Phyllanthus emblica medicinal material quality grading method based on pharmacodynamic theory

Abstract

The invention belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a phyllanthus emblica quality grading method based on pharmacodynamic theory, which comprises the following steps: S1, identifying a phyllanthus emblica liver protection quality marker group; s2, performing one-time quantitative analysis on the phyllanthus emblica quality marker group; s3, constructing an emblic leafflower fruit quality grading model; and S4, application of the emblic leafflower fruit quality grading model. According to the invention, by combining mass spectrometry and network analysis, six mass markers with liver protection activity in phyllanthus emblica are excavated for the first time; the established quality marker quantitative method is good in specificity and high in accuracy; the obtained grading model is excellent in effect, the phyllanthus emblica medicinal material can be divided into three grades according to the liver protection activity of the phyllanthus emblica, the problems of high subjectivity and poor traceability of an existing shape-distinguishing and quality-discovering evaluation standard are solved, establishment of a high-quality and high-price system of the phyllanthus emblica medicinal material is promoted, and high-quality development of the traditional Chinese medicine industry is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of quality control of traditional Chinese medicines, and specifically relates to a quality grading method for Phyllanthus emblica medicinal materials based on pharmacodynamics, more precisely, to a quality grading method for Phyllanthus emblica using pharmacodynamic quality markers as discriminant variables. Background Art

[0002] As a powerful means to ensure the quality, safety and effectiveness of Chinese medicinal materials and preparations, the quality evaluation of traditional Chinese medicine is of great significance to maintaining the physical and mental health of the people and promoting the high-quality development of the traditional Chinese medicine industry.

[0003] Phyllanthus emblica belongs to the genus Phyllanthus in the Euphorbiaceae family. It is a traditional Chinese medicinal material with a profound historical background. It has the effects of protecting the liver, relieving cough, and diuresis. However, due to its large habitat differences and high genetic diversity, as well as the poor stability and easy transformation of its main component polyphenols, the quality of Phyllanthus emblica on the market is uneven. At present, the industry generally distinguishes the grades of Chinese medicine based on appearance characteristics, cross-sectional characteristics, texture, weight, length, diameter and thickness, as well as smell, that is, "distinguishing appearance and judging quality". It is generally believed in the industry that the quality of Phyllanthus emblica after frosting is better, and there is a saying that "those that are frosted are better". However, this method has the problems of strong subjectivity of the evaluation criteria and poor traceability. Therefore, it is urgent to construct a method that is simple to operate, low-cost, and can scientifically and effectively evaluate the quality grade of Phyllanthus emblica. Summary of the invention

[0004] In view of the lack of existing quality grading methods for Phyllanthus emblica medicinal materials, the present invention proposes a set of quality grading methods for Phyllanthus emblica medicinal materials based on the principle of pharmacodynamics. Compared with the traditional method of "quality based on symptoms", the quality grading of medicinal materials based on "quality based on pharmacodynamics" in the present invention is guided by pharmacodynamics, is closely related to clinical efficacy, and can more accurately reflect the actual quality of Phyllanthus emblica medicinal materials.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] A quality grading method for Phyllanthus emblica medicinal materials based on efficacy, comprising the following steps:

[0007] S1. Identification of hepatoprotective quality markers in Phyllanthus emblica: Use time-of-flight high-resolution mass spectrometry to analyze the blood-entering components of Phyllanthus emblica; construct a "component-target-pathway" network to screen the hepatoprotective quality markers in Phyllanthus emblica;

[0008] S2. One-time quantitative analysis of the quality marker group of Phyllanthus emblica: Establish an HPLC method for simultaneously determining the liver-protective quality marker group in Phyllanthus emblica; Determine the content of liver-protective quality markers in different batches of Phyllanthus emblica;

[0009] S3. Construction of Phyllanthus emblica quality grading model: Test the in vitro liver-protecting biological activities of Phyllanthus emblica from different batches; classify Phyllanthus emblica from different batches according to the liver-protecting activity, take the content of liver-protecting quality markers as discriminant variables, and construct a discriminant model for Phyllanthus emblica quality grading;

[0010] S4. Application of the discriminant analysis model for Phyllanthus emblica quality grading.

[0011] Optionally, the specific operation steps of step S1 are as follows: Use a time-of-flight high-resolution mass spectrometer to identify the components of Phyllanthus emblica entering the blood by comparing the aqueous extract of Phyllanthus emblica, blank serum, and drug-containing serum, and comparing the secondary spectra of reference substances and samples; use the Swiss Target Prediction website to predict the action targets of the components entering the blood, collect alcohol-induced liver injury-related targets from the GeneCards and OMIM databases to obtain the intersection target genes of components-diseases, use the DAVID Bioinformatics Resources platform for KEGG pathway enrichment, use Cytoscape software to construct a "component-target-pathway" network, use the CytoNCA plug-in to analyze the topological parameters of this network, and select the components with a Degree value greater than the median in the network as potential quality markers.

[0012] Optionally, the Phyllanthus emblica liver-protecting quality marker group includes at least one of gallic acid, methyl gallate, quercitrin, ellagic acid, chebulagic acid, and corilagin.

[0013] Optionally, the specific operation steps of step S2 are as follows: Prepare a mixed reference substance solution with gallic acid, methyl gallate, quercitrin, ellagic acid, chebulagic acid, and corilagin; prepare a test solution from Phyllanthus emblica; perform ultra-high performance liquid chromatography detection on the mixed reference substance solution and the test solution respectively, substitute the peak areas corresponding to each component into the corresponding standard curves of each component, and calculate the contents of gallic acid, methyl gallate, quercitrin, ellagic acid, chebulagic acid, and corilagin in Phyllanthus emblica respectively.

[0014] Optionally, in the mixed reference substance solution, the concentration of gallic acid is 200.0 - 400.0 μg·mL -1 , the concentration of methyl gallate is 5.00 - 10.00 μg·mL -1 , the concentration of quercitrin is 3.00 - 6.00 μg·mL -1 , the concentration of ellagic acid is 20.00 - 40.00 μg·mL -1 , the concentration of chebulagic acid is 20.00 - 40.00 μg·mL -1 and the concentration of corilagin is 20.00 - 40.00 μg·mL -1 .

[0015] Optionally, the preparation of the Phyllanthus emblica test sample solution specifically includes: heating and refluxing with 25-95% methanol for 1 h, cooling, making up the weight loss, filtering, and the material-liquid ratio of Phyllanthus emblica to methanol is 0.1 g:50 mL.

[0016] Optionally, the ultra-high performance liquid chromatography detection specifically includes: the chromatographic column is an octadecylsilane-bonded silica gel chromatographic column, using 0.05 vol% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase, and the flow rate is 0.6 mL min -1 , the detection wavelength is 254 nm, the column temperature is 30 °C, the injection volume is 5 μL, and gradient elution is used for elution; among them, the elution program is: 0-7 min, 98% → 97% mobile phase A, 2% → 3% mobile phase B; 7-12 min, 97% → 90% mobile phase A, 3% → 10% mobile phase B; 12-22 min, 90% → 89% mobile phase A, 10% → 11% mobile phase B; 22-25 min, 89% → 83% mobile phase A, 11% → 17% mobile phase B; 25-35 min, 83% → 79% mobile phase A, 17% → 21% mobile phase B; 35-38 min, 79% → 10% mobile phase A, 21% → 90% mobile phase B.

[0017] Optionally, the corresponding standard curves of the respective components are specifically as follows: the standard curve corresponding to gallic acid is y = 12358x - 8.9826, R = 0.9998; the standard curve corresponding to methyl gallate is y = 14672x - 0.9726, R = 0.9998; the standard curve corresponding to quercitrin is y = 34335x + 0.2465, R = 0.9999; the standard curve corresponding to ellagic acid is y = 67737x - 23.921, R = 0.9997; the standard curve corresponding to chebulic acid is y = 9990.2x - 10.29, R = 0.9995, and the standard curve corresponding to corilagin is y = 11494x - 4.2252, R = 0.9997.

[0018] Optionally, the gallic acid content in different batches of Phyllanthus emblica is measured to be 1.473-3.920 μg / mL, the methyl gallate content is 0.0494-0.1059 μg / mL, the quercitrin content is 0.0318-0.0925 μg / mL, the ellagic acid content is 0.1214-1.468 μg / mL, the chebulic acid content is 0.1450-0.9050 μg / mL, and the corilagin content is 0.2055-0.6772 μg / mL.

[0019] Optionally, in step S3, the specific operation steps for testing the in vitro hepatoprotective biological activity of different batches of Phyllanthus emblica are: taking HepG2 cells, at 2×10 5Inoculate at a ratio of [[ID=]] cells / mL in a 96-well plate and culture it in an incubator at 37 °C with 5% CO₂; when the cells grow to 70% - 90% of the culture dish, aspirate the culture medium; set up a model control group, a drug administration group, and a blank control group respectively. The drug administration group is pretreated with Phyllanthus emblica extract for 12 h, and then the model control group and the drug administration group are respectively treated with 500 mmol / L ethanol for 12 h. Use an ALT test kit to measure the ALT activity in the cell supernatant of each group, and calculate the inhibition rate of Phyllanthus emblica extract on the release of ALT induced by alcohol in HepG2 cells;

[0020] Inhibition rate of ALT release (%) = (ALT activity 模型组 - ALT activity 给药组 ) / ALT activity 模型组 .

[0021] Optionally, the preparation method of the Phyllanthus emblica extract specifically includes: ultrasonically treat with pure water for 30 min, and the material-liquid ratio of Phyllanthus emblica to water is 0.1 g:50 mL, and then filter.

[0022] Optionally, the inhibition rate range of Phyllanthus emblica extracts from different batches on the release of ALT induced by alcohol in HepG2 cells is 9.43% - 38.13%.

[0023] Optionally, in step S3, the specific operation steps for constructing the discrimination model for the quality grading of Phyllanthus emblica are as follows: according to the inhibition rate of Phyllanthus emblica extract on the release of ALT induced by alcohol in HepG2 cells, different batches of Phyllanthus emblica are divided into 3 quality grades: excellent, good, and poor. Among them, the inhibition rate > 24% is excellent, the inhibition rate in the range of 12% - 24% is good, and the inhibition rate < 12% is poor, and response values 3, 2, and 1 are respectively assigned; use the discriminant analysis algorithm to establish the functional relationship between the contents of 6 hepatoprotective active ingredients in each sample and the quality grade (response value), and use SPSS software to solve the parameters of the discrimination model.

[0024] Optionally, in step S3, the discrimination model for the quality grading of Phyllanthus emblica specifically includes: taking the chemical component concentrations of chebulagic acid, quercitrin, ellagic acid, methyl gallate, corilagin, and gallic acid in Phyllanthus emblica as discriminant variables to distinguish different quality grades; in the discriminant formulas of different quality grades, the coefficients (weights) and constant terms of each chemical component are different; by multiplying the content of each component by its weight and summing them up, a linear combination is formed; by calculating the values of three discriminant functions (y 优 , y 良 , y 差 ), the quality of Phyllanthus emblica can be classified into a certain grade.

[0025] Optionally, the specific operation steps of step S4 are as follows: Substitute the measured values of the contents of gallic acid, methyl gallate, quercitrin, ellagic acid, chebulagic acid, and corilagin in Phyllanthus emblica fruits from different batches into the discriminant formula, and the grade corresponding to the maximum y value is the determination result to determine the quality grade of Phyllanthus emblica fruits.

[0026] The present invention also discloses the application of the hepatoprotective quality markers of Phyllanthus emblica fruits in evaluating the quality grade of Phyllanthus emblica fruits, and the hepatoprotective quality markers of Phyllanthus emblica fruits include at least one of gallic acid, methyl gallate, quercitrin, ellagic acid, chebulagic acid, and corilagin.

[0027] Implementing the embodiments of the present invention will have the following beneficial effects:

[0028] Compared with the traditional method of "evaluating quality by appearance", the quality grading method of Phyllanthus emblica fruits based on "evaluating quality by efficacy" proposed by the present invention takes efficacy as the core guiding principle, is closely related to clinical efficacy, and conforms to the purpose of using the medicinal materials. Through the organic combination of mass spectrometry analysis and network analysis, the quality markers that can comprehensively reflect the efficacy of Phyllanthus emblica fruits can be quickly screened out; the established quantitative method for quality markers has good specificity and high accuracy; the discriminant formula constructed with the content of quality markers as the discriminant variable can divide Phyllanthus emblica fruits into three grades according to the hepatoprotective activity of Phyllanthus emblica fruits, which is conducive to promoting the establishment of the "high quality, high price" system of Phyllanthus emblica fruits and promoting the high-quality development of the traditional Chinese medicine industry. Description of the Drawings

[0029] Figure 1 It is the total ion current chromatogram of the Phyllanthus emblica fruit extract and the plasma sample containing the drug under positive and negative ion modes; among them, Figure A is the Phyllanthus emblica fruit extract sample under positive ion mode; Figure B is the Phyllanthus emblica fruit extract sample under negative ion mode; Figure C is the plasma sample containing the drug under positive ion mode; Figure D: The plasma sample containing the drug under negative ion mode.

[0030] Figure 2 It is the "component-target-pathway" network of Phyllanthus emblica fruits against alcoholic liver injury.

[0031] Figure 3 It is the chromatogram of the blank solvent, mixed reference substance, and sample for quantitatively determining 6 quality markers in Phyllanthus emblica fruits at one time by HPLC method established by the present invention.

[0032] Figure 4 The standard curve graph for quantitatively determining 6 quality markers in Phyllanthus emblica fruits at one time by HPLC established by the present invention; among them, Figures A-F are the standard curve graphs of gallic acid, methyl gallate, quercitrin, ellagic acid, chebulagic acid, and corilagin respectively.

[0033] Figure 5 It is the inhibition rate of different batches of Phyllanthus emblica fruits on the release of ALT in HepG2 cells.

[0034] Figure 6 The figure is a scatter plot of the discriminant model for quality grading of Phyllanthus emblica established in the present invention. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0036] Embodiment 1:

[0037] The method for grading the quality of Phyllanthus emblica medicinal materials based on the pharmacodynamics of the present embodiment comprises the following steps:

[0038] (1) Identification of hepatoprotective quality markers of Phyllanthus emblica

[0039] a. Preparation of Phyllanthus emblica extract: Weigh an appropriate amount of Phyllanthus emblica powder, heat and reflux with 20 times the amount of 25-95% methanol for 1 hour. Let cool, shake well, centrifuge, filter with a 0.22μm microporous membrane, and store in a 4℃ refrigerator for later use.

[0040] b. Preparation of intragastric solution: The solution in (1) was concentrated at low temperature until the solvent was completely evaporated, and water was added to prepare a solution containing 94 mg of crude drug per 1 mL to obtain the intragastric solution of Phyllanthus emblica.

[0041] c. Dosage regimen: Six healthy male SD rats were selected and fed in an SPF animal laboratory for 7 days before the experiment. Rats were given emblica emblica liquid (940 mg kg -1 ), continuously gavage for 7 days.

[0042] d. Collection of plasma samples: Blood was collected from the orbital vein of rats 2 h before the first dose and 0.5 h, 1 h, and 2 h after the last dose. The blood was placed in a heparinized EP tube and immediately centrifuged at 4°C and 4000 rpm for 10 min. The supernatant was stored in a -80°C refrigerator for later use.

[0043] e. Plasma sample pretreatment: The plasma samples of rats at each time point after the last administration were mixed, 1 mL of the mixed plasma sample was taken, 500 μL of 1% formic acid and 4 mL of methanol were added, vortexed for 3 min, and centrifuged for 5 min (4°C, 12000 rpm). The supernatant was blown under nitrogen flow, the residue was re-dissolved with 50 μL of methanol, vortexed for 3 min, ultrasonicated for 5 min, centrifuged for 5 min (4°C, 12000 rpm), and the supernatant was taken for analysis.

[0044] f. Chromatographic and mass spectrometry conditions

[0045] The chromatographic column is an octadecylsilyl-bonded silica gel column. A 0.01 vol% formic acid aqueous solution (A) - acetonitrile (B) is used as the mobile phase, and the flow rate is 0.6 mL / min -1 , the column temperature is 30 °C, the injection volume is 4 μL, and gradient elution is carried out; among them, the elution program is: 0 - 7 min, 98% → 97% mobile phase A, 2% → 3% mobile phase B; 7 - 12 min, 97% → 90% mobile phase A, 3% → 10% mobile phase B; 12 - 25 min, 90% → 83% mobile phase A, 10% → 17% mobile phase B; 25 - 35 min, 83% → 79% mobile phase A, 17% → 21% mobile phase B; 35 - 38 min, 79% → 10% mobile phase A, 21% → 90% mobile phase B; 38 - 42 min, 10% mobile phase A, 90% mobile phase B; 42 - 45 min, 10% → 98% mobile phase A, 90% → 2% mobile phase B; 45 - 50 min, 98% mobile phase A, 2% mobile phase B.

[0046] The ion source is an electrospray ionization source (ESI source); the detection mode is positive and negative ion detection; spray voltage: 5500 V (+) / 4500 V (-); ion source temperature: 550 °C; nebulizing gas (Gas1): 50 psi; auxiliary gas (Gas 2): 50 psi; curtain gas: 30 psi; the nebulizing gas and other auxiliary gases are all nitrogen. The scanning range in TOF MS mode is m / z 50 - 1000 Da; declustering voltage (DP): ±80 V; collision energy (CE): ±10 V. The scanning range in TOF MS / MS mode is m / z 50 - 1000 Da; declustering voltage (DP): ±80 V; collision energy (CE): ±35 V.

[0047] Under the above conditions, high-resolution mass spectrometry analysis is carried out. The total ion current chromatograms of the Phyllanthus emblica extract and the plasma samples containing drugs in positive and negative ion modes are shown in Figure 1 .

[0048] g. Qualitative analysis process

[0049] First, analyze the mass spectrometry results of Phyllanthus emblica extract. Use the multi-characteristic fragment ion filtering technique (mPIF) and neutral loss filtering technique (NLF) in the mass spectrometry data processing software to screen out compounds containing the same daughter ions or the same neutral loss molecules, thereby excluding a large amount of interference information of other non-target compounds. Among the remaining valid information, based on the precursor ion mass accuracy, mass spectrometry fragmentation rules, and chromatographic retention behavior, conduct compound structure identification and construct an in vitro compound database of Phyllanthus emblica. Subsequently, analyze the results of plasma samples after intragastric administration of Phyllanthus emblica to rats, screen out the ions that do not exist in blank biological samples but exist in drug-containing biological samples, and use the established in vitro compound database of Phyllanthus emblica to screen and confirm the prototype components of Phyllanthus emblica entering the blood.

[0050] Under the above conditions, qualitative analysis was carried out, and 12 prototype components entering the blood in Phyllanthus emblica were obtained, including gallic acid, methyl gallate, quercitrin, ellagic acid, chebulagic acid, corilagin, geraniin, digalloyl glucose, trigalloyl glucose, vitamin C, ellagic acid deoxyhexoside, and malic acid.

[0051] h. Construction of the "component-target-pathway" network: Use the Swiss TargetPrediction website to predict the targets related to the prototype components of Phyllanthus emblica entering the blood, collect the targets related to alcoholic liver injury from the GeneCards and OMIM databases, and obtain the intersection target genes of components and diseases; use the DAVID Bioinformatics Resources platform to perform KEGG pathway enrichment on the intersection target genes, and use Cytoscape software to construct the "component-target-pathway" network; use the CytoNCA plug-in to analyze the network topology parameters, and select the components with a Degree value greater than the median in the network as potential quality markers.

[0052] The "component-target-pathway" network obtained by the above scheme is shown as Figure 2 , and the present invention obtains a group of hepatoprotective quality markers of Phyllanthus emblica, including gallic acid, methyl gallate, quercitrin, ellagic acid, chebulagic acid, corilagin, etc.

[0053] (2) Simultaneous quantitative analysis of the group of quality markers of Phyllanthus emblica

[0054] a. Preparation of the reference substance solution: Take appropriate amounts of reference substances of gallic acid, methyl gallate, quercitrin, ellagic acid, chebulagic acid, and corilagin, accurately weigh them, dissolve them with methanol and dilute them to concentrations of 200.0 - 400.0 μg·mL -1 , 5.00 - 10.00 μg·mL -1 , 3.00 - 6.00 μg·mL -1 , 20.00 - 40.00 μg·mL-1 、20.00 - 40.00 μg·mL -1 、20.00 - 40.00 μg·mL -1 of the mixed reference solution.

[0055] b. Preparation of the reference solution: Weigh accurately about 0.1 g of Phyllanthus emblica powder (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 mL of 25 - 95% methanol, weigh, heat under reflux for 1 h, cool, weigh again, make up the weight loss with 25 - 95% methanol, shake well, filter, and take the subsequent filtrate, that is, obtain.

[0056] c. Chromatographic conditions: The chromatographic column is an octadecylsilane - bonded silica gel column, using 0.05 vol% phosphoric acid aqueous solution (A) - acetonitrile (B) as the mobile phase, the flow rate is 0.6 mL / min -1 , the detection wavelength is 254 nm, the column temperature is 30 °C, the injection volume is 5 μL, and gradient elution is carried out; among them, the elution program is: 0 - 7 min, 98% → 97% mobile phase A, 2% → 3% mobile phase B; 7 - 12 min, 97% → 90% mobile phase A, 3% → 10% mobile phase B; 12 - 22 min, 90% → 89% mobile phase A, 10% → 11% mobile phase B; 22 - 25 min, 89% → 83% mobile phase A, 11% → 17% mobile phase B; 25 - 35 min, 83% → 79% mobile phase A, 17% → 21% mobile phase B; 35 - 38 min, 79% → 10% mobile phase A, 21% → 90% mobile phase B

[0057] d. Take reference solutions with different concentrations and perform ultra - high performance liquid chromatography detection under the above chromatographic conditions to obtain the ultra - high performance liquid chromatograms corresponding to the reference solutions with different concentrations, and obtain the corresponding peak areas for the determination of the concentrations of each component.

[0058] According to the concentrations of each component and the corresponding peak areas for the determination of the concentrations of the corresponding components, draw the standard curves for different components, and use the concentrations of each component in the reference solution as the abscissa (i.e., x, unit: μg / mL), and the peak area (A) corresponding to the determination of the concentration of the corresponding component as the ordinate (i.e., y), and find its linear regression equation;

[0059] The standard curves drawn using reference solution series with different concentrations are as follows:

[0060] The standard curve for gallic acid is y = 12358x - 8.9826, R = 0.9998, and the linear range is 11.50 - 230.0 μg / mL, see Figure 3 A;

[0061] The standard curve corresponding to methyl gallate is y = 14672x - 0.9726, R = 0.9998, and the linear range is 0.5200 - 10.40 μg / mL, as shown in Figure 3 B;

[0062] The standard curve corresponding to quercitrin is y = 34335x + 0.2465, R = 0.9999, and the linear range is 0.2400 - 4.320 μg / mL, as shown in Figure 3 C;

[0063] The standard curve corresponding to ellagic acid is y = 67737x - 23.921, R = 0.9997, and the linear range is 1.500 - 30.00 μg / mL, Figure 3 D;

[0064] The standard curve corresponding to chebulagic acid is y = 9990.2x - 10.29, R = 0.9995, and the linear range is 1.520 - 30.40 μg / mL, as shown in Figure 3 E;

[0065] The standard curve corresponding to corilagin is y = 11494x - 4.2252, R = 0.9997, and the linear range is 1.160 - 23.20 μg / mL, as shown in Figure 3 F;

[0066] Take the test solution under the above chromatographic conditions for ultra-high performance liquid chromatography detection to obtain the ultra-high performance liquid chromatogram of the test solution, as shown in Figure 4 ;

[0067] Meanwhile, from the ultra-high performance liquid chromatogram of the test solution, obtain the peak areas corresponding to each component in the test solution, substitute the peak areas of the corresponding components into the corresponding standard curves, and calculate the concentrations C 供试品-相应成分 .

[0068] The content of the corresponding component in Phyllanthus emblica = C 供试品-相应成分 ×V / m, and calculate the content of each component in Phyllanthus emblica.

[0069] Using the above method, the gallic acid content in different batches of Phyllanthus emblica was measured to be between 1.473 - 3.920 μg / mL, the methyl gallate content was between 0.0494 - 0.1059 μg / mL, the quercitrin content was between 0.0318 - 0.0925 μg / mL, the ellagic acid content was between 0.1214 - 1.468 μg / mL, the chebulagic acid content was between 0.1450 - 0.9050 μg / mL, and the corilagin content was between 0.2055 - 0.6772 μg / mL.

[0070] (3) Construction of the quality grading model for Phyllanthus emblica

[0071] a. Determination of in vitro liver - protecting biological activity of Phyllanthus emblica fruits from different batches: Take HepG2 cells and inoculate them into 96 - well plates at a ratio of 2×10 5 cells / mL, and place them in an incubator at 37°C and 5% CO2 for culture. When the cells grow to 70% - 90% of the culture dish, aspirate the culture medium. The treatment group is pretreated with the extract of Phyllanthus emblica for 12 h. After 12 h, the model control group and the treatment group are respectively treated with 500 mmol / L ethanol for 12 h, and the blank control group is not treated. Use an ALT test kit to measure the ALT activity in the cell supernatant of each group, and calculate the inhibition rate of the extract of Phyllanthus emblica on the release of ALT in alcohol - induced HepG2 cells.

[0072] Using the above - mentioned protocol, the inhibition rates of Phyllanthus emblica fruits from different batches on the release of ALT in HepG2 cells were measured to be between 9.43% and 38.13%, as shown in Figure 5 .

[0073] b. Model construction: According to the in vitro liver - protecting biological activity of Phyllanthus emblica fruits, fruits from different batches are divided into three quality grades: excellent (inhibition rate > 24%), good (inhibition rate between 12% and 24%), and poor (release inhibition rate < 12%), and response values 3, 2, and 1 are respectively assigned. The discriminant analysis algorithm is used to establish the functional relationship between the contents of 6 liver - protecting active components in each sample and the quality grade (response value), and the SPSS software is used to solve the parameters of the discriminant analysis model.

[0074] Using the above - mentioned protocol, the discriminant formula for the quality grading of Phyllanthus emblica fruits is obtained as follows:

[0075] y 优 = 44.003*C 诃黎勒酸 + 613.556*C 槲皮苷 + 18.449*C 鞣花酸 - 7.853*C 没食子酸甲酯 + 21.879*C 柯里拉京 + 25.234*C 没食子酸 - 90.451

[0076] y 良 = 5.921*C 诃黎勒酸 + 260.236*C 槲皮苷 + 3.559*C 鞣花酸 + 429.156*C 没食子酸甲酯 + 10.998*C 柯里拉京 + 11.294*C 没食子酸 - 40.024

[0077] y 差 = 15.933*C 诃黎勒酸 + 301.579*C槲皮苷 +4.712*C 鞣花酸 +89.224*C 没食子酸甲酯 +17.644*C 柯里拉京 +13.629*C 没食子酸 -28.994

[0078] The above discriminant uses the concentrations of multiple chemical components in Phyllanthus emblica (chebulagic acid, quercitrin, ellagic acid, methyl gallate, corilagin, gallic acid) as discriminant variables to distinguish different quality grades (excellent, good, poor); in the discriminants for different quality grades, the coefficients (weights) and constant terms of each chemical component are different; by multiplying the concentration of each component by its weight and summing them up, a linear combination is formed; by calculating the values of three discriminant functions (y 优 , y 良 , y 差 ), the quality of Phyllanthus emblica can be classified into a certain grade.

[0079] (4) Application of the quality grading model of Phyllanthus emblica

[0080] Substitute the measured values of the contents of gallic acid, methyl gallate, quercitrin, ellagic acid, chebulagic acid and corilagin in different batches of Phyllanthus emblica into the above discriminant analysis expression, so as to determine the quality grade of Phyllanthus emblica (the grade corresponding to the maximum y value is the judgment result). As Figure 6 shown, the Phyllanthus emblica samples of the same grade show clustering behavior, and the Phyllanthus emblica samples of different grades are clearly separated, indicating that the established discriminant model can effectively distinguish Phyllanthus emblica samples of different grades.

[0081] The above embodiments only represent several implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A method for grading the quality of Phyllanthus emblica medicinal materials based on pharmacodynamics, characterized in that: The following steps are involved: S1. Identification of hepatoprotective quality markers in Phyllanthus emblica: Use time-of-flight high-resolution mass spectrometry to analyze the blood-entering components of Phyllanthus emblica; construct a "component-target-pathway" network to screen the hepatoprotective quality markers in Phyllanthus emblica; S2. One-time quantitative analysis of the quality marker group of Phyllanthus emblica: Establish an HPLC method for simultaneously determining the liver-protective quality marker group in Phyllanthus emblica; Determine the content of liver-protective quality markers in different batches of Phyllanthus emblica; S3. Construction of a quality grading model for Phyllanthus emblica: Test the in vitro hepatoprotective biological activity of different batches of Phyllanthus emblica; grade different batches of Phyllanthus emblica according to their hepatoprotective activity, and construct a discriminant model for the quality grading of Phyllanthus emblica using the content of hepatoprotective quality markers as discriminant variables; S4. Application of discriminant analysis model for quality grading of Phyllanthus emblica.

2. The method for grading the quality of Phyllanthus emblica medicinal materials based on the pharmacodynamics according to claim 1, characterized in that: The specific operation steps of step S1 are as follows: using a time-of-flight high-resolution mass spectrometer, by comparing the water extract of Phyllanthus emblica, blank serum and drug-containing serum, and by comparing the secondary spectra of the reference substance with the sample, the blood-entering components of Phyllanthus emblica were identified; using the Swiss TargetPrediction website to predict the targets of the blood-entering components, collecting alcoholic liver injury-related targets from the GeneCards and OMIM databases, obtaining component-disease intersection target genes, using the DAVID Bioinformatics Resources platform to enrich the KEGG pathway, using the Cytoscape software to construct a "component-target-pathway" network, using the CytoNCA plug-in to analyze the network topology parameters, and selecting components with Degree values ​​greater than the median in the network as potential quality markers.

3. The method for grading the quality of Phyllanthus emblica medicinal materials based on the pharmacodynamics of claim 1, characterized in that: The group of hepatoprotective quality markers of Phyllanthus emblica includes at least one of gallic acid, methyl gallate, quercetin, ellagic acid, cheleic acid and corilagin.

4. The method for grading the quality of Phyllanthus emblica medicinal materials based on the pharmacodynamics of claim 1, characterized in that: The specific operation steps of step S2 are: Prepare a mixed reference solution by taking gallic acid, methyl gallate, quercetin, ellagic acid, cheleic acid and corilagin; Take Phyllanthus emblica to prepare the test solution; The mixed reference solution and the test solution are respectively subjected to ultra-high performance liquid chromatography detection, and the peak area corresponding to each component is substituted into the corresponding standard curve of each component to calculate the contents of gallic acid, methyl gallate, quercetin, ellagic acid, cheleic acid and corilagin in Phyllanthus emblica.

5. The method for grading the quality of Phyllanthus emblica medicinal materials based on the pharmacodynamics of claim 4, characterized in that: In the mixed reference solution, the concentration of gallic acid is 200.0 to 400.0 μg mL -1 , the concentration of methyl gallate is 5.00~10.00μg·mL -1 The concentration of quercetin is 3.00~6.00μg·mL -1 , the concentration of ellagic acid is 20.00~40.00μg·mL -1 The concentration of cheleic acid is 20.00~40.00μg·mL -1 The concentration of corilagin is 20.00~40.00μg·mL -1 ; The method of preparing a test solution from the emblica buds includes: heating with 25-95% methanol under reflux for 1 hour and cooling to make up for the weight loss and filtering, wherein the material-liquid ratio of the emblica buds to methanol is 0.1 g:50 mL; The ultra-high performance liquid chromatography detection specifically includes: the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, and 0.05 vol% phosphoric acid aqueous solution (A)-acetonitrile (B) is used as the mobile phase, and the flow rate is 0.6 mL min -1 , the detection wavelength was 254 nm, the column temperature was 30°C, the injection volume was 5 μL, and elution was performed in a gradient elution manner; wherein, the elution program was: 0-7 min, 98%→97% mobile phase A, 2%→3% mobile phase B; 7-12 min, 97%→90% mobile phase A, 3%→10% mobile phase B; 12-22 min, 90%→89% mobile phase A, 10%→11% mobile phase B; 22-25 min, 89%→83% mobile phase A, 11%→17% mobile phase B; 25-35 min, 83%→79% mobile phase A, 17%→21% mobile phase B; 35-38 min, 79%→10% mobile phase A, 21%→90% mobile phase B.

6. The method for grading the quality of Phyllanthus emblica medicinal materials based on the pharmacodynamics of claim 1, characterized in that: In step S3, the specific operation steps of testing the in vitro hepatoprotective biological activity of different batches of Phyllanthus emblica are as follows: HepG2 cells were taken and 2×10 5 The cells were inoculated into a 96-well plate at a ratio of 100 cells / mL and cultured in a 37°C, 5% CO2 incubator; when the cells grew to 70% to 90% of the culture dish, the culture medium was aspirated; a model control group, a drug-treated group, and a blank control group were set up respectively, and the drug-treated group was pre-treated with the extract of Phyllanthus emblica for 12 hours, and then the model control group and the drug-treated group were treated with 500mmol / L ethanol for 12 hours, respectively, and the ALT activity in the cell supernatant of each group was determined using an ALT test kit, and the inhibition rate of the extract of Phyllanthus emblica on the ALT release of HepG2 cells induced by alcohol was calculated; ALT release inhibition rate (%) = (ALT activity 模型组 -ALT Vitality 给药组 ) / ALT Vitality 模型组 ; The preparation method of the emblica extract specifically comprises: using pure water for ultrasonic treatment for 30 minutes, the solid-liquid ratio of emblica to water is 0.1g:50mL, and then filtering; The inhibition rates of different batches of Phyllanthus emblica extract on alcohol-induced ALT release in HepG2 cells ranged from 9.43% to 38.13%.

7. The method for grading the quality of Phyllanthus emblica medicinal materials based on the pharmacodynamics of claim 1, characterized in that: In step S3, the specific operation steps of constructing the discriminant model for quality grading of Phyllanthus emblica are as follows: according to the inhibition rate of Phyllanthus emblica extract on alcohol-induced ALT release of HepG2 cells, different batches of Phyllanthus emblica are divided into three quality grades: excellent, good and poor, wherein an inhibition rate greater than 24% is excellent, an inhibition rate in the range of 12% to 24% is good, and an inhibition rate less than 12% is poor, and response values ​​of 3, 2, and 1 are assigned respectively; a discriminant analysis algorithm is used to establish a functional relationship between the content of 6 liver-protecting active ingredients in each sample and the quality grade (response value), and SPSS software is used to solve the discriminant model parameters.

8. The method for grading the quality of Phyllanthus emblica medicinal materials based on the pharmacodynamics of claim 7, characterized in that: In step S3, the quality grading discriminant model of Phyllanthus emblica specifically includes: using the concentrations of the chemical components of chebulic acid, quercetin, ellagic acid, methyl gallate, corilagin and gallic acid in Phyllanthus emblica as discriminant variables to distinguish different quality grades; in the discriminant formulas of different quality grades, the coefficients (weights) and constant terms of each chemical component are different; a linear combination is formed by multiplying the content of each component by its weight and summing them; by calculating the three discriminant function values ​​(y 优 ,y 良 ,y 差 ), the quality of Phyllanthus emblica can be classified into a certain grade.

9. The method for grading the quality of Phyllanthus emblica medicinal materials based on the pharmacodynamics of claim 8, characterized in that: The specific operation steps of step S4 are: substituting the measured values ​​of gallic acid, methyl gallate, quercetin, ellagic acid, cheleic acid and corilagin in different batches of emblica into the discriminant formula, and the grade corresponding to the maximum y value is the judgment result to determine the quality grade of emblica.

10. Application of the hepatoprotective quality marker of Phyllanthus emblica in evaluating the quality grade of Phyllanthus emblica, characterized in that: The hepatoprotective quality marker of Phyllanthus emblica includes at least one of gallic acid, methyl gallate, quercetin, ellagic acid, cheleic acid and corilagin.

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