracted by adopting the process has good antioxidant activity, and 1mL of the cercis chinensis anthocyanin extracting solution with the concentration of 1OD has the antioxidant activity equivalent to 2.733mg of L-ascorbic acid.

Drawings

FIG. 1 is a graph showing the results of the assay of the relationship between the dilution of cyanidin and the antioxidant activity of cercis chinensis.

FIG. 2 is a graph showing the results of examining the influence of petal state on the anthocyanin content.

FIG. 3 is a graph showing the results of the detection of the influence of the kind of the extractant on the anthocyanin content and the antioxidant activity thereof.

FIG. 4 is a graph showing the results of the measurement of the influence of the pH of the extractant on the extraction rate of cyanidin from Vitex negundo L.

FIG. 5 is a graph showing the results of examining the influence of extraction time on the extraction rate of anthocyanins.

FIG. 6 is a graph showing the results of measurement of the effect of feed liquid ratio on the anthocyanin extraction yield.

FIG. 7 is a graph showing the results of examining the influence of the leaching temperature on the extraction rate of anthocyanins.

FIG. 8 is a graph showing the results of the measurement of the relationship between the dilution of the anthocyanin extract and the antioxidant activity thereof.

FIG. 9 is a 3D plot of the response of leaching time versus leaching temperature.

FIG. 10 is a 3D plot of leaching time versus liquor to feed ratio response surface.

FIG. 11 is a 3D plot of leaching temperature versus liquid-to-feed ratio response surface.

FIG. 12 is a standard graph of the antioxidant activity of L-ascorbic acid solutions.

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.

The ABTS working solution used in the embodiment of the invention is prepared at present, and specifically comprises the following components: distilled water is used as a solvent, and 2, 2' -hydrazine-bis (3-ethylbenzthiazoline-6-sulfonic acid) diamine salt (ABTS) is added to prepare a solution. Weighing 0.01g of ABTS, dissolving in 10mL of distilled water, and shaking up until the ABTS is completely dissolved; weighing (NH)₄)₂S₂O₈Dissolving 0.052g of the mixture in 40mL of distilled water, and shaking up until the mixture is completely dissolved; mixing the two solutions, standing for 12-16 hr, and making into ABTS working solution.

Example 1 results of measurement of drying Rate

Weighing the collected fresh cercis negundo petals by weight (M), naturally airing the fresh cercis negundo petals, weighing the fresh cercis negundo petals by weight (W), repeating the operation for three times according to the drying rate of W/M multiplied by 100%, and calculating the average value of the drying rates of three fresh cercis negundo flowers to obtain the drying rate of the fresh cercis negundo flowers of 9.85% (Table 1).

TABLE 1 dry-breaking rate of fresh folium Cercis chinensis

Example 2 detection of anthocyanin absorption peaks from Cercis negundo

Taking 0.1g of crushed natural air-dried flowers of cercis negundo, adding 20mL of distilled water, soaking for 20min at 25 ℃, filtering, and repeating the operation for three times. The wavelength is averaged over three scans using an ultraviolet-visible spectrophotometer to determine the average wavelength. The obtained cercis chinensis anthocyanin has an absorption peak wavelength of 523nm by scanning.

Example 3 correlation of Cercis negundo anthocyanin concentration with its antioxidant Activity

Taking a certain anthocyanin extracting solution, respectively diluting by 2, 4, 8 and 16 times, determining the anti-oxidation activity of the acacia mearnsii anthocyanin extracting solution with each dilution time through an ABTS free radical scavenging experiment, and repeating the operation for three times. Making a scatter diagram of the cercis chinensis anthocyanin and the antioxidant activity thereof according to the average value of the data, and solving R²To obtain the figure 1.

FIG. 1 shows that R²>0.9, the cercis chinensis anthocyanin and the antioxidant activity have linear correlation. In subsequent experiments, the concentration of the cercis chinensis anthocyanin is only measured, the optimal process is optimized, and then the antioxidant activity is measured.

Example 4 Effect of petal State on the content of extracted anthocyanins

Weighing Chinese redbud respectively: 1g of fresh and naturally fallen flowers on the ground, flowers on trees, 0.1g of naturally fallen flowers on the ground which are preserved for months, adding a proper amount of liquid nitrogen, grinding into powder, soaking for 20min at 25 ℃ by using water as an extracting solution and with the liquid-material ratio of 200:1(mL/g), filtering, and measuring the light absorption value at 523 nm. Three parallel experiments were performed per group and the results are shown in figure 2.

FIG. 2 shows that the above-ground dried flowers have similar anthocyanin content to the flowers on the trees, but all have lower anthocyanin content than the above-ground flowers. This may be associated with a portion of the above ground flowers having lost water at the time of collection, resulting in a higher content of anthocyanins extracted therefrom. Considering the factors that fresh flowers are not easy to preserve, are limited by the flowering period, damage the ornamental value of the picked flowers and the like, the fallen flowers (dried flowers on the ground) which are naturally dried are taken as the best raw materials.

Example 5 Effect of extractant type on the content of extracted anthocyanins and their antioxidant Activity

Weighing 24 parts of 0.1g of naturally dried flowers which are crushed by a crusher and stored for months, respectively adding the crushed flowers into 20mL (liquid-material ratio 200:1(mL/g) of distilled water, 30% ethanol, 50% ethanol, 60% ethanol, 75% ethanol solution, absolute ethanol and 0.1% and 0.3% HCl solution by mass fraction, leaching for 20min at 25 ℃, filtering, setting three parallel experiments per group, taking 3mL of cercis chinensis anthocyanin extract, measuring absorbance at 523nm, and obtaining the antioxidant activity through an ABTS free radical scavenging experiment, thereby obtaining a graph 3.

Fig. 3 shows that, although the anthocyanin content extracted with 60% ethanol is relatively high, the antioxidant activity of the anthocyanin does not differ significantly from that of all treatments, which may be related to the influence of the presence of ethanol on the antioxidant activity of the anthocyanin. Distilled water is the best extractant in view of cost saving, environmental protection and convenience for later product development.

EXAMPLE 6 Effect of extractant pH on Jacquard anthocyanin content

Weighing 21 parts of 0.1g of natural dried flowers of cercis negundo crushed by a crusher and stored for months, adding 6mL (liquid-material ratio 60:1(mL/g)) of distilled water with the pH value adjusted to 2, 3, 4, 5, 6, 7 and 8 respectively as an extracting agent, leaching for 20min at 25 ℃, filtering, and measuring the light absorption value at 523 nm. Three parallel experiments were performed per group, resulting in FIG. 4.

FIG. 4 shows that the content of cyanidin extracted from cercis negundo is not greatly different when the extractant is acidic and neutral, and is higher when the extractant is alkaline. However, according to the literature, the anthocyanin is more stable under the acidic condition, so that the anti-oxidation activity of the cercis negundo anthocyanin extract with the pH of 6, 7 and 8 is measured, and the anti-oxidation activity of the cercis negundo anthocyanin is obviously lower than that of the cercis negundo anthocyanin under the acidic and neutral conditions when the leaching solution is alkaline. Neutral distilled water with a pH close to 7 is therefore the best extractant.

EXAMPLE 7 Effect of leach time on Jacquard Green content

27 parts of 0.1g of naturally air-dried flowers of cercis negundo crushed by a crusher and stored for several months were weighed, and then extracted at 25 ℃ for 5, 10, 15, 20, 25, and 30min in 50mL conical flasks each with 10mL of distilled water (liquid-to-material ratio 100:1(mL/g)), followed by filtration, and the absorbance was measured at 523 nm. Three parallel experiments were performed per group, resulting in fig. 5.

FIG. 5 shows that the extraction time is not less than 15min, and the anthocyanin extraction rate is kept stable.

Example 8 Effect of liquid to Material ratio on Jacquard Green content

Weighing 18 parts of 0.1g of natural dried flowers of cercis negundo crushed by a crusher and stored for months, adding 5, 8, 10, 15, 20 and 25mL of distilled water into 50mL conical flasks respectively at the liquid-material ratio of 1:50, 1:80, 1:100, 1:150, 1:200 and 1:250(g/mL), extracting for 10min at 25 ℃, filtering, and measuring the light absorption value at 523 nm. Three parallel experiments were performed per group, resulting in FIG. 6.

FIG. 6 shows that the extracted anthocyanin content is highest when the liquid-material ratio is 50:1, and the extraction rate of the cercis chinensis anthocyanin is relatively stable after the liquid-material ratio is 80: 1.

EXAMPLE 9 Effect of leach temperature on Jacquard Green content

Weighing 18 parts of 0.1g of naturally dried flowers of cercis negundo crushed by a crusher and stored for months, adding 8mL of distilled water into 15mL of centrifuge tubes respectively, wherein the liquid-material ratio is 80:1(mL/g), vacuumizing the centrifuge tubes by using a vacuum machine, shaking the leaching liquor uniformly, leaching at the leaching temperatures of 25 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ for 15min, filtering, and measuring the light absorption value at 523 nm. Three replicates of each set were set up to give 7 (in the figure, room temperature conditions at 25 ℃).

Fig. 7 shows that the extracted anthocyanin content is highest at 60 c, and then decreases, which is related to the decomposition of anthocyanin under high temperature conditions, the higher the temperature, the faster the decomposition speed.

Example 10 Effect of anthocyanin extract color on absorbance

Diluting certain anthocyanin extract solution by 2 times, 5 times, 10 times and 15 times respectively, taking the same volume (3mL) of each dilution, measuring absorbance at the wavelength of the anthocyanin absorption peak of cercis negundo, carrying out ABTS free radical scavenging experiment, and measuring the antioxidant activity of each dilution to obtain figure 8.

Fig. 8 shows that the antioxidant activity is linearly related to the anthocyanin content when the stock solution is diluted by more than five times, and as the ABTS working solution is green and the cercis chinensis anthocyanin extract is dark brown, the antioxidant activity of the stock solution is measured after the stock solution is diluted by 10 times in consideration of the influence of color on the measured value, the anthocyanin extract after the stock solution is diluted by ten times is nearly transparent, and the influence of the anthocyanin extract color on the experiment is almost eliminated.

Example 11

After the single-factor experiment is completed, the experimental result is analyzed, the naturally dried flower drop is used as a raw material, water is used as an extracting agent, the center combination principle of Box-Behnken Design (BBD) is utilized, three-factor three-level experimental Design is carried out on three factors of leaching time, leaching temperature and leaching liquor material ratio, and the experimental factor level selection and Design are shown in Table 2.

TABLE 2 response surface experiment coding table

Example 12

By selecting leaching time, leaching temperature and leaching liquor material ratio as influencing factors, and taking the absorbance of anthocyanin extracted from cercis red-ocean as a response value, a mathematical model is established by applying central combined test design on the basis of a single-factor test, and response surface analysis is carried out, wherein the response surface design and result are shown in table 3, the variance analysis is shown in table 4, and the fitting degree is shown in table 5.

TABLE 3 response surface protocol and results

TABLE 4 ANOVA TABLE

Note that the difference is significant (P < 0.05), the difference is very significant (P < 0.01), and the difference is not significant when P > 0.05.

Performing multiple regression analysis on the factors and the response values to obtain a corresponding quadratic fitting equation (namely the extraction rate X of the cercis chinensis anthocyanin) of which X is 7.86456+0.046173 xA-0.159535 xB-0.483250 xC-0.00112 xAB +0.004925 xAC +0.000462 xBC-0.000246 xA²+0.001432×B²+0.10419×C²

From the results in the table, the P value of the model is <0.0001, which proves that the regression effect is very significant; the P value of the mismatching item is greater than 0.05, no mismatching factor exists, and therefore, a regression equation can be used for replacing a real experimental point to analyze an experimental result.

According to the analysis result of variance, C, AB, AC and B are obtained²To this endSignificant factors of the model, C, AB, B²The effect is very significant and the AC effect is significant.

The major and minor sequence of the influence of the three factors on the extraction rate of the cercis chinensis anthocyanin in the experiment can be seen through the value of F: c (liquid-to-material ratio) > A (leaching time) > B (leaching temperature).

TABLE 5 degree of Fit

This experiment Adjusted R²＝0.9779(>0.8), the coefficient of variation C.V. is 4.06%, the model is suitable, the fitting degree is good, and the method can be used for preliminary analysis and prediction of the optimal cercis negundo anthocyanin extraction process.

Example 13

By observing the contour lines of the response surface and the 3D map of the response surface, the magnitude of the influence can be analyzed. In the contour diagram, when the interaction between the two is not obvious, the contour line is circular; when the interaction is significant, the contour lines are elliptical or horseshoe-shaped. In the 3D map, the steeper the gradient, the more the response value is affected by the factor; conversely, the smaller the response value is affected by the condition factor.

Fig. 9, 10, and 11 show how strongly the 3 factors in this experiment interact with the extraction rate of cercis negundo anthocyanin. As can be seen from the graph, the slope of the response surface formed by the leaching time and the leaching temperature is the steepest in the three response surface graphs, which shows that the strongest interaction is the leaching time and the leaching temperature, the second time the leaching time and the liquid-material ratio, and the weakest interaction is the leaching temperature and the liquid-material ratio, which is not different from the result of the anova.

Example 14 response surface optimization and verification

The physical meaning of the response value in the experiment is the extraction rate of the cercis chinensis anthocyanin, so that the extraction rate reaches the maximum value by optimizing three factors, and the optimal extraction process of the cercis chinensis anthocyanin predicted by the system is shown in table 6.

Optimizing the optimized process conditions to be as follows according to the practical situation of experimental operation: the extraction time is 5min, the extraction temperature is 70 ℃, and the liquid-material ratio is 1:30 (g/mL). The predicted value of absorbance of the extract of cercis negundo anthocyanidin under this optimal extraction condition was 2.315 (table 7). Three groups of parallel extraction experiments are carried out according to the process for verification, the determined extraction content of the cercis chinensis anthocyanin is stable, the deviation is small, the average value is 2.988, and the system prediction value is superior, so that the model is reliable, and the result reliability is high.

TABLE 6

TABLE 7 methods (find 100 sets of methods)

Example 15ABTS free radical scavenging assay to determine antioxidant Activity of Cercis chinensis anthocyanin extract

Taking 2mL of ABTS working solution, measuring and recording the absorbance at the wavelength of 734nm, adding 1mL of diluted tenfold folium cercis chinensis anthocyanin extracting solution (extracted by adopting the optimized process obtained in the example 14), uniformly mixing, processing for 3min in a dark place, and measuring the absorbance at the wavelength of 734 nm. When S is ═ A₀-A)/A₀100% ABTS radical clearance (S), where A₀The absorbance of the ABTS solution is shown, and A is the absorbance of the anthocyanin extracting solution added. The results are shown in Table 8.

TABLE 8 antioxidant Activity of Cercis chinensis anthocyanin extract from optimal Process

L-ascorbic acid was prepared in a gradient of 0.01, 0.02, 0.05, 0.1g/L, and its antioxidant activity was measured by ABTS free radical scavenging test, and a standard curve of L-ascorbic acid antioxidant activity was plotted, and the results are shown in FIG. 12.

FIG. 12 shows that the antioxidant activity of 1mL of the C.maritima anthocyanin extract solution with a concentration of 1OD extracted by the optimum extraction process obtained in example 14 is 2.733mg of L-ascorbic acid.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.