A color-based method for rapidly grading tripterygium wilfordii hook

Abstract

The application discloses a kind of based on color threeleaf ginseng fast grade division method, including collecting different batches of threeleaf ginseng sample, obtains threeleaf ginseng component content and color data, by total flavone, total polysaccharide and total phenolic acid content, weight coefficient corresponding to index is determined using AHP-entropy weight method, the calculation formula of threeleaf ginseng grade division is obtained, and threeleaf ginseng is graded, selects the threeleaf ginseng of each grade and carries out efficacy difference verification, and efficacy result and grade division present same trend, I grade efficacy is significantly better than II, III grade.Color difference meter measured data L*, a*, b*, E*ab and grading result are comprehensively analyzed, obtain the non-standard canonical discriminant function with color information as variable, threeleaf ginseng grade can be discriminated based on color information, it is favorable for threeleaf ginseng fast grade division, and threeleaf ginseng efficacy is preliminarily fuzzy evaluated through color information.

Description

TECHNICAL FIELD

[0001] The present application relates to the quality control field of Radix Tetrastigme, and in particular to a color-based rapid grade classification method of Radix Tetrastigme. BACKGROUND

[0002] CN113720931A discloses a quality evaluation method of Radix Tetrastigme, which uses the content of total flavonoids in Radix Tetrastigme, the relative content of rutin, quercetin and kaempferol in total flavonoids, the characteristic peaks in the total flavonoid fingerprint of Radix Tetrastigme, the proliferation inhibition rate of total flavonoids in Radix Tetrastigme on pancreatic cancer cells, and the apoptosis rate of total flavonoids in Radix Tetrastigme on pancreatic cancer cells to evaluate the quality of Radix Tetrastigme. However, this patent needs to use high-performance liquid chromatography, cell anti-proliferation experiments, etc. to realize, which is complex in operation, high in cost, time-consuming, difficult to apply in the circulation of Radix Tetrastigme market, and has poor practicability. At the same time, it mainly scores the total flavonoids in Radix Tetrastigme, ignoring other important active ingredients such as polysaccharides and phenolic acids in Radix Tetrastigme.

[0003] The total flavonoids, total polysaccharides and total phenolic acids in Radix Tetrastigme are three important chemical components in Radix Tetrastigme. The content of flavonoids in Radix Tetrastigme is extremely rich, and more than 50 kinds of flavonoids have been identified in the tubers and aboveground parts, mainly including flavones, flavonoid glycosides, dihydroflavones, etc. Radix Tetrastigme polysaccharides have great clinical application potential due to their unique pharmacological activity, and have pharmacological activities such as antioxidant and blood lipid regulation. Radix Tetrastigme contains a variety of special and complex phenolic acids, which have the effects of anti-tumor, anti-bacterial, anti-inflammatory, liver protection, and blood lipid reduction.

[0004] The epidermis ratio of Radix Tetrastigme and the drying method in the processing process all have an impact on the color of Radix Tetrastigme and have a great impact on the content of active substances in Radix Tetrastigme, so color can be used for rapid evaluation of Radix Tetrastigme. Compared with traditional methods, the rapid grade classification of Radix Tetrastigme using color is non-destructive and efficient, and can play an important role in the fields of medicinal material processing and procurement. SUMMARY

[0005] The present application aims at the deficiencies of the prior art and proposes a color-based rapid grade classification method of Radix Tetrastigme.

[0006] The purpose of the present application is achieved by the following technical solution: The technical solution adopted by the present application includes the following steps: a color-based rapid grade classification method of Radix Tetrastigme, which includes a component-based grade classification method and a non-standardized canonical discriminant function taking color information as a variable;

[0007] The grading method calculates the weight score of the multiple batches of three-leaf green sample to grade the three-leaf green, associates the graded three-leaf green with color information, constructs a non-standardized canonical discriminant function taking color information as a variable, and realizes rapid grading of the three-leaf green based on the constructed function.

[0008] Further, the contents of flavonoids, polysaccharides and phenolic acids in the multiple batches of three-leaf green samples are determined; the content of flavonoids is determined by the NaNO2-Al(NO3)3-NaOH method at 510 nm, the content of polysaccharides is determined by the sulfuric acid-phenol method at 485 nm, and the content of phenolic acids is determined by the Folin phenol method at 750 nm.

[0009] Further, the weight indexes corresponding to the total flavonoid content, the total polysaccharide content and the total phenolic acid content are determined by the AHP-entropy weight method, and a calculation formula is obtained: score = 0.3573*total flavonoid content + 0.5651*total polysaccharide content + 0.0775*total phenolic acid content.

[0010] Further, the component calculation results determined by the AHP-entropy weight method are graded to make the component calculation results normally distributed, and the pharmacodynamics of different grades presents obvious differences.

[0011] Further, the three-leaf green is divided into I, II and III grades based on the score, <15 is divided into III grade, 15-30 is divided into II grade, and >30 is divided into I grade.

[0012] Further, the results of the grade division are subjected to collinearity analysis with the color information, the variables with collinearity statistics greater than 10 are removed, and the remaining variables are subjected to discriminant analysis to obtain a non-standardized canonical discriminant function about color information and grades.

[0013] Further, the non-standardized canonical discriminant function taking color information as a variable is y = -0.015E*ab + 1.378a* + 0.287b*-10.148.

[0014] Further, the non-standardized canonical discriminant function outputs a corresponding y value, when the y value is close to 1, the San Ye Qing grade is I grade; when the y value is close to 2, the San Ye Qing grade is II grade; when the y value is close to 3, the San Ye Qing grade is III grade. The beneficial effects of the present application: the present application provides a color-based San Ye Qing rapid grade division method, first, the San Ye Qing sample is scored and graded by including the content index of the component related to the drug effect, then the grading results are associated with the color information, and the non-standardized canonical discriminant function y = -0.015E*ab + 1.378a* + 0.287b*-10.148 about the grade is obtained, which can be based on color information to distinguish the grade of San Ye Qing, which is beneficial to the construction of San Ye Qing quality control system and the popularization and application of San Ye Qing. Compared with the traditional method, the rapid grade division of San Ye Qing by color is lossless and efficient, and can play an important role in the fields of medicinal material processing, procurement and the like. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0016] Figure 1 Oil red O staining effect diagram for each group.

[0017] Figure 2 AST(A) and ALT(B) level diagram for each group. DETAILED DESCRIPTION

[0018] The present application will be further described below in conjunction with specific embodiments, but the protection scope of the present application is not limited to this:

[0019] Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific technology or condition is not indicated in the examples, it is carried out according to the technology or condition described in the literature in the art or according to the product instruction. If the reagent or instrument is not indicated by the manufacturer, it is a conventional product that can be obtained by market purchase. The reagents and raw materials used in the following examples can be obtained by market purchase.

[0020] Example 1

[0021] San Ye Qing sample collection: different batches of San Ye Qing dry tubers were collected, crushed and passed through a No. 4 sieve. The production places include Zhejiang, Jiangxi, Fujian, Yunnan, Guangxi and the like, the crushed and sieved products meet the requirements of "Fujian Province Chinese Medicine Decoction Specification" in moisture content, ash content and the like.

[0022] E*ab value determination: take a suitable amount of Radix Scrophulariae sample powder, evenly spread in the colorimeter powder test box until full, cover the cover glass, light source is D65, aperture 8mm, LED blue light excitation, sample measurement mode is set to SCI, color space is CIELab, observer angle is 10°, instrument error value is less than 0.4, after correcting the instrument with black and white board, sample determination is carried out, record color value (L*, a*, b*), and calculate 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 Radix Scrophulariae

[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 test solution: accurately weigh 2.0g of Radix Scrophulariae sample powder, put it in a conical flask, add 50mL of 80% methanol, weigh, ultrasonic for 60min, cool, supplement the weight loss with methanol, filter to obtain the test solution.

[0026] Determination of total flavonoid content: using rutin as standard, draw the total flavonoid standard curve, using NaNO2-Al(NO3)3-NaOH colorimetric method, determine the absorbance at 510nm in the ultraviolet visible spectrophotometer, use external standard method to determine the total flavonoid content of different batches of Radix Scrophulariae.

[0027] Determination of total polysaccharide content: using anhydrous glucose as standard, draw the total polysaccharide standard curve, using sulfuric acid-phenol colorimetric method, determine the absorbance at 485nm in the ultraviolet visible spectrophotometer, use external standard method to determine the total polysaccharide content of different batches of Radix Scrophulariae.

[0028] Determination of total phenolic acid content: using gallic acid as standard, draw the total phenolic acid standard curve, using Folin phenol colorimetric method, determine the absorbance at 750nm in the ultraviolet visible spectrophotometer, use external standard method to determine the total phenolic acid of different batches of Radix Scrophulariae.

[0029] The results of total flavonoid content, total polysaccharide content and total phenolic acid content of different batches of Radix Scrophulariae are shown in Table 2.

[0030] Table 2 Results of total flavonoid, total polysaccharide and total phenolic acid content of different batches of Radix Scrophulariae

[0031]

[0032]

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

[0034] Table 3 Content indexes and weight coefficients of Sanqie

[0035] Total flavonoids Total polysaccharides Total phenolic acids 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 Sanqie were calculated according to the finally obtained AHP-entropy weight coefficients, and the calculation formula was comprehensive score = 0.3573*total flavonoid content + 0.5651*total polysaccharide content + 0.0775*total phenolic acid content. According to the comprehensive scores of Sanqie, Sanqie was classified into three grades, <15 was classified as grade III, 15-30 was classified as grade II, and >30 was classified as grade I. The comprehensive scores and classification of different batches of Sanqie are shown in Table 4. Among the 20 batches of Sanqie, 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 Sanqie

[0038]

[0039]

[0040] The study of Sanqie found that it had significant hepatoprotective activity, so 1 batch of grade I, grade II and grade III Sanqie were randomly selected respectively, and an acute liver injury model of zebrafish induced by alcohol was used for in vivo pharmacodynamic verification.

[0041] Establishment of acute liver injury model of zebrafish induced by alcohol: 4 dhf AB strain wild type zebrafish were selected and given 2% ethanol by volume for modeling, and the modeling lasted for 32 h. 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 Sanqie administration group: the test sample solution obtained by ultrasonic extraction with 80% methanol was rotary evaporated to remove organic solvents, and freeze-dried to obtain Sanqie extract freeze-dried powder of corresponding grade. 4 dpf AB strain wild type zebrafish were selected, and the test sample solution was prepared using embryo culture medium containing 2% ethanol by volume, and the modeling lasted for 32 h.

[0042] The lipid deposition in zebrafish can be observed obviously after oil red O staining. The alcohol-induced liver function damage can cause the lipid deposition in liver area, and also can cause the abnormal yolk sac absorption. After the modeling and drug administration, the zebrafish are anesthetized by tricaine, washed by PBS, selected by 1,2-propanediol gradient dehydration, dyed by 0.5% oil red O solution for 1 hour, eluted by 1,2-propanediol, washed by PBS, and then stored in 80% glycerol. 15 zebrafish are randomly selected in each group for photographing under a microscope.

[0043] Liver biochemical index (AST, ALT) determination: 4dpf AB line wild-type zebrafish with consistent growth and development are selected in a 6-well plate, and randomly divided into a blank group, a model group, a positive drug group (glutathione), and San Ye Qing I, II and III groups, 15 zebrafish per well, and 3 replicates are set. The modeling and drug administration are performed at the same time, and the whole process lasts for 32 hours. After the modeling, the zebrafish are anesthetized by tricaine, a certain volume of physiological saline is added, the tissue is homogenized, and then centrifuged at 3000 rpm / min for 10 minutes at 4°C. The supernatant is used to determine the protein concentration. The AST and ALT levels in the supernatant of the tissue homogenate are determined by using an AST and ALT kit (Beijing Solaybao), and the detailed steps of the kit detection are as follows:

[0044] (1) Standard dilution: distilled water is used to dilute the standard, and a series of gradient concentration standard solutions are obtained;

[0045] (2) Standard sample addition: 30 μL of each concentration standard solution is taken in a 96-well plate;

[0046] (3) Sample addition: sample determination holes and sample control holes are set, 5 μL of the sample to be tested and 25 μL of reagent one are added to the sample determination holes, and 25 μL of reagent one is added to the sample control holes;

[0047] (4) Incubation: after mixing, react for 30 minutes at 37°C (for mammals) or 25°C (for other species);

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

[0049] (6) Incubation: after mixing, react for 20 minutes at 37°C (for mammals) or 25°C (for other species);

[0050] (7) Add reagent three: add 240 μL of reagent three to all groups;

[0051] (8) Incubation: mix well, and place at room temperature for 10 minutes;

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

[0053] The pharmacodynamic verification data analysis results are X±SD (mean±standard deviation), and the data results are analyzed by Image J and Graphpad Prism 9.5, and ANOVA analysis is used for comparison between groups.

[0054] The oil red O staining results are shown in Table 5, and the staining effects are shown in Figure 1 , and the 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, and **** indicates p<0.0001 compared with the model group.

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

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

[0057] The oil red O staining and liver biochemical index levels all show that, by comparison with the model group, the liver lipid deposition of the positive drug group (glutathione) is significantly reduced, the yolk sac absorption is normal, and the AST and ALT levels also have a very significant difference from the model group, indicating that glutathione has a good relieving effect on alcohol-induced acute liver injury in zebrafish. At the same time, the I, II and III grade administration groups of San Ye Qing all show obvious improvement in liver gray value, yolk sac absorption abnormality, AST and ALT levels. There is also a significant difference in the liver protection effect of different grades of San Ye Qing. The improvement effect of the I grade administration group in liver gray value, yolk sac area and other aspects is obviously better than that of the II and III grades. In combination with various pharmacodynamic indexes, the overall liver protection effect shows I grade>II grade>III grade, indicating that the grade classification method of the San Ye Qing is reasonable and reliable, and can classify the pharmacodynamic effect of San Ye Qing through quantifiable indexes.

[0058] The data is analyzed by SPSS Statistics software for collinearity analysis. The L* collinearity statistics (VIF) is greater than 10, and the VIF values of the remaining variables are all less than 10, which can be used for subsequent discriminant analysis. The results of the characteristic values of the cumulative discriminant function are shown in Table 6. The function 1 has a high proportion of typical correlation, and has a significant statistical difference, so the non-standardized canonical discriminant function obtained is y=-0.015E*ab+1.378a*+0.287b*-10.148. In the subsequent grade classification of San Ye Qing, the color difference meter can be used to collect the color data of San Ye Qing powder. The obtained data is substituted into the non-standardized canonical discriminant function, and the corresponding y value is output. When the y value is close to 1, the grade of the San Ye Qing is I grade; when the y value is close to 2, the grade of the San Ye Qing is II grade; and when the y value is close to 3, the grade of the San Ye Qing is III grade.

[0059] Table 6 Discriminant function characteristic value results

[0060]

[0061] The present application can grade Radix Tetrastigme by quantifiable content indicators, and the efficacy verification shows that the grading is scientific and reliable. The grading is associated with the non-destructive color information, and a non-standard canonical discriminant function is obtained. The color information of Radix Tetrastigme can be collected and substituted into the non-standard canonical discriminant function for calculation, and the output data can be used for grading, so that Radix Tetrastigme can be quickly graded. The grading results can also be used as a basis for preliminary screening of efficacy, and provide a new idea for quality control of Radix Tetrastigme.

[0062] Five batches of unknown grade Radix Tetrastigme samples were selected, and the content of ingredients and color information were determined. The belonging grade was determined by the Radix Tetrastigme grade division calculation formula and the non-standard canonical discriminant function, and the results are shown in Tables 7-8. The accuracy rate of Radix Tetrastigme grade discrimination using color information is 80%, and the score of WZ4 non-standard canonical discriminant function is at the edge of II-III grade, and the overall accuracy is high. In summary, the color-based rapid grading method of Radix Tetrastigme is scientific and reliable, and the quality of Radix Tetrastigme can be divided by color information, which is conducive to the preliminary screening of Radix Tetrastigme efficacy and the quality control and application of Radix Tetrastigme.

[0063] Table 7 Content of ingredients and grade division of Radix Tetrastigme

[0064]

[0065] Table 8 Color information and grade division of Radix Tetrastigme

[0066]

[0067]

[0068] The above examples are used to explain and illustrate the present application, but not to limit the present application. Any modification and change made to the present application within the spirit and protection scope of the claims of the present application, falls into the protection scope of the present application.

Claims

1. A color-based rapid grading method for Tripterygium wilfordii, characterized in that: The method includes a component-based hierarchical classification method and a non-standardized canonical discriminant function with color information as a variable; The contents of flavonoids, polysaccharides and phenolic acids in multiple batches of Tripterygium wilfordii samples were determined, and the weight indexes corresponding to the three indicators of total flavonoids content, total polysaccharide content and total phenolic acid content were determined by the AHP-entropy weight method, and the calculation formula was obtained: score = 0.3572*total flavonoids content + 0.5651*total polysaccharide content + 0.0775*total phenolic acid content; the component calculation results determined by the AHP-entropy weight method were graded so that the component calculation results were normally distributed, and the pharmacodynamics of different grades showed obvious differences; based on the score, Tripterygium wilfordii was divided into grades I, II and III, <15 was divided into grade III, 15-30 was divided into grade II, and >30 was divided into grade I; The color information of the powder of the three-leaf green sample was measured, the chromaticity values ​​L*, a*, and b* were measured, and the total color difference value E*ab was calculated. The grade classification results and the color information were subjected to collinearity analysis. After removing the variables with collinearity statistics greater than 10, the remaining variables were subjected to discriminant analysis to obtain a non-standardized canonical discriminant function for color information and grade. The non-standardized canonical discriminant function with color information as the variable is y=-0.015E*ab+1.378a*+0.287b*-10.148; the non-standardized canonical discriminant function outputs the corresponding y value. When y≤1.50, the grade of the three-leaf green is Grade I; when y is in the range of 1.50-2.50, the grade of the three-leaf green is Grade II; when y≥2.50, the grade of the three-leaf green is Grade III.

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

Images