Color-based rapid grade division method for radix tetrastigme

Abstract

The invention discloses a color-based radix tetrastigme rapid grading method, which comprises the following steps: collecting different batches of radix tetrastigme samples, obtaining the component content and color data of radix tetrastigme, determining weight coefficients corresponding to indexes by using an AHP-entropy weight method according to the contents of total flavonoids, total polysaccharides and total phenolic acids, and obtaining a calculation formula of radix tetrastigme grading. Tetrastigma hemsleyanum Diels et Gilg is classified, Tetrastigma hemsleyanum Diels et Gilg of each grade is selected for drug effect difference verification, the drug effect result presents the same trend as grade division, and the drug effect of the grade I is obviously superior to that of the grade II and the grade III And comprehensively analyzing the measured data L *, a *, b * and E * ab of the colorimeter and the grading result to obtain a non-standardized canonical discrimination function taking color information as a variable, so that the grade of the radix tetrastigme can be discriminated based on the color information, rapid grade division of the radix tetrastigme is facilitated, and preliminary fuzzy evaluation is performed on the efficacy of the radix tetrastigme through the color information.

Description

Technical Field

[0001] The invention relates to the technical field of quality control of cloverleaf green, and in particular to a color-based cloverleaf green rapid grading method. Background Art

[0002] CN113720931A discloses a quality evaluation method for the medicinal material of Sanyeqing, using the content of total flavonoids in Sanyeqing; the relative content of rutin, quercetin and kaempferol in total flavonoids; the characteristic peaks in the fingerprint of Sanyeqing total flavonoids are used to perform the main component comprehensive scoring; the proliferation inhibition rate of Sanyeqing total flavonoids on pancreatic cancer cells; the apoptosis rate of pancreatic cancer cells by Sanyeqing total flavonoids are five indicators to evaluate the quality of Sanyeqing, but the patent needs to be implemented by high-performance liquid chromatography, cell anti-proliferation experiments, etc., which is complicated to operate, costly, time-consuming, and difficult to apply in the circulation of Sanyeqing market, and the practicality is poor. At the same time, the scoring is mainly based on the total flavonoids of Sanyeqing, ignoring other important active ingredients such as polysaccharides and phenolic acids in Sanyeqing.

[0003] Total flavonoids, total polysaccharides, and total phenolic acids are the three major important chemical components of Tripterygium wilfordii. Tripterygium wilfordii is extremely rich in flavonoid compounds, with more than 50 flavonoid compounds identified in the tuberous roots and aerial parts, mainly including flavonoids, flavonoid glycosides, dihydroflavonoids, etc. Tripterygium wilfordii polysaccharides have great clinical application potential due to their unique pharmacological activity, and have pharmacological activities such as antioxidant and lipid-regulating activities. Tripterygium wilfordii contains a variety of special and complex phenolic acids, which have anti-tumor, antibacterial, anti-inflammatory, liver-protecting, and lipid-lowering effects.

[0004] The percentage of the epidermis of Sanyeqing and the drying method during the processing process have an impact on the color of Sanyeqing, and also have a great impact on the content of effective substances in Sanyeqing. Therefore, color can be used to quickly evaluate Sanyeqing. Compared with traditional methods, the rapid grading of Sanyeqing by color is non-destructive and efficient, and can play an important role in the processing and procurement of medicinal materials. Summary of the invention

[0005] The present invention aims to address the deficiencies of the prior art and to propose a color-based rapid grading method for Tripterygium wilfordii.

[0006] The object of the present invention is achieved by the following technical scheme: The technical scheme adopted by the present invention comprises the following steps: a color-based rapid grading method for clover, the method comprising a component-based grading method and a non-standardized canonical discriminant function with color information as a variable;

[0007] The grading method calculates the weight scores of multiple batches of Tripterygium wilfordii samples to grade Tripterygium wilfordii, associates the graded grades with their colors, constructs a non-standardized canonical discriminant function with color information as a variable, and implements rapid grading of Tripterygium wilfordii based on the constructed function.

[0008] Furthermore, the contents of flavonoids, polysaccharides and phenolic acids in multiple batches of Tripterygium wilfordii samples were determined; the NaNO2-Al(NO3)3-NaOH method was used to measure the absorbance at 510 nm to obtain the flavonoid content, the sulfuric acid-phenol method was used to measure the absorbance at 485 nm to obtain the polysaccharide content, and the Folin-phenol method was used to measure the absorbance at 750 nm to obtain the phenolic acid content.

[0009] Furthermore, the weight indexes corresponding to the three indicators of total flavonoids content, total polysaccharides content and total phenolic acid content were determined by the AHP-entropy weight method, and the calculation formula was obtained: score = 0.3573*total flavonoids content + 0.5651*total polysaccharides content + 0.0775*total phenolic acid content.

[0010] Furthermore, the calculation results of the components determined by the AHP-entropy weight method were graded so that the calculation results of the components were normally distributed and the pharmacodynamics of different grades showed obvious differences.

[0011] Furthermore, based on the scores, the three-leaf green was divided into grades I, II, and III, with scores <15 being graded as III, 15-30 being graded as II, and >30 being graded as I.

[0012] Furthermore, the collinearity analysis was performed on the grade classification results and the color information. The variables with collinearity statistics greater than 10 were removed, and the remaining variables were subjected to discriminant analysis to obtain the non-standardized canonical discriminant function for color information and grades.

[0013] Furthermore, the unstandardized canonical discriminant function with color information as a variable is y=-0.015E*ab+1.378a*+0.287b*-10.148.

[0014] Furthermore, the non-standardized canonical discriminant function outputs a corresponding y value. When the y value is close to 1, the grade of the three-leaf green is grade I; when the y value is close to 2, the grade of the three-leaf green is grade II; when the y value is close to 3, the grade of the three-leaf green is grade III. Beneficial effects of the present invention: The present invention provides a color-based rapid grading method for three-leaf green. First, the three-leaf green samples are scored and graded by including component content indicators related to efficacy, and then the grading results are associated with color information to obtain a non-standardized canonical discriminant function y=-0.015E*ab+1.378a*+0.287b*-10.148 about the grade. The grade of three-leaf green can be discriminated based on color information. This method is conducive to the construction of the three-leaf green quality control system and the promotion and application of three-leaf green. Compared with traditional methods, the rapid grading of three-leaf green by color is non-destructive and efficient, and can play an important role in the fields of medicinal material preparation and procurement. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 These are the Oil Red O staining effects of each group.

[0017] Figure 2 Schematic diagram of AST (A) and ALT (B) levels in each group. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0019] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are conventional products that can be obtained commercially without specifying the manufacturer. The reagents and raw materials used in the following examples can be obtained commercially.

[0020] Example 1

[0021] Sample collection of Sanyeqing: Collect dried tubers of Sanyeqing from different batches, crush them, and pass through a No. 4 sieve. The origins include Zhejiang, Jiangxi, Fujian, Yunnan, Guangxi, etc., crush them, pass through a No. 4 sieve, and the moisture and ash content are all in accordance with the "Fujian Province Traditional Chinese Medicine Preparation Specifications".

[0022] E*ab value determination: Take an appropriate amount of Sanyeqing sample powder, evenly spread it in the colorimeter powder test box until it is full, cover it with a cover glass, set the light source to D65, aperture 8mm, LED blue light excitation, set the sample measurement mode to SCI, color space to CIELab, observer angle to 10°, instrument error value less than 0.4, calibrate the instrument with a black and white plate, perform sample measurement, record the chromaticity values ​​(L*, a*, b*), and calculate the total color difference value (E*ab): E*ab=(L* 2 +a* 2 +b* 2 ) 1 / 2 ), the results are shown in Table 1.

[0023] Table 1 Color information of different batches of Sanyeqing

[0024] batch L* a* b* E*ab S1 67.56 6.40 14.27 69.35 S2 69.79 5.47 13.01 71.20 S3 82.55 3.37 11.01 83.35 S4 64.13 6.07 16.33 66.45 S5 68.85 6.98 14.49 70.71 S6 71.32 5.05 11.67 72.44 S7 69.19 6.11 14.38 70.93 S8 73.09 3.97 10.50 73.95 S9 73.11 3.47 10.88 74.00 S10 72.31 4.10 10.75 73.22 S11 68.18 5.67 12.58 69.56 S12 73.31 4.35 10.71 74.22 S13 65.21 6.41 14.94 67.20 S14 67.71 5.08 12.31 69.00 S15 63.79 6.04 14.46 65.69 S16 67.75 6.21 14.70 69.60 S17 68.89 6.87 14.89 70.82 S18 74.39 5.94 14.63 76.04 S19 69.61 6.25 12.52 71.00 S20 74.07 4.80 12.85 75.33

[0025] Preparation of the test solution: Accurately weigh 2.0 g of the Tripterygium wilfordii sample powder, place it in a conical flask, add 50 mL of 80% methanol by volume, weigh it, ultrasonicate for 60 min, cool, make up the weight loss with methanol, filter, and obtain the test solution.

[0026] Determination of total flavonoids content: Using rutin as the standard, a total flavonoids standard curve was drawn, and the absorbance was determined at a wavelength of 510 nm in a UV-visible spectrophotometer using the NaNO2-Al(NO3)3-NaOH colorimetric method. The total flavonoids content of different batches of Tripterygium wilfordii was determined using the external standard method.

[0027] Determination of total polysaccharide content: Using anhydrous glucose as the standard, a total polysaccharide standard curve was drawn, and the absorbance was measured at a wavelength of 485 nm in a UV-visible spectrophotometer using a sulfuric acid-phenol colorimetric method. The total polysaccharide content of different batches of Tripterygium wilfordii was determined using the external standard method.

[0028] Determination of total phenolic acid content: Using gallic acid as the standard, a total phenolic acid standard curve was drawn, and the absorbance was measured at a wavelength of 750 nm in a UV-visible spectrophotometer using the folin phenol colorimetric method. The total phenolic acid of different batches of Tripterygium wilfordii was determined using the external standard method.

[0029] The results of total flavonoids content, total polysaccharide content and total phenolic acid content of different batches of Tripterygium wilfordii are shown in Table 2.

[0030] Table 2 Results of total flavonoids, total polysaccharides and total phenolic acid contents in different batches of Tripterygium wilfordii

[0031]

[0032]

[0033] The weight coefficients of the subjective and objective combination corresponding to the three indicators are obtained by the AHP-entropy weight method. The three content indicators and their corresponding weight coefficients are shown in Table 3.

[0034] Table 3 Content index and weight coefficient of Tripterygium wilfordii

[0035] Total flavonoids Total polysaccharides Total phenolic acid AHP coefficient 0.3333 0.3333 0.3334 Entropy weight coefficient 0.3582 0.5664 0.0754 AHP-Entropy Weight Coefficient 0.3573 0.5651 0.0775

[0036] The comprehensive scores of different batches of Sanyeqing were calculated according to the final AHP-entropy weight coefficient, and the calculation formula was comprehensive score = 0.3573*total flavonoids content + 0.5651*total polysaccharide content + 0.0775*total phenolic acid content. According to the comprehensive score of Sanyeqing, Sanyeqing was graded, <15 was divided into grade III, 15-30 was divided into grade II, and >30 was divided into grade I. The comprehensive scores and grading of different batches of Sanyeqing are shown in Table 4. Among the 20 batches of Sanyeqing, there were 6 batches of grade III, 9 batches of grade II, and 5 batches of grade I. Grade II accounted for the highest proportion, and the overall distribution was normal.

[0037] Table 4 Comprehensive scores and classification of different batches of Sanyeqing

[0038]

[0039]

[0040] Research on Tripterygium wilfordii found that it has significant hepatoprotective activity, so one batch of each of grade I, II, and III Tripterygium wilfordii were randomly selected and the alcohol-induced zebrafish acute liver injury model was used for in vivo pharmacological verification.

[0041] Establishment of alcohol-induced acute liver injury model in zebrafish: 4dhf AB wild-type zebrafish were selected and given 2% ethanol by volume for modeling, and the modeling lasted for 32 hours. Compared with the blank group, the model group showed obvious pericardial edema, pigment deposition, delayed yolk sac absorption and other phenomena, indicating that the modeling was successful. I, II, and III grade clover administration groups: The test solution obtained by ultrasonic extraction with 80% methanol by volume was evaporated to remove the organic solvent, and freeze-dried to obtain the corresponding grade of clover extract freeze-dried powder. 4dpfAB wild-type zebrafish were selected, and the test solution was prepared using embryo culture medium containing 2% ethanol by volume, and the modeling lasted for 32 hours.

[0042] After staining with Oil Red O, lipid deposition in zebrafish can be clearly observed. Alcohol-induced liver damage can lead to lipid deposition in the liver region and abnormal yolk sac absorption. After modeling and drug administration, the fish were killed by tricaine anesthesia, washed with PBS, dehydrated with 1,2-propylene glycol gradient, stained with 0.5% Oil Red O dye for 1 hour, eluted with 1,2-propylene glycol gradient, washed with PBS, and stored in 80% glycerol by volume. Fifteen fish were randomly selected from each group and photographed under a microscope.

[0043] Determination of liver biochemical indicators (AST, ALT): 4dpf AB wild-type zebrafish with consistent growth and development conditions were selected in a 6-well plate and randomly divided into a blank group, a model group, a positive drug group (glutathione), and three-level drug administration groups of Sanyeqing I, II, and III, with 15 fish in each well and 3 replicates. Modeling and drug administration were carried out simultaneously and lasted for a total of 32 hours. After the modeling was completed, the fish were anesthetized with tricaine, a certain volume of physiological saline was added, the tissues were homogenized, and centrifuged at 4°C 3000rpm / min for 10 minutes. The supernatant was taken to determine the protein concentration. The AST and ALT levels in the supernatant of the tissue homogenate were determined using the AST and ALT kits (Beijing Solebao). The detailed steps of the kit detection are as follows:

[0044] (1) Dilution of standard: dilute the standard with distilled water to obtain a series of standard solutions with gradient concentrations;

[0045] (2) Addition of standard samples: 30 μL of standard solution of each concentration was placed in a 96-well plate;

[0046] (3) Sample addition: Set up sample measurement wells and sample control wells respectively, add 5 μL of the sample to be tested and 25 μL of reagent 1 to the sample measurement wells, and add 25 μL of reagent 1 to the sample control wells;

[0047] (4) Incubation: After mixing, incubate at 37°C (for mammals) or 25°C (for other species) for 30 min.

[0048] (5) Add reagent 2: add 25 μL of reagent 2 to each standard well and sample well, and add 5 μL of the sample to be tested and 25 μL of reagent 2 to the sample blank well;

[0049] (6) Incubation: After mixing, incubate at 37°C (for mammals) or 25°C (for other species) for 20 min.

[0050] (7) Add reagent 3: 240 μL of reagent 3 was added to all groups;

[0051] (8) Incubation: Mix well and incubate at room temperature for 10 min;

[0052] (9) Determination: Measure the absorbance of each tube at a wavelength of 505 nm.

[0053] The results of pharmacodynamic validation data analysis were expressed as X±SD (mean±standard deviation). Image J and GraphpadPrism 9.5 were used to analyze the data results, and ANOVA analysis was used for comparison between groups.

[0054] The results of Oil Red O staining are shown in Table 5, and the staining effect is shown in Figure 1 , AST and ALT level results are shown in Figure 2 , * indicates p<0.05 compared with the model group, ** indicates p<0.01 compared with the model group, *** indicates p<0.001 compared with the model group, **** indicates p<0.0001 compared with the model group.

[0055] Table 5 Effects of different drug administration groups on zebrafish liver and yolk sac

[0056] Group Gray value of liver area Yolk sac area Yolk sac delayed absorption inhibition rate (%) Model Group 35563±3910 17637±4414 / Blank Group <![CDATA[15068±4853 **** ]]> <![CDATA[7586±1531 **** ]]> / Positive drug group <![CDATA[17932±7082 **** ]]> <![CDATA[10346±2744 **** ]]> 72.54 Level I <![CDATA[18597±6251 **** ]]> <![CDATA[10424±3175 **** ]]> 71.76 Level II <![CDATA[23745±8097 ** ]]> <![CDATA[13794±4250 ** ]]> 38.24 Level III <![CDATA[25695±7872 * ]]> <![CDATA[14931±3626 * ]]> 26.92

[0057] Oil red O staining and liver biochemical index levels showed that, by comparing with the model group, the positive drug group (glutathione) had significantly reduced liver lipid deposition, normal yolk sac absorption, and extremely significant differences in AST and ALT levels compared with the model group, indicating that glutathione has a good alleviating effect on alcohol-induced acute liver injury in zebrafish. At the same time, the three-leaf green I, II, and III grade administration groups all showed significant improvements in liver gray value, abnormal yolk sac absorption, and AST and ALT levels. There are also significant differences in the hepatoprotective effects of different grades of three-leaf green. The improvement effect of the grade I administration group in terms of liver gray value and yolk sac area is significantly better than that of grades II and III. Comprehensively combining various efficacy indicators, it presents an overall hepatoprotective effect of grade I> grade II> grade III, indicating that the grading method of the three-leaf green is reasonable and reliable, and the efficacy of the three-leaf green can be graded by quantifiable indicators.

[0058] SPSS Statistics software was used to analyze the data for collinearity. The L* collinearity statistic (VIF) was greater than 10, and the VIF values ​​of the remaining variables were all less than 10, which could be used for subsequent discriminant analysis. The results of the eigenvalues ​​of each cumulative discriminant function are shown in Table 6. Function 1 has a high proportion of typical correlation and has significant statistical differences. Therefore, the obtained non-standardized canonical discriminant function is y = -0.015E*ab+1.378a*+0.287b*-10.148. In the subsequent classification of Sanyeqing, a colorimeter can be used to collect the color data of Sanyeqing powder, and the obtained data can be substituted into the non-standardized canonical discriminant function to output the corresponding y value. When the y value is close to 1, the Sanyeqing grade is I; when the y value is close to 2, the Sanyeqing grade is II; when the y value is close to 3, the Sanyeqing grade is III.

[0059] Table 6 Results of discriminant function eigenvalues

[0060]

[0061] The present invention can classify Sanyeqing by quantifiable content indicators, and the efficacy verification shows that the classification is scientific and reliable, and the classification is associated with the color information that can be obtained non-destructively, and a non-standardized canonical discriminant function is obtained. The color information of Sanyeqing medicinal materials can be collected, substituted into the non-standardized canonical discriminant function for calculation, and the classification is performed according to the output data. The Sanyeqing medicinal materials can be quickly classified, and the classification results can also be used as the basis for preliminary screening of efficacy, providing a new idea for the quality control of Sanyeqing.

[0062] Five batches of unknown grade of Tripterygium wilfordii samples were selected, and the component content and color information were measured respectively. The grade was determined by the Tripterygium wilfordii grade calculation formula and the standardized canonical discriminant function. The results are shown in Tables 7 and 8. The accuracy of using color information to discriminate the grade of Tripterygium wilfordii reached 80%, and the score of the WZ4 non-standardized canonical discriminant function was at the edge of grade II-III, with a high overall accuracy. In summary, the color-based rapid grading method of Tripterygium wilfordii is scientific and reliable. The quality of Tripterygium wilfordii can be divided by color information, which is conducive to the preliminary screening of the efficacy of Tripterygium wilfordii, and is conducive to the quality control and application of Tripterygium wilfordii.

[0063] Table 7 Content and grade classification of three-leaf green ingredients

[0064]

[0065] Table 8 Color information and grade classification of Sanyeqing

[0066]

[0067]

[0068] The above embodiments are used to illustrate the present invention rather than to limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A color-based rapid grading method for Tripterygium wilfordii, characterized in that: The method includes a component-based ranking method and a non-standardized canonical discriminant function with color information as a variable; The grading method calculates the weight scores of multiple batches of Tripterygium wilfordii samples to grade Tripterygium wilfordii, associates the graded grades with their colors, constructs a non-standardized canonical discriminant function with color information as a variable, and implements rapid grading of Tripterygium wilfordii based on the constructed function.

2. The color-based rapid grading method for Tripterygium wilfordii according to claim 1, characterized in that: The contents of flavonoids, polysaccharides and phenolic acids in multiple batches of Tripterygium wilfordii samples were determined; the NaNO2-Al(NO3)3-NaOH method was used to measure the absorbance at 510 nm to obtain the flavonoid content, the sulfuric acid-phenol method was used to measure the absorbance at 485 nm to obtain the polysaccharide content, and the Folin-phenol method was used to measure the absorbance at 750 nm to obtain the phenolic acid content.

3. The color-based rapid grading method for Tripterygium wilfordii according to claim 2, characterized in that: The AHP-entropy weight method was used to determine the weight indexes corresponding to the three indicators of total flavonoid content, total polysaccharide content and total phenolic acid content, and the calculation formula was obtained: score = 0.3572*total flavonoid content + 0.5651*total polysaccharide content + 0.0775*total phenolic acid content.

4. The method for rapid grading of clover based on color according to claim 3 is characterized in that: The calculation results of the components determined by the AHP-entropy weight method were graded so that the calculation results of the components were normally distributed, and the pharmacodynamics of different grades showed obvious differences.

5. The method for rapid grading of clover based on color according to claim 4, characterized in that: Based on the scores, the three-leaf green tree is divided into grades I, II, and III. <15 is divided into grade III, 15-30 is divided into grade II, and >30 is divided into grade I.

6. The method for rapid grading of clover based on color according to claim 1, characterized in that: The collinearity analysis was performed on the grade classification results and the color information. The variables with collinearity statistics greater than 10 were removed, and the remaining variables were subjected to discriminant analysis to obtain the non-standardized canonical discriminant function of color information and grade.

7. The method for rapid grading of clover based on color according to claim 6, characterized in that: The unstandardized canonical discriminant function with color information as a variable is y=-0.015E*ab+1.378a*+0.287b*-10.

148.

8. The method for rapid grading of clover based on color according to claim 7, characterized in that: The output of the non-standardized canonical discriminant function corresponds to the y value. When y≤1.50, the grade of the three-leaf green is I; when y is in the range of 1.50-2.50, the grade of the three-leaf green is II; when y≥2.50, the grade of the three-leaf green is III.

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