CN117350600A - Comprehensive evaluation method of kudzu vine root semi-bionic enzymatic extraction process - Google Patents
Comprehensive evaluation method of kudzu vine root semi-bionic enzymatic extraction process Download PDFInfo
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Abstract
The invention discloses a comprehensive evaluation method of a kudzuvine root semi-bionic enzymatic extraction process, which relates to the field of traditional Chinese medicines and comprises the following steps of: s1: determining a factor set affecting the radix puerariae extraction process; s2: determining an evaluation set according to the factor set, wherein the evaluation set is a comprehensive score of an evaluation index; the evaluation index is the content of the main component of the kudzuvine root, and the weight in the comprehensive scoring formula is determined according to the importance of the kudzuvine root; s3: and optimizing the kudzu root extraction process according to the comprehensive score to obtain an optimal factor set. The invention adopts the semi-bionic enzymatic method to extract the pueraria isoflavone component, optimizes the semi-bionic enzymatic extraction process by taking the comprehensive score based on puerarin, soyabean glycoside and total isoflavone content as an index, has the advantages of environmental protection, low cost, mild condition and comprehensive evaluation index in the whole extraction process, is suitable for large-scale production, has higher ultrasonic extraction yield than the conventional ethanol extraction method by the semi-bionic enzymatic method, and provides a new way for efficiently extracting the effective components of the pueraria.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines, in particular to a comprehensive evaluation method of a kudzuvine root semi-bionic enzymatic extraction process.
Background
Traditional methods for extracting the pueraria isoflavone comprise a water decoction method, an alcohol heating reflux method, an ultrasonic extraction method and the like, however, compared with modern novel extraction methods such as an enzymatic method, a semi-bionic enzymatic method and the like, the traditional methods have the defects of high loss of active ingredients, lower extraction rate and the like. The semi-bionic enzymatic extraction is an emerging extraction method of plant active ingredients, and the principle simulates the process of oral administration and the transportation of medicines through gastrointestinal tracts. The plant cell wall components are subjected to enzymolysis by introducing biological enzymes such as cellulase and pectase, so that the cell wall structure is destroyed, and the release of active components is accelerated. Meanwhile, by using solvents of different pH to perform continuous extraction, an active mixture with higher content of active ingredients can be obtained, thereby improving extraction efficiency.
The semi-bionic enzymatic extraction is widely applied to extraction of plant active ingredients, has relevant research reports on extraction of active ingredients such as flavonoids, glycosides, saponins, alkaloids, phenolic acids and the like, and has certain advantages in extraction of different active ingredients. However, at present, single indexes such as total flavone yield are mostly adopted as evaluation indexes, comprehensive scores based on the mixed component yields of monomer component yields and activity are less considered for evaluation, and researches on the use of a semi-bionic enzymatic method for pueraria isoflavone component extraction are freshly reported, so that whether the semi-bionic enzymatic method can effectively extract pueraria isoflavone components and improve the in-vitro antioxidant activity of the pueraria isoflavone crude extract is not clear by taking the comprehensive scores based on puerarin, daidzin and total isoflavone contents as indexes.
Disclosure of Invention
The invention aims to at least solve one of the technical problems in the prior art and provides a comprehensive evaluation method of a kudzuvine root semi-bionic enzymatic extraction process.
The technical scheme of the invention is as follows:
a comprehensive evaluation method of a kudzuvine root semi-bionic enzymatic extraction process comprises the following steps:
s1: determining a factor set affecting the radix puerariae extraction process;
s2: determining an evaluation set according to the factor set, wherein the evaluation set is a comprehensive score of an evaluation index; the evaluation index is determined according to the content of main components in the kudzuvine root, the different main component contents have different dimension and magnitude, the different main components are subjected to dimensionless treatment, so that all data are mapped between 0 and 1, the content of each main component is compared and analyzed on the same dimension, the normalization treatment of the data is realized by adopting an index dimensionless treatment method of normalized value = original value/maximum value, and the weight in a comprehensive scoring formula is distributed according to the importance of the content of the main component of the kudzuvine root;
the comprehensive scoring formula is as follows:
comprehensive score = [ (puerarin content/puerarin maximum content of each treatment group) ×0.4+ (daidzin content/daidzin maximum content of each treatment group) ×0.2+ (total isoflavone content/total isoflavone maximum content of each treatment group) ×0.4] ×100; s3: and (5) optimizing the kudzu root extraction process according to the comprehensive score to obtain an optimal factor set.
Preferably, in step S1, the extraction process adopts a semi-bionic enzymatic method, and the factor set includes a type of biological enzyme, an enzyme dosage, an enzymolysis temperature, and a feed-liquid ratio of radix Puerariae powder to disodium hydrogen phosphate-citric acid buffer solution in the semi-bionic enzymatic method.
Preferably, the semi-bionic enzymatic method comprises the following steps:
step one: adding radix Puerariae powder into disodium hydrogen phosphate-citric acid buffer solution with pH of 2.0, mixing, reflux extracting in water bath at 50-70deg.C, and filtering;
step two: adding the filter residue obtained in the step one into disodium hydrogen phosphate-citric acid buffer solution with the pH value of 3.5-4.8, simultaneously adding biological enzyme, carrying out enzymolysis, and filtering;
step three: adding the filter residue obtained in the second step into disodium hydrogen phosphate-citric acid buffer solution with the pH of 7.5, uniformly mixing, carrying out reflux extraction in a water bath with the temperature of 50-70 ℃, and filtering to obtain filtrate; and (3) adding disodium hydrogen phosphate-citric acid buffer solution with pH of 8.3 into the filter residue again, uniformly mixing, extracting in a water bath at 50-70 ℃ under reflux, filtering, combining the filtrates obtained in the first step, the second step and the third step, and finely filtering to obtain the product.
Preferably, in step S2, the evaluation index includes puerarin, daidzin, and total isoflavone content.
Preferably, in step S3, the optimal condition of the radix Puerariae extraction process is determined according to the comprehensive score of each evaluation index, and the optimal factor set is obtained.
Preferably, the method further comprises step S4: the extract obtained by the extraction process of the optimal factor set is applied to the preparation of antioxidant active foods, health-care products or cosmetics.
The beneficial effects of the invention are as follows: the invention takes the kudzuvine root as a raw material, adopts a semi-bionic enzymatic method to extract isoflavone components, takes the comprehensive score based on the content of puerarin, daidzin and total isoflavone as an index, and optimizes the semi-bionic enzymatic extraction process; compared with the traditional ultrasonic extraction method, the content of the main isoflavone component and the in-vitro antioxidant activity obtained by the semi-bionic enzymatic extraction are greatly improved, and the in-vitro antioxidant activity is excellent; the whole extraction process has the advantages of environment friendliness, low cost, mild condition and comprehensive evaluation index, is suitable for large-scale production, provides a new way for efficient extraction of the kudzuvine root effective components, and provides theoretical basis for comprehensive development and utilization of the kudzuvine root.
Drawings
FIG. 1 is a graph showing the effect of enzyme type biological enzymes on the extraction effect of main isoflavone components of radix Puerariae; the different letters (a, b, c) in the figure indicate that there is a significant difference between the dataP＜0.05）。
FIG. 2 is a graph showing the effect of enzyme dosage on the extraction effect of main isoflavone components of kudzuvine root; the different letters (a, b, c, d, e) in the figure represent the obvious difference between the dataP< 0.05), where "de" in D in fig. 2 indicates that the data is not significantly different from both "D" and "e", i.e., there is no significant difference between the 20 mg pectinase dosage and the 10 mg and 30 mg pectinase dosage data.
FIG. 3 is a graph showing the influence of enzymolysis temperature on the extraction effect of main isoflavone components of radix Puerariae; the different letters (a, b, c, d, e) in the figure represent the obvious difference between the dataP＜0.05）。
FIG. 4 is a graph showing the effect of liquid-to-liquid ratio on the extraction effect of the main isoflavone component of radix Puerariae; in the figure, different letters (a, b, c, d, e and f) represent that there is a significant difference between dataP< 0.05), wherein "bc" in fig. 4 indicates that the data is not significantly different from both "b" and "C", i.e., feed to liquid ratio 1:10 to feed liquid ratio 1:14 and 1: there was no significant difference between 8 data.
FIG. 5 is a graph showing the relationship between the DPPH radical scavenging ability of the crude extract of Pueraria isoflavone; in the figure, different letters (a, b and c) show that obvious difference exists between different extraction modes and positive controlP＜0.05）。
FIG. 6 is a graph showing the relationship between the ability of crude extract of pueraria isoflavone to scavenge ABTS free radicals; in the figure, different letters (a, b and c) show that obvious difference exists between different extraction modes and positive controlP＜0.05）。
Detailed Description
The technical scheme of the invention is as follows: a comprehensive evaluation method of a kudzuvine root semi-bionic enzymatic extraction process comprises the following steps:
s1: determining a factor set affecting the radix puerariae extraction process;
s2: determining an evaluation set according to the factor set, wherein the evaluation set is a comprehensive score of an evaluation index; the evaluation index is determined according to the content of the main component in the kudzuvine root, and the weight in the comprehensive scoring formula is distributed according to the distribution of the content of the main component in the kudzuvine root;
s3: and (5) optimizing the kudzu root extraction process according to the comprehensive score to obtain an optimal factor set.
Preferably, in step S1, the extraction process adopts a semi-bionic enzymatic method, and the factor set includes a type of biological enzyme, an enzyme dosage, an enzymolysis temperature, and a feed-liquid ratio of radix Puerariae powder to disodium hydrogen phosphate-citric acid buffer solution in the semi-bionic enzymatic method.
Preferably, the semi-bionic enzymatic method comprises the following steps:
step one: adding radix Puerariae powder into disodium hydrogen phosphate-citric acid buffer solution with pH of 2.0, mixing, reflux extracting in water bath at 50-70deg.C, and filtering;
step two: adding the filter residue obtained in the step one into disodium hydrogen phosphate-citric acid buffer solution with the pH value of 3.5-4.8, simultaneously adding biological enzyme, carrying out enzymolysis, and filtering;
step three: adding the filter residue obtained in the second step into disodium hydrogen phosphate-citric acid buffer solution with the pH of 7.5, uniformly mixing, carrying out reflux extraction in a water bath with the temperature of 50-70 ℃, and filtering to obtain filtrate; and (3) adding disodium hydrogen phosphate-citric acid buffer solution with pH of 8.3 into the filter residue again, uniformly mixing, extracting in a water bath at 50-70 ℃ under reflux, filtering, combining the filtrates obtained in the first step, the second step and the third step, and finely filtering to obtain the product.
Preferably, in step S2, the evaluation index includes puerarin, daidzin, and total isoflavone content.
Preferably, the comprehensive scoring formula is as follows:
comprehensive score = [ (puerarin content/puerarin maximum content of each treatment group) ×0.4+ (daidzein content/daidzein maximum content of each treatment group ] ×0.2+ (total isoflavone content/total isoflavone maximum content of each treatment group) ×0.4) ×100.
Preferably, in step S3, the optimal condition of the radix Puerariae extraction process is determined according to the comprehensive score of each evaluation index, and the optimal factor set is obtained.
Specific embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The raw materials used in the following examples are as follows:
pueraria decoction pieces (lot number 2211001) were purchased from An Guoshi changda Chinese herbal decoction pieces limited; puerarin (purity 98.98%) and daidzin (purity 98.77%) were purchased from Chengdu Angstrom Biotechnology Co., ltd; cellulase (enzyme activity 100000U/g), pectase (enzyme activity 100000U/g) were purchased from Ningxia and the wall biotechnology company, inc.; disodium hydrogen phosphate dodecahydrate (AR), citric acid monohydrate (AR) was purchased from the company of the sciences of the schlempe; phosphoric acid (chromatographic grade) was purchased from Shanghai Meilin Biochemical technologies Co., ltd; acetonitrile (chromatographic grade) was purchased from Thermo Fisher Scientific company; DPPH free radical scavenging ability detection kit and ABTS free radical scavenging ability detection kit are purchased from Beijing Soy Bao technology Co.
The purity is specifically a peak area ratio of the peak area of the peak in the entire chromatogram.
The instruments and equipment used in the following examples are as follows:
acquity H-Class Plus ultra-high performance liquid chromatography (Waters), CORTECS UPLC C18 column (Waters, 2.1 mm ×150 mm,1.6 μm), UV-1900i UV-visible spectrophotometer (Shimadzu), HWS-26 electrothermal thermostatic waterbath (Shanghai-Hemsleyakulare Co., ltd.), DFT-200A hand high speed pulverizer (Wen Lingshi Lin big machinery Co., ltd.), milli-Q IQ7005 pure water/ultra-pure water all-in-one system (Millipore Co., ltd.), S220 pH meter, XSR105 ten parts per million balance, ME303 thousandth balance (Metrer-Toli group).
The following test involves the following analysis methods:
the mean, error and composite scores were calculated using Microsoft Excel, the significance analysis (Duncan) using IBM SPSS 26, and the plot using Origin 2022.
Example 1
Determining a factor set influencing the radix puerariae extraction process, wherein the extraction process comprises the following steps of:
according to the bionics principle, the pH value of the body fluid of human stomach, small intestine and large intestine is 2.0,7.5 and 8.3.
Weighing radix Puerariae powder 5.0. 5.0 g, placing in conical flask, adding 60 mL disodium hydrogen phosphate-citric acid buffer solution with pH of 2.0, shaking sufficiently to form uniform phase, placing in water bath at 60deg.C, reflux extracting for 0.5 h, filtering, and retaining filtrate; adding disodium hydrogen phosphate-citric acid buffer solution 60 mL with pH of 3.5 into the filter residue, adding 50 mg biological enzyme, sufficiently and uniformly shaking, performing enzymolysis at 50deg.C for 1 h, filtering, and retaining filtrate; sequentially adding the filter residues into disodium hydrogen phosphate-citric acid buffer solutions with pH values of 7.5 and 8.3, respectively extracting under reflux in water bath at 60 ℃ for 0.5 h, respectively retaining the filtrate; mixing the above 4 filtrates, constant volume with distilled water to 250 mL, and filtering with 0.22 μm filter membrane.
And (3) respectively carrying out single-factor optimization tests on biological enzyme types (cellulase, pectase and complex enzyme (the cellulase and the pectase are compounded according to the mass ratio of 1:1)), enzyme dosage (10, 20, 30, 40, 50, 60 and mg), enzymolysis temperature (35, 40, 45, 50, 55 and 60 ℃), and the feed liquid ratio (1:6, 1:8, 1:10, 1:12, 1:14 and 1:16 g/mL) of kudzuvine root powder and buffer solution, and observing the influence of the four factors (factor set) on the extraction effect of main isoflavone components of kudzuvine root.
The puerarin, the daidzin and the total isoflavone content in the kudzuvine root are used as evaluation indexes, the comprehensive score of each test is calculated, and then the extraction effect of the semi-bionic enzyme method on the main isoflavone components of the kudzuvine root is examined, wherein the comprehensive score is = [ (puerarin content/puerarin maximum content obtained by each treatment group) ×0.4+ (daidzin content/daidzin maximum content obtained by each treatment group ] ×0.2+ (total isoflavone content/total isoflavone maximum content) ×0.4) ×100.
The method comprises the steps of obtaining an index with different dimensions, wherein in order to eliminate the influence of the evaluation index on the index, the index is comparable, and the normalization processing of data is realized by adopting a common index dimensionless processing method of 'normalized value=original value/maximum value'; the weight is determined according to the importance of puerarin, soyasaponin and total isoflavone in the radix puerariae, specifically, the isoflavone component occupies the main part in the radix puerariae, the content of the isoflavone component is highest in the existing plants, and the total mass fraction of the pueraria lobata isoflavone can reach 6.2% -17.0%. Puerarin, daidzein and the like are main active ingredients in kudzuvine root, and isoflavone ingredients with high content of puerarin, daidzein and the like are used for evaluating the quality of the kudzuvine root. Puerarin is used as a marker for evaluating the quality of kudzuvine root, the content of the puerarin accounts for a large proportion in the isoflavone of kudzuvine root, the mass fraction of the puerarin reaches 1.58% -7.68%, and the mass fraction of the puerarin in the kudzuvine root raw material is definitely specified in the 2020 edition of Chinese pharmacopoeia is not lower than 2.4%. Based on the above description, weights of puerarin, daidzin and total isoflavone were given to 0.4, 0.2 and 0.4, respectively.
The test method is as follows:
(1) Method for measuring content of puerarin and daidzin
Determining puerarin and soyabean glycoside content in radix Puerariae extract by UPLC method, and performing chromatographic conditions: CORTECS UPLC C18 chromatographic column (2.1 mm ×150 mm,1.6 μm), mobile phase acetonitrile and 0.1% (v/v) phosphoric acid solution, detection wavelength 254 nm, flow rate 0.2 mL/min, column temperature 35 ℃, sample injection amount 1. Mu.L, gradient elution procedure as shown in Table 1:
TABLE 1 gradient elution procedure
| Time (min) | Acetonitrile (%) | 0.1% phosphoric acid solution (%) |
| 0 | 10 | 90 |
| 10 | 10 | 90 |
| 16 | 20 | 80 |
| 20 | 25 | 75 |
| 25 | 35 | 65 |
| 29 | 80 | 20 |
| 30 | 10 | 90 |
| 40 | 10 | 90 |
Note that: % is volume percent.
The content of puerarin and daidzin in the puerarin extract is determined by standard curve method, and the standard curve regression equation of puerarin is y= 1.180914 ×10 7 x+1.202859×10 6 ，R 2 Linear range = 0.9990140.6-3.0 mg/mL; standard curve regression equation of daidzin is y= 3.923131 ×10 7 x-1.044829×10 6 ，R 2 = 0.994212, linear range 0.06 to 0.3 mg/mL.
(2) Method for measuring total isoflavone content
The standard puerarin is used as a reference substance, and the direct ultraviolet spectrophotometry is adopted for determination. Precisely sucking the reference substance, and diluting with 30% (v/v) ethanol solution to 0.01, 0.02, 0.03, 0.04, 0.05 mg/mL to obtain puerarin standard solution. Taking 30% (v/v) ethanol solution as blank control, measuring absorbance at 250 nm, calculating to obtain total isoflavone content in radix Puerariae extract according to standard curve regression equation, wherein the standard curve regression equation of total isoflavone is y=43.3740x+0.03253120,R 2 = 0.99946, linear range 0.01 to 0.05 mg/mL.
The single factor optimization test results are as follows:
(1) The results of the biological enzyme type test are shown in figure 1, and the cellulase and the pectase are widely used in the extraction of flavonoid and isoflavone components, and can decompose the components of plant cell walls, such as cellulose, pectin and the like, so as to destroy the cell structure, so that the total flavonoid and the total isoflavone components are easier to release, and the extraction efficiency is improved. In this example, the effect of cellulase, pectase and its complex enzyme on the extraction of main isoflavone components of radix Puerariae was examined. As can be seen from FIG. 1, the puerarin content obtained by the treatment of the cellulase and the pectase is significantly higher than that of the compound enzyme treatment group of the cellulase and the pectasePLess than 0.05), and the content of the soyasaponin obtained by pectase treatment is obviously higher than that of the cellulose and complex enzyme treatment groupP< 0.05). In addition, the content of the total isoflavone obtained by the cellulase treatment is also obviously higher than that of the pectinase and compound enzyme treatment groupP< 0.05). The results based on the composite score (D in FIG. 1) showed that the pectinase treatment group was optimal, the cellulase treatment group was inferior, and the complex enzyme treatment group was the worst.
(2) The screening test result of the biological enzyme dosage is shown in fig. 2, and as shown in fig. 2, the puerarin content tends to be stable after increasing along with the increase of the enzyme dosage, and reaches the maximum value when the enzyme dosage is 50 and mg; the content of the soyabean glycoside is increased and then reduced along with the increase of the enzyme dosage, and the maximum value is reached when the enzyme dosage is 50 and mg; the total isoflavone content also tended to increase and decrease with increasing enzyme usage, reaching a maximum at enzyme usage 40 mg. These trends may occur because at low concentrations, the efficiency with which the cell walls of kudzuvine root are decomposed gradually increases with increasing amounts of pectinase, thereby allowing more kudzuvine root isoflavone components to be eluted; however, when the amount of pectase is too high, part of the enzyme and decomposed pectin, cellulose and the like adhere to the surface of the kudzuvine root powder, which is not conducive to dissolution of the active ingredients, and further the content of the extracted kudzuvine root isoflavone is reduced. According to the result of the comprehensive score (D in FIG. 2), the comprehensive score was highest when the pectase amount was 50 mg, and the effect of extracting the main isoflavone component from the radix Puerariae by the enzyme amount was best, so that three levels of enzyme amounts 40 mg, 50 mg and 60 mg were selected for orthogonal experiment optimization.
(3) Referring to FIG. 3, it can be seen from FIG. 3 that the puerarin content, the daidzein content and the total isoflavone content show a tendency of increasing and then decreasing with increasing of the enzymolysis temperature, and reach maximum values at the enzymolysis temperature of 55 ℃, 40 ℃ and 55 ℃ respectively. At a lower temperature, the thermal movement of molecules can be accelerated along with the rise of the temperature, and simultaneously the activity of pectase is increased along with the rise of the temperature, so that the dissolution of the pueraria isoflavone component is promoted; however, when the temperature is too high, the pectase activity decreases, the efficiency of cell wall decomposition decreases, resulting in a decrease in the release rate of the active ingredient, and the high temperature may damage the structure of the isoflavone ingredient, resulting in a decrease in the content thereof. According to the result of the comprehensive score (D in FIG. 3), the highest comprehensive score is obtained at 55 ℃, and the effect of extracting the main isoflavone component of the kudzuvine root at the enzymolysis temperature is best, so that three levels of 50 ℃, 55 ℃ and 60 ℃ of the enzymolysis temperature are selected in the orthogonal test to further optimize the extraction conditions.
(4) Referring to FIG. 4, as can be seen from FIG. 4, the puerarin content, the daidzein content and the total isoflavone content basically show a trend of increasing and then decreasing with decreasing feed liquid ratio, and reach maximum values when the feed liquid ratio is 1:14 g/mL, 1:14 g/mL and 1:12 g/mL respectively. When the feed liquid is relatively large, the contact area of the kudzuvine root particles and the solvent is small, and part of isoflavone components are not dissolved out effectively; along with the continuous reduction of the feed-liquid ratio, the concentration of the substrate is reduced, the combination of enzyme and the substrate is not facilitated, and the dissolution of isoflavone components is further reduced. According to the result of the comprehensive score (D in FIG. 4), the comprehensive score is highest when the feed-liquid ratio is 1:14 g/mL, and the effect of extracting the main isoflavone component of the kudzuvine root by the feed-liquid ratio is best at the moment, so that three levels of 1:12 g/mL, 1:14 g/mL and 1:16 g/mL of the feed-liquid ratio are selected for orthogonal experiment optimization.
According to the single factor optimization test result, selecting proper level for each factor, adopting orthogonal table (see table 2) to develop L 9 (3 4 ) And (5) carrying out an orthogonal test, and obtaining the optimal extraction process of the main isoflavone components of the kudzuvine root according to the comprehensive score.
TABLE 2 level of orthogonal test factors
| Horizontal level | A pectase dosage (mg) | B enzymolysis temperature (. Degree. C.) | C feed liquid ratio (g/mL) | D blank |
| 1 | 40 | 50 | 1:12 | / |
| 2 | 50 | 55 | 1:14 | / |
| 3 | 60 | 60 | 1:16 | / |
Based on the single factor test result, carrying out orthogonal test on 3 factors of pectase dosage (A), enzymolysis temperature (B) and feed liquid ratio (C), wherein a blank column is set as a factor D. The content of the main isoflavone components of the radix puerariae extracted by the 9 groups of tests is shown in table 3, and the comprehensive scores of the main isoflavone components are calculated to obtain the orthogonal test results (table 4). From the result of the range analysis (K 1 、K 2 、K 3 ) The order of influence of 3 factors on the composite score is known as follows: enzymolysis temperature>Feed-to-liquid ratio>Pectase dosage, optimal extraction process combination is A 2 B 2 C 2 (not among the 9 combinations in Table 3), i.e., enzyme dosage 50 mg, enzymatic hydrolysis temperature 55 ℃, feed-to-liquid ratio 1:14 g/mL. Therefore, 3 repeated verification tests are carried out on the optimal combination, the puerarin content is 63.287 +/-0.220 mg/g, the daidzin content is 5.177 +/-0.047 mg/g, the total isoflavone content is 221.583 +/-0.079 mg/g, the comprehensive score is 98.887, the comprehensive score is higher than that of each test number in the table 3, the optimal process is verified to obtain a result higher than that of 9 groups of orthogonal test results, the process condition is stable and reliable, and the comprehensive evaluation method is feasible.
TABLE 3 extraction of major isoflavone content and comprehensive score from Pueraria lobata by orthogonal test
| Test number | Puerarin content (mg/g) | Soy glycoside content (mg/g) | Total isoflavone content (mg/g) | Comprehensive scoring |
| 1 | 59.700 | 4.271 | 209.414 | 90.941 |
| 2 | 63.404 | 4.802 | 221.652 | 97.499 |
| 3 | 56.694 | 4.658 | 212.936 | 91.180 |
| 4 | 63.228 | 4.759 | 221.445 | 97.182 |
| 5 | 63.349 | 5.085 | 213.163 | 97.097 |
| 6 | 56.066 | 4.242 | 219.605 | 90.310 |
| 7 | 62.744 | 4.908 | 213.220 | 96.031 |
| 8 | 60.620 | 4.443 | 229.633 | 95.718 |
| 9 | 58.044 | 4.549 | 209.937 | 91.078 |
TABLE 4 orthogonal test results of the extraction Process of the major isoflavone component of Pueraria lobata
| Test number | A pectase dosage (mg) | B enzymolysis temperature (. Degree. C.) | C feed liquid ratio (g/mL) | D blank | Comprehensive scoring |
| 1 | 1 | 1 | 1 | 1 | 90.941 |
| 2 | 1 | 2 | 2 | 2 | 97.499 |
| 3 | 1 | 3 | 3 | 3 | 91.180 |
| 4 | 2 | 1 | 2 | 3 | 97.182 |
| 5 | 2 | 2 | 3 | 1 | 97.097 |
| 6 | 2 | 3 | 1 | 2 | 90.310 |
| 7 | 3 | 1 | 3 | 2 | 96.031 |
| 8 | 3 | 2 | 1 | 3 | 95.718 |
| 9 | 3 | 3 | 2 | 1 | 91.078 |
| K 1 | 279.620 | 284.155 | 276.970 | 279.116 | |
| K 2 | 284.589 | 290.314 | 285.760 | 283.840 | |
| K 3 | 282.828 | 272.568 | 284.307 | 284.080 | |
| k 1 | 93.207 | 94.718 | 92.323 | 93.039 | |
| k 2 | 94.863 | 96.771 | 95.253 | 94.613 | |
| k 3 | 94.276 | 90.856 | 94.769 | 94.693 | |
| R | 1.656 | 5.915 | 2.930 | 1.655 |
Comparative example 1
The kudzuvine root powder is extracted by adopting an agricultural industry standard NY/T4307-2023 high performance liquid chromatography-tandem mass spectrometry for measuring flavonoid compounds in kudzuvine root. Weighing radix Puerariae powder 0.5. 0.5 g, adding 25 mL 30% (v/v) ethanol solution, ultrasonically extracting at room temperature for 45 min, centrifuging, and collecting supernatant. Washing the precipitate with 30% (v/v) ethanol solution for 2 times, 5-mL each time, centrifuging, mixing the supernatants, fixing volume of 30% (v/v) ethanol solution to 50-mL, and filtering with 0.22 μm filter membrane.
While for the optimal conditions (A) in example 1 2 B 2 C 2 ) The prepared samples and comparative example 1 were subjected to the main isoflavone content and comprehensive score test, and the test results are shown in Table 5.
TABLE 5 content of major isoflavones of Pueraria lobata by different extraction methods and comprehensive score
| Extraction mode | Puerarin content (mg/g) | Soy glycoside content (mg/g) | Total isoflavone content (mg/g) | Comprehensive scoring |
| Semi-bionic-enzyme extraction method | 63.287±0.220a | 5.177±0.047a | 221.583±0.079a | 98.887 |
| Ultrasonic extraction method of 30% ethanol | 61.740±0.072b | 4.528±0.018b | 209.233±0.319b | 93.206 |
Note that: in the same column, different letters (a, b) represent that there is a significant difference between the dataP＜0.05）。
As is clear from Table 5, the puerarin, the daidzin and the total isoflavone which are obtained by 30% (v/v) ethanol ultrasonic extraction method have the contents of 61.740 +/-0.072 mg/g, 4.528 +/-0.018 mg/g and 209.233 +/-0.319 mg/g respectively, and the comprehensive score is 93.206, which is obviously lower than that of the extraction by the semi-bionic enzymatic methodPLess than 0.05), the extraction effect of the semi-bionic enzymatic method for extracting the main isoflavone components of the kudzuvine root is better than that of a 30% (v/v) ethanol ultrasonic extraction method.
Example 2: application of pueraria isoflavone crude extract in-vitro antioxidation activity
Under the optimum conditions (A) 2 B 2 C 2 ) Extracting radix Puerariae with 30% (v/v) ethanol under ultrasonic, concentrating the extractive solution in rotary evaporator at 60deg.C, concentrating to a certain volume, adding 250 mL 70% (v/v) ethanol, and precipitating with ethanol at 4deg.C in refrigerator for 6 h. After the alcohol precipitation is finished, placing the mixture into a 8000 rpm centrifuge for centrifugation at room temperature for 10 min, taking supernatant, concentrating and freeze-drying to obtain a semi-bionic enzymatic extraction and 30% (v/v) ethanol ultrasonic extraction of the crude pueraria isoflavone extract respectively for later use.
(1) Method for measuring DPPH free radical scavenging ability
According to the experimental method of the DPPH free radical scavenging capacity detection kit instruction, respectively preparing semi-bionic enzymatic extraction with mass concentration of 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL and coarse extraction liquid of pueraria isoflavone extracted by 30% (v/v) ethanol ultrasonic extraction, respectively taking 10 mu L of the coarse extraction liquid and 190 mu L of DPPH working liquid, mixing, shaking uniformly, and taking the mixture as a sample group. The blank group uses the same amount of the self-contained extracting solution of the kit to replace the coarse extracting solution of the pueraria isoflavone, the control group uses the same amount of absolute ethyl alcohol to replace the DPPH working solution, and the positive control group uses the Vc solution with the same mass concentration to replace the coarse extracting solution of the pueraria isoflavone. Mixing, shaking, standing at room temperature in dark for 30 min, and measuring absorbance at 515 and nm.
DPPH radical scavenging Rate of sample group (%)；
DPPH radical clearance of the positive control group (%)；
Wherein: a is that 0 Absorbance for the blank group; a is that 1 Absorbance for the sample group; a is that 2 Absorbance for control group; a is that 3 Absorbance was used as the positive control.
The test result is shown in fig. 5, the dpph free radical is a relatively safe, stable and cheap free radical reagent, plays a role according to an in vivo free radical stabilization mechanism, is one of important indexes for measuring the antioxidant capacity of a sample, and is widely used in the research of antioxidant foods, medicines and health care products. As can be seen from FIG. 5The scavenging ability of the coarse pueraria isoflavone extract obtained by semi-bionic enzymatic extraction, 30% (v/v) ethanol ultrasonic extraction and Vc positive control on DPPH free radicals is enhanced along with the increase of mass concentration within the concentration range of 0.2-3.2 mg/mL, and reaches the maximum value when the mass concentration is 3.2 mg/mL, and the DPPH free radical scavenging rates are 63.51%, 60.31% and 93.26%, respectively, and IC 50 The values are 1.538 mg/mL, 2.409 mg/mL and 0.2830 mg/mL respectively, and the DPPH free radical scavenging capability of the crude extract of the pueraria isoflavone obtained by the semi-bionic-enzyme extraction method is obviously higher than that of the crude extract of the pueraria isoflavone obtained by ultrasonic extraction of 30 percent ethanolP< 0.05), but significantly lower than the positive control VcP< 0.05). Wei Lei et al (Wei Lei, wang Wei, houying, et al, research on the optimization of extraction process and antibacterial and antioxidant ability of flavonoids of Ge Shezong [ J ]]Indonesia agriculture science, 2021, 49 (14): 152-161.) ultrasonic extraction of Ge Shezong flavone with 85% (v/v) ethanol, and its extract has IC with DPPH free radical scavenging ability 50 The value is 1.58 mg/mL, and the specific method is as follows: weigh 1.00 g Ge Shefen in a 100 mL conical flask according to a liquid to material ratio of 48:1 (mL/g) is added with 85% (v/v) ethanol solution, ultrasonic extraction is carried out for 90min at 73 ℃, the ultrasonic extraction is repeated for 3 times, the filtration is carried out after the filtration is combined, 2 times of volume of absolute ethanol is added after concentration, the mixture is left standing overnight at low temperature to remove most of polysaccharide, and Ge Shecu flavone is obtained after vacuum freeze drying for measuring the DPPH free radical scavenging effect. Wu Di et al (Wu Di, liu Pingping, li Meng, et al. Evaluation of in vitro antioxidant and antiaging effects of aqueous extract of Pueraria lobata and fermentation broth of Pueraria lobata [ J ]]Research of food industry science and technology, 2019, 40 (12): 285-294.) shows that the aqueous extract of radix Puerariae has IC for scavenging DPPH free radical 50 The value is 3.51 mg/mL, which is higher than that of the semi-bionic-enzyme extraction method in the invention, and the specific method in the document is as follows: selecting 50-mesh radix Puerariae powder 20 g, adding deionized water 300 mL according to a feed-liquid ratio of 1:15 (g/mL), extracting 3 h in a 70 ℃ water bath shaking table, cooling, centrifuging at 4900 r/min for 10 min, and collecting supernatant for measuring DPPH free radical scavenging effect. The method shows that the coarse pueraria isoflavone extract obtained by the optimized semi-bionic enzymatic extraction method has stronger DPPH free radical scavenging effect.
(2) Method for measuring ABTS free radical scavenging ability
According to the experimental method of the specification of the ABTS free radical scavenging capacity detection kit, respectively preparing semi-bionic enzymatic extraction with mass concentration of 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL and crude pueraria isoflavone extraction liquid with 30% (v/v) ethanol ultrasonic extraction, respectively taking 10 mu L and 170 mu L of ABTS working liquid and 20 mu L of reagent-carrying working liquid of the kit, mixing uniformly, and taking the mixture as a sample group. The blank group is characterized in that the equivalent distilled water is used for replacing the crude extract of the pueraria isoflavone, the equivalent reagent-carrying agent of the kit is used for replacing the ABTS working solution and the reagent-carrying agent of the kit is used for replacing the four working solutions of the kit, and the positive control group is used for replacing the Vc solution with the equivalent mass concentration for replacing the crude extract of the pueraria isoflavone. Mixing, shaking, standing at room temperature in dark for 6 min, and measuring absorbance at 405 and nm.
Sample group ABTS radical clearance (%)；
Positive control group ABTS radical clearance (%)；
Wherein: a is that 0 Absorbance for the blank group; a is that 1 Absorbance for the sample group; a is that 2 Absorbance for control group; a is that 3 Absorbance was used as the positive control.
As shown in FIG. 6, in the concentration range of 0.2-3.2 mg/mL, the scavenging ability of the crude extract of pueraria isoflavone obtained by semi-bionic enzymatic extraction and 30% (v/v) ethanol ultrasonic extraction in terms of resisting ABTS free radicals is enhanced along with the increase of mass concentration, and reaches the maximum value when the mass concentration is 3.2 mg/mL, wherein the scavenging rate of DPPH free radicals is 69.16% and 68.41%, and the scavenging rate of IC is respectively 50 Values 0.7486 mg and 0.9927 mg/mL, respectively; the Vc positive control has the ABTS free radical scavenging rate rapidly increased within the concentration range of 0.2-0.4 mg/mL, and then the ABTS free radical scavenging rate reaches 90.01% at the time of 3.2 mg/mL along with the increase of Vc mass concentration, and the ABTS free radical scavenging capability of the crude extract of the pueraria isoflavone obtained by the semi-bionic-enzyme extraction method is higher than 30% (v/v) ethanol ultrasonicThe extraction is obviously lower than the positive control VcP< 0.05). In addition, semi-bionic-enzyme extraction method is used for IC with ABTS free radical scavenging capability 50 The value 0.7486 mg/mL is lower than Wei Lei (Wei Lei, wang Wei, houying, et al, ge Shezong, study on the antibacterial and antioxidant capacity of flavone]Ge Shezong flavone IC measured by Anhui agricultural science, 2021, 49 (14): 152-161.) 50 The value of 0.86 mg/mL indicates that the crude extract of the pueraria isoflavone obtained by the semi-bionic enzyme extraction method has stronger ABTS free radical scavenging effect.
From the analysis, the invention carries out comprehensive research on the in-vitro antioxidant activity of the pueraria isoflavone crude extract by the extraction process for extracting the pueraria main isoflavone by the semi-bionic enzymatic method. The comprehensive score based on puerarin, daidzin and total isoflavone content is used as an index, the defect that in the traditional mode, single total flavone yield is used as an evaluation index is overcome, the known flavone is nearly ten thousand, and can be subdivided into flavone, flavonols, dihydroflavonols, isoflavones, dihydroisoflavones, flavan-3-ols, flavan-3, 4-diols, anthocyanidins, orange ketones, chalcones, dihydrochalcones and the like according to the parent nuclear structure and the conformation of the known flavone. Through single factor and orthogonal tests, the optimal extraction process condition of the main isoflavone of the kudzuvine root is that the dosage of pectase is 50 mg, the enzymolysis temperature is 55 ℃, and the feed-liquid ratio is 1:14 g/mL. Under the condition, the puerarin content is 63.287 mg/g, the daidzein content is 5.177 mg/g, the total isoflavone content is 221.583 mg/g, and the calculated comprehensive score is 98.887 minutes, which is obviously higher than that of the traditional 30% (v/v) ethanol ultrasonic extraction methodP< 0.05). The in vitro antioxidant activity research results show that the crude extract of the pueraria isoflavone extracted by the semi-bionic enzymatic method has better scavenging ability to DPPH and ABTS free radicals, and when the mass concentration is 3.2 mg/mL, the scavenging rate of DPPH and ABTS free radicals is 63.51 percent and 69.16 percent respectively, and IC is high 50 The values are 1.538 mg/mL and 0.7486 mg/mL respectively, which are obviously superior to the traditional 30% (v/v) ethanol ultrasonic extraction method. The invention shows that the semi-bionic-enzyme extraction method has certain advantages in the extraction of the main isoflavone of the kudzuvine root, and the crude extract of the isoflavone of the kudzuvine root has stronger antioxidant activity and is the kudzuvine rootFurther development and utilization of antioxidant products provides a reference. The foregoing examples have shown only the preferred embodiments of the invention, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be pointed out that various other corresponding changes and modifications can be made by those skilled in the art in light of the above description of the technical solution and the idea, and all such changes and modifications are intended to be within the scope of the invention as defined in the appended claims.
Claims (6)
1. A comprehensive evaluation method of a kudzuvine root semi-bionic enzymatic extraction process is characterized by comprising the following steps:
s1: determining a factor set affecting the radix puerariae extraction process;
s2: determining an evaluation set according to the factor set, wherein the evaluation set is a comprehensive score of an evaluation index; the evaluation index is determined according to the content of main components in the kudzuvine root, the different main component contents have different dimension and magnitude, the different main components are subjected to dimensionless treatment, so that all data are mapped between 0 and 1, the content of each main component is compared and analyzed on the same dimension, the normalization treatment of the data is realized by adopting an index dimensionless treatment method of normalized value = original value/maximum value, and the weight in a comprehensive scoring formula is distributed according to the importance of the content of the main component of the kudzuvine root;
the comprehensive scoring formula is as follows:
comprehensive score = [ (puerarin content/puerarin maximum content of each treatment group) ×0.4+ (daidzin content/daidzin maximum content of each treatment group) ×0.2+ (total isoflavone content/total isoflavone maximum content of each treatment group) ×0.4] ×100;
s3: and (5) optimizing the kudzu root extraction process according to the comprehensive score to obtain an optimal factor set.
2. The comprehensive evaluation method of the extraction process of the kudzuvine root semi-bionic enzymatic method according to claim 1, wherein in the step S1, the extraction process adopts the semi-bionic enzymatic method, and the factor set comprises the type of biological enzyme, the enzyme dosage, the enzymolysis temperature and the feed-liquid ratio of kudzuvine root powder to disodium hydrogen phosphate-citric acid buffer solution in the semi-bionic enzymatic method.
3. The comprehensive evaluation method of a kudzuvine root semi-bionic enzymatic extraction process according to claim 2, wherein the semi-bionic enzymatic method comprises the following steps:
step one: adding radix Puerariae powder into disodium hydrogen phosphate-citric acid buffer solution with pH of 2.0, mixing, reflux extracting in water bath at 50-70deg.C, and filtering;
step two: adding the filter residue obtained in the step one into disodium hydrogen phosphate-citric acid buffer solution with the pH value of 3.5-4.8, simultaneously adding biological enzyme, carrying out enzymolysis, and filtering;
step three: adding the filter residue obtained in the second step into disodium hydrogen phosphate-citric acid buffer solution with the pH of 7.5, uniformly mixing, carrying out reflux extraction in a water bath with the temperature of 50-70 ℃, and filtering to obtain filtrate; and (3) adding disodium hydrogen phosphate-citric acid buffer solution with pH of 8.3 into the filter residue again, uniformly mixing, extracting in a water bath at 50-70 ℃ under reflux, filtering, combining the filtrates obtained in the first step, the second step and the third step, and finely filtering to obtain the product.
4. The comprehensive evaluation method of a semi-bionic enzymatic extraction process of radix Puerariae according to claim 1, wherein in step S2, the evaluation index comprises puerarin, daidzein and total isoflavone content.
5. The comprehensive evaluation method of the kudzuvine root semi-bionic enzymatic extraction process according to claim 1, wherein in the step S3, the optimal condition of the kudzuvine root extraction process is determined according to the comprehensive score of each evaluation index, and the optimal factor set is obtained.
6. The comprehensive evaluation method of the kudzuvine root semi-bionic enzymatic extraction process according to claim 1, further comprising the step of S4: the extract obtained by the extraction process of the optimal factor set is applied to the preparation of antioxidant active foods, health-care products or cosmetics.
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