Gastrodia elata grading method based on Q-marker and application of gastrodia elata grading method

Abstract

The invention provides a gastrodia elata grading method based on Q-marker and application of the gastrodia elata grading method, and belongs to the technical field of traditional Chinese medicine genuine medicinal material grading evaluation. The method comprises the following steps: firstly, determining six candidate compounds as gastrodin, p-hydroxybenzyl alcohol, parishin A, parishin B, parishin C and parishin E; and verifying that the six candidate compounds are Q-markers with the antioxidant activity of the gastrodia elata by integrating multi-dimensional data such as contents of the six candidate compounds, in-vitro anti-free radical (ABTS) and in-vivo antioxidant (nematode H2O2 damage resistance) biological activity. According to the method, a Q-marker coefficient integrating candidate compound content, in-vitro chemical activity and in-vivo biological activity is constructed by using a radar map area, an integrated quantity-effect model for rapidly predicting the antioxidant activity of gastrodia elata through the Q-marker content is established, a result shows that a model predicted value is consistent with an experiment, and the score of high-quality gastrodia elata with good antioxidant and anti-aging activity is greater than or equal to 8.79.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine (TCM) genuine medicinal material grading evaluation, in particular to a Gastrodia elata grading method based on Q-marker and application thereof. BACKGROUND

[0002] Traditional TCM quality control methods are mostly based on the appearance of medicinal materials, which are not accurate enough; modern methods focus on chemical composition control, which is accurate but difficult to directly reflect the fundamental requirements of TCM decoction pieces efficacy. Bioactivity-based TCM potency evaluation can be associated with the clinical efficacy of drugs, but TCM potency evaluation has problems such as high experimental requirements, long cycle, poor precision and reproducibility. Therefore, it is very important to establish a chemical-biological potency related evaluation system. Academician Liu Changxiao proposed the concept of TCM quality marker (Q-Marker), which closely combines efficacy and chemical composition and has received nationwide attention. However, how to integrate multiple Q-marker components into a complete quality evaluation system for the grading evaluation of genuine medicinal materials has rarely been reported.

[0003] Gastrodia elata is a traditional Chinese precious medicinal material, which has the traditional effects of relieving convulsions, calming liver yang, and expelling wind and dredging collaterals. Modern research has found that Gastrodia elata has good anti-inflammatory, antioxidant, anti-aging, anticonvulsant and sedative effects. The activity of Gastrodia elata is mainly derived from its rich phenolic acid compounds, among which gastrodin (Gas) has a sleep-inducing effect, and bartzin A has a DOX-induced cardiotoxicity relieving effect. Traditional quality control methods for Gastrodia elata use appearance evaluation, and modern methods mostly use chemical fingerprint and multi-component content determination methods of gastrodin and bartzin to carry out effective component quality control research, which can be accurately determined, but cannot directly reflect the biological activity of Gastrodia elata.

[0004] How to quickly predict the antioxidant activity of Gastrodia elata to realize the comprehensive evaluation of chemical components and biological activity, and determine the quality grading limit of Gastrodia elata through a scoring system is a work worth studying, and has certain reference value for the research of other TCMs. SUMMARY

[0005] The present application aims at the above-mentioned deficiencies of the prior art, and provides a Gastrodia elata grading method based on Q-marker and application thereof. Taking the antioxidant activity of Gastrodia elata as an example, a comprehensive scoring system of Q-marker coefficient is established, so that the antioxidant activity of Gastrodia elata can be quantitatively scored and predicted by determining the content of Q-marker components, and the accuracy of the model is verified in real samples.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: The first objective of this invention is to provide a Q-marker-based method for grading Gastrodia elata, comprising the following steps: S1. Determine the content of 6 candidate compounds in the Gastrodia elata sample, wherein the candidate compounds are gastrodin, p-hydroxybenzyl alcohol, barisonoside A, barisonoside B, barisonoside C and barisonoside E; ABTS free radical scavenging test was performed on Gastrodia elata samples; Oxidative stress experiment of Caenorhabditis elegans on Gastrodia elata samples; S2. The contributions of the six candidate compounds to the ABTS· scavenging ability of Gastrodia elata and the oxidative stress protection ability of Caenorhabditis elegans were calculated by the change of the "dose-effect" slope, as shown in Equations (1) and (2): (1); (2), in Scoring the contribution of different candidate compounds to ABTS· scavenging ability; Six different candidate compounds; To determine the concentration of the added candidate compound; To add ABTS clearance rate at certain concentrations; The ABTS scavenging rate without the addition of candidate compounds; The number of samples added to the monomer; Scoring was assigned to the contribution of different candidate compounds to the survival rate of Caenorhabditis elegans. To add Survival rate of *C. elegans* at different concentrations; Survival rate of *C. elegans* without the addition of candidate compounds; S3. Data normalization was used to process the content distribution trends, ABTS scavenging ability contribution, and oxidative stress protection contribution of the six candidate compounds in Gastrodia elata, and normalized values ​​of the content of different candidate compounds in Gastrodia elata were obtained. Normalized scores of the contribution of different candidate compounds to ABTS· scavenging ability Normalized scores of the contribution of different candidate compounds to the survival rate of Caenorhabditis elegans ; S6, will , and The radar chart is drawn in three dimensions as a three-dimensional radar chart. After the area of ​​the radar chart is normalized, the Q-Marker coefficients of different candidate compounds are obtained. S7、According to the obtained Q-Marker coefficient, the antioxidant score of the Gastrodia sample is calculated according to formula (9), and the Gastrodia is graded according to the antioxidant score; (9) In the formula, is the antioxidant score of the Gastrodia sample, is the Q-Marker coefficient of the candidate compound, is the content of a single candidate compound in the Gastrodia sample; S8, the Gastrodia sample is divided into two categories of high quality and ordinary by using a support vector machine model, and a grading boundary QS is calculated and determined 界限 , and it is determined that the antioxidant score of high-quality Gastrodia should be no less than QS 界限 .

[0007] Further, the calculation formula of the Q-Marker coefficient is: (3), In the formula, is the normalized value of the content dimension of different candidate compounds in Gastrodia; are 6 different candidate compounds; is the average content of a single candidate compound in the Gastrodia sample; is the maximum value of the average content of the 6 candidate compounds.

[0008] Further, the calculation formula of the Q-Marker coefficient is: (4), In the formula, is the normalized value of the contribution score of different candidate compounds to the ABTS· clearance ability; is the contribution degree of the ABTS· clearance ability of a single candidate compound in the Gastrodia sample, is the maximum value of the contribution degree of the ABTS· clearance ability of the 6 candidate compounds.

[0009] Further, the calculation formula of the Q-Marker coefficient is: (5), In the formula, is the normalized value of the contribution score of different candidate compounds to the survival rate of C. elegans; is the contribution degree of the C. elegans oxidative stress protection ability of a single candidate compound in the Gastrodia sample; is the maximum value of the contribution degree of the C. elegans oxidative stress protection ability of the 6 candidate compounds.

[0010] Further, the area of the radar chart is​​​ , calculated by formula (6): (6); wherein, (7), wherein, is the normalized value of different candidate compounds in the content dimension of Gastrodia elata; is the normalized value of the contribution degree score of different candidate compounds to the ABTS· scavenging ability; is the normalized value of the contribution degree score of different candidate compounds to the survival rate of C. elegans.

[0011] Further, the method for normalizing the area of the radar chart is: (8) wherein, is the normalized value of different candidate compounds , which is also the Q-Marker coefficient of the six different candidate compounds; is the highest value of in the six candidate compounds.

[0012] Further, the method for determining the content of the six candidate compounds in the Gastrodia elata sample is: high performance liquid chromatography is used for determination, a chromatographic column with octadecylsilane bonded silica gel as the filler, 0.1% formic acid aqueous solution and acetonitrile as the mobile phase, gradient elution is performed, the elution program is: 0-8 min, 3-10% acetonitrile; 8-15 min, 10-12% acetonitrile; 15-25 min, 15-18% acetonitrile; 25-35 min, 18% acetonitrile; 35-35.1 min, 18-95% acetonitrile, the column temperature is 30 DEG C, and the detection wavelength is 220 nm.

[0013] Further, in the ABTS· free radical scavenging experiment, vitamin C is used as a positive drug for comparison, the absorbance is detected at 734 nm by using an enzyme marker, non-linear regression is used for analysis, and the IC 50 value of the sample to be tested is calculated.

[0014] Further, in the C. elegans oxidative stress experiment, a H2O2 oxidative damage model is used to evaluate the antioxidant effect of the Gastrodia elata extract.

[0015] The second object of the application provides the application of the above method in the quality control of Gastrodia elata.

[0016] Compared with the prior art, the application has the following beneficial effects: (1) The application provides a Gastrodia elata grading method based on Q-marker, which integrates Q-marker content, in vitro anti-free radical (ABTS) and in vivo antioxidant (anti-nematode H2O2 damage) biological activity and other multi-dimensional data, and creates a quantitative model for predicting antioxidant activity by determining Q-marker content. Briefly, the contents of 6 main blood-entering chemical components in 27 batches of GE: Gastrodin (Gas), p-hydroxybenzyl alcohol (HBA), Parishin A (PA), Parishin B (PB), Parishin C (PC) and Parishin E (PE), IC 50 values of ABTS· clearance, using principal component analysis (PCA) to divide Gastrodia elata into two grades of "high quality" and "qualified", calculating the correlation degree of the six components and the antioxidant activity of Gastrodia elata, and then verifying that the six components are Q-marker of the antioxidant activity of Gastrodia elata through ABTS· clearance experiment and H2O2 Caenorhabditis elegans aging model. Finally, the Q-marker coefficient integrating the content of chemical components, in vitro chemical activity and in vivo biological activity is constructed by using radar chart area, and an integrated quantitative-effect model for quickly predicting the antioxidant activity of Gastrodia elata by Q-marker content is established, and the experiment is verified in real samples. The results show that the model prediction value is consistent with the experiment (R 2 =0.8062), and it is considered that the high-quality Gastrodia elata with good antioxidant aging activity has a score greater than or equal to 8.79.

[0017] (2) The application provides a Gastrodia elata grading method based on Q-marker, which can quickly predict the antioxidant activity of Gastrodia elata by determining the content of Q-marker within 1 hour compared with 5 days required by H2O2 Caenorhabditis elegans aging experiment, realizes the comprehensive evaluation of chemical components and biological activity, and determines the quality grading limit of Gastrodia elata based on the activity through a scoring system, which provides a new method for quality control of Gastrodia elata and has certain reference value for research on other traditional Chinese medicinal materials. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the standard fingerprint of 6 candidate compounds in Gastrodia elata, 1: Gas; 2: HBA; 3: PE; 4: PB; 5: PC; 6: PA; Figure 2 is the structural formula of 6 candidate compounds in Gastrodia elata; Figure 3 is a typical dose-effect curve of ABTS· clearance experiment of Gastrodia elata extract; Figure 4Figure for the result of PCA correlation calculation between ABTS· scavenging capacity of Gastrodia elata extract and content of 6 components; Figure 5 Correlation coefficient between ABTS· scavenging capacity of Gastrodia elata extract and content of 6 candidate compounds; Figure 6 Figure for the change of ABTS· scavenging capacity of Gastrodia elata sample after adding 6 candidate compounds, (A) for adding Gas, (B) for adding HBA, (C) for adding PA, (D) for adding PB, (E) for adding PC, and (F) for adding PE; Figure 7 Figure for the change of protective capacity of Gastrodia elata sample on C.E. H2O2 damage model after adding 6 candidate compounds, (A) for adding Gas, (B) for adding HBA, (C) for adding PA, (D) for adding PB, (E) for adding PC, and (F) for adding PE; Figure 8 Figure for the content distribution trend of 6 candidate compounds in 60 batches of Gastrodia elata; Figure 9 Figure for the contribution degree of 6 candidate compounds to ABTS· scavenging capacity of Gastrodia elata extract; Figure 10 Figure for the contribution degree of 6 candidate compounds to protection of C.E. stimulated by H2O2; Figure 11 Figure for the multi-dimensional evaluation system of 6 candidate compounds by establishing visual radar chart through content-in vivo activity-in vitro activity three-dimensional; Figure 12 Figure for the Q-Marker scoring system obtained by calculating the area of visual radar chart of 6 candidate compounds; Figure 13 Figure for the survival rate of C.E. after using 7 different batches of Gastrodia elata extract to protect C.E. for 24 h, and then stimulating C.E. with H2O2 for 1 h; Figure 14 Figure for the linear fitting of C.E. survival rate in Figure 13 and antioxidant score, Y is the survival rate of C.E., and X is the antioxidant score of Gastrodia elata; Figure 15 Figure for the result visualization of SVM classifying Gastrodia elata into high-quality and ordinary two categories. DETAILED DESCRIPTION

[0019] In order to make the objects, technical solutions, and advantages of the present application clearer, the embodiments of the present application are described in detail below, examples of which are shown in the drawings, wherein identical or similar labels represent identical or similar elements or elements with identical or similar functions throughout. The embodiments described below by referring to the drawings are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0020] The materials, reagents, and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0021] The fresh Gastrodia elata samples collected in the present application are from five provinces of Yunnan, Guizhou, Hubei, Anhui, and Shaanxi, and are identified as rhizomes of Gastrodia elata of Orchidaceae. The fresh Gastrodia elata samples are steamed, cut into 2 mm slices, dried in an oven at 50 ℃ for 36 h, and then ground and sieved to obtain 87 batches of Gastrodia elata powder samples.

[0022] Example 1 The present embodiment provides a Gastrodia elata grading method based on Q-marker, and the specific steps are as follows: 1. Chemical component analysis Take the powder (0.2 g) of each batch of Gastrodia elata sample and transfer it to a 10 mL volumetric flask. Extract it with 50% methanol aqueous solution at 25 ℃ for 30 min, cool it to room temperature, and then dilute it to the calibration mark with 50% methanol aqueous solution. Then, centrifuge the sample solution at 4 ℃ and 3500 rpm for 15 min. Take the supernatant after centrifugation, filter it, and then analyze it by HPLC-DAD.

[0023] Perform chromatographic analysis on an Agilent 1290 HPLC-DAD system, and the fingerprint map conditions and content determination conditions: perform gradient elution on a ZORBAX SB-C18 reversed-phase column (4.6 × 250 mm, 5 μm) at 30 ℃, with 0.1% phosphoric acid aqueous solution (A) and acetonitrile (B) as the mobile phase, a flow rate of 0.800 mL / min, and the following gradient elution program: 0-8 min, 3-10% B; 8-15 min, 10-12% B; 15-25 min, 15-18% B; 25-35 min, 18% B; 35-35.1 min, 18-95% B. The detection wavelength is set to 220 nm. The injection amount is 10 μL.

[0024] The fingerprint map is analyzed by Agilent Data Analyse and Chinese medicine chromatographic fingerprint similarity evaluation system software, and the similarity of 87 batches ranges from 0.835 to 0.997.

[0025] The blood component analysis was performed according to the experimental method of Lee et al. with some modifications (Lee et al., 2023). Twelve male mice were randomly divided into blank and drug groups (n = 6). After fasting for 12 h, the mice were given a gavage of Gastrodia extract at a dose of 1.7 g / kg (crude drug / body weight). Blood plasma was collected before administration and at 0, 1, 1.5, 2, 2.5, and 3 h after administration and stored at -80°C. The plasma samples were prepared by mixing the plasma of the blank and drug groups at different times (about 1.0 ml), adding 3 times the volume of ice methanol, vortexing, centrifuging at 4°C at 15000 rpm for 30 min, taking the supernatant, vacuum centrifuging to dryness, redissolving with 100 ml of 50% methanol-water solution, filtering with a 0.22 mm filter, and then analyzing by HPLC-UV. The analysis conditions were as follows: Gradient elution was performed on a ZORBAX SB-C18 reversed-phase column (4.6 × 250 mm, 5 μm) at 30°C with a mobile phase consisting of 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.800 mL / min. The following gradient elution program was used: 0-5 min, 5% B; 5-50 min, 5-17.1% B; 50-63 min, 17.1-90% B. The detection wavelength was set to 220 nm. The injection volume was 10 μL.

[0026] Based on the HPLC-DAD method, multi-component quantitative analysis of Gastrodia extract was performed, and the typical chromatogram of the sample solution is shown in Figure 1 Through detection of mouse serum samples, six common candidate compounds in Gastrodia were determined (as shown in Figure 2 ), which were Gastrodin (Gas), p-hydroxybenzyl alcohol (HBA), Parishin A (PA), Parishin B (PB), Parishin C (PC), and Parishin E (PE).

[0027] Based on the established HPLC-DAD method, 6 candidate compounds in 60 batches of Gastrodia were detected and quantified. The content of these candidate compounds was distributed between 0.0226-1.4725%. The specific content was as follows: GAS was 0.0537-0.4687%; HBA was 0.0319-0.3656%; PA was 0.0957%-0.8700%; PB was 0.1555-0.6280%; PC was 0.0226-0.1859%; and PE was 0.1727-1.4725%. The content of GAS and HBA was similar to that of Du et al. (Du et al., 2022), indicating that the Gastrodia in this study was representative 2. ABTS·radical scavenging experiment From 87 batches of samples, 27 samples were selected for ABTS test. The research method referred to the research method of Re et al. (Re et al., 1999), and the absorbance was detected at 734 nm by enzyme label instrument. Nonlinear regression (GraphPad Prism 9 software) was used for analysis, and the IC 50 value of the sample to be tested was calculated. VC was used as a positive drug to examine the detection method, and the results showed that R 2 = 0.99. The typical ABTS·scavenging ability of Gastrodia samples is shown in Figure 3 .

[0028] The IC 50 values of the ABTS·scavenging rate of 27 batches of Gastrodia samples and the content of 6 candidate compounds were subjected to unsupervised classification by principal component analysis (PCA) (as shown in Figure 4 ). The 27 batches of Gastrodia were clearly divided into two categories, which suggested that the quality grading of Gastrodia might be two levels of high quality and ordinary, and the correlation coefficients of 6 candidate compounds and ABTS·radical scavenging ability of Gastrodia were obtained in the classification process, which revealed the influence degree of 6 candidate compounds on the ABTS·radical scavenging ability of Gastrodia extract. In the result analysis, it was found that HBA, PA and GAS had strong correlation with ABTS·radical scavenging ability, and the correlation coefficients were -1, 0.57644 and 0.30702, respectively. Among them, HBA showed a strong negative correlation with ABTS·radical scavenging ability, while PA and GAS showed a strong positive correlation; the correlation coefficients of the remaining 3 candidate compounds PB, PB and PC were 0.29449, 0.21805 and 0.08521, respectively (as shown in Figure 5 ).

[0029] The Pearson correlation coefficients of the 6 candidate compounds on PCA classification were used to predict their contribution to antioxidant activity. Based on the correlation coefficients of the candidate compounds in Gastrodia elata obtained by PCA, the ABTS· scavenging experiment was used to verify whether they were Q-markers of Gastrodia elata antioxidant. From 87 batches of samples, the NO37 sample with the lowest content of 6 components was selected as the matrix (the contents of Gas, HBA, PA, PB, PC and PE were 0.0537%, 0.0319%, 0.0957%, 0.1555%, 0.0226% and 0.1727% respectively), and the highest content as the endpoint (the contents of 6 components were 0.4687%, 0.3656%, 0.8700%, 0.6280%, 0.1859% and 1.4725% respectively), and the monomer compounds were added step by step to determine the ABTS· scavenging rate of different monomer added samples.

[0030] The results are shown in Figure 6 HBA was consistent with the results predicted by PCA, and was negatively correlated with ABTS· scavenging rate, Gas and PB were positively correlated with ABTS· scavenging rate, PC and PE had little effect on ABTS· scavenging rate, and PA showed an interesting phenomenon, that is, with the increase of PA concentration in the sample, the ABTS· scavenging rate of the sample would first rise to a certain limit and then decrease.

[0031] 3. Oxidative damage experiment of Caenorhabditis elegans The Caenorhabditis elegans used in the experiment were wild type and were donated by the laboratory of Yunnan University. The Caenorhabditis elegans were synchronized before the experiment. The oxidative damage model of H2O2 was used to evaluate the antioxidant effect of Gastrodia elata extract. Briefly, the synchronized L4 stage Caenorhabditis elegans were washed into centrifuge tubes with M9 buffer, rinsed 3 times, and then adjusted to 10 μL of worm solution with 15-20 worms, and then added 10 μL of worm solution, 80 μL of sample solution to be tested, 1.5 μL of 5-FuDR (5 mg / mL), 2 μL of ampicillin (5 mg / mL) to each well of the 96-well plate, and finally diluted to a total volume of 90 μL per well with M9 buffer. After incubating at 20 ℃ for 24 h, 10 μL of hydrogen peroxide (200 mM) was added to each well, and after 1 h, the survival rate of Caenorhabditis elegans was observed, and statistical analysis was performed using One-way ANOVA (GraphPad Prism 9).

[0032] The sample selection scheme of the Caenorhabditis elegans oxidative stress experiment was consistent with that of the ABTS· scavenging experiment. The high concentration was the highest concentration when the monomer compound was added, the low concentration was without the addition of monomer compound, and the medium concentration was the median value of the low and high concentrations. The monomer compounds were added step by step to determine the survival rate of different monomer added samples. The results are shown in Figure 7As shown, the trend of the protective effect of the 6 candidate compounds on H2O2-stimulated C. elegans was basically consistent with the results of the ABTS· experiment, in which HBA was negatively correlated with antioxidant capacity, and the survival rate of C. elegans increased significantly with the increase of the concentration of Gas; with the increase of the concentration of PA, the survival rate of C. elegans first increased to a certain limit and then decreased; with the increase of the concentration of PB and PC, the survival rate of C. elegans slightly increased at high concentrations, but was not obvious; PE had basically no effect on the survival rate of C. elegans.

[0033] 4. Establishing an antioxidant quality evaluation system for Gastrodia elata by multidimensional data analysis Due to the wide range of active ingredients in traditional Chinese medicinal materials, we selected three aspects, including content, ABTS· scavenging capacity and C. elegans oxidative stress protection capacity, to comprehensively predict the antioxidant quality markers of Gastrodia elata. Comprehensive analysis of the quality markers in Gastrodia elata from three dimensions can avoid analysis bias caused by higher content but weaker activity or stronger activity but lower content.

[0034] First, in order to more clearly and intuitively show the ABTS· scavenging capacity of the 6 candidate compounds and the C. elegans oxidative stress protection capacity, the contribution of the 6 candidate compounds to the ABTS· scavenging capacity of Gastrodia elata and the C. elegans oxidative stress protection capacity was calculated by the change of “dose-effect” slope, as shown in formula (1) and (2).

[0035] (1); (2), wherein is the scoring value of the contribution of different candidate compounds to the ABTS· scavenging capacity; is the 6 different candidate compounds; is the concentration of the added candidate compound; is the ABTS· scavenging rate at the concentration of the added candidate compound; is the ABTS· scavenging rate without adding the candidate compound; is the number of single-added samples.

[0036] is the scoring value of the contribution of different candidate compounds to the survival rate of C. elegans, is the survival rate of C. elegans at the concentration of the added candidate compound; is the survival rate of C. elegans without adding the candidate compound.

[0037] ​​In order to eliminate the data bias caused by large data fluctuations, the content distribution trend, ABTS· scavenging ability contribution and Caenorhabditis elegans oxidative stress protection ability contribution of the six candidate compounds were processed by data normalization method, and calculated by formula (3), (4) and (5).

[0038] (3), (4), (5), is the normalized value of the content dimension of different candidate compounds in Gastrodia elata; Gas, HBA, PA, PB, PC and PE are six different candidate compounds; is the average content of a single candidate compound in 87 batches of Gastrodia elata samples; is the maximum value of the average content of the six candidate compounds, and the results are shown in Table 1, the values of Gas, HBA, PA, PB, PC and PE are 0.414, 0.297, 0.803, 0.879, 0.195 and 1.000, respectively. Figure 8

[0039] is the scoring and normalization value of the contribution of different candidate compounds to the ABTS· scavenging ability, and the results are shown in Table 2, the values of Gas, HBA, PA, PB, PC and PE are 0.531, -0.449, 0.194, 1.000, 0.614 and 0.092, respectively. Figure 9

[0040] is the scoring and normalization value of the contribution of different candidate compounds to the survival rate of Caenorhabditis elegans, and the results are shown in Table 3, the values of HBA, PA, PB, PC and PE are 1.000, -0.180, 0.201, 0.044, 0.280 and 0.012, respectively. Figure 10

[0041] and , and are plotted as three-dimensional radar charts, and the results are shown in Figure 1, the area of the radar chart is normalized to obtain the Q-Marker coefficient of the candidate compounds under multi-dimensional data analysis, the higher the Q-Marker coefficient, the higher the importance of the compound in the antioxidant activity of Gastrodia elata, and the greater the possibility of becoming Q-Marker. The area of the radar chart is calculated by the following formula: Figure 11 ​​​​​​​​ (6); in, (7), In the formula, Normalized values ​​for the content of different candidate compounds in Gastrodia elata; Normalized scores were assigned to the contributions of different candidate compounds to the ABTS· scavenging ability; Normalized scores were assigned to the contributions of different candidate compounds to the survival rate of Caenorhabditis elegans.

[0042] (8) For different candidate compounds The normalized value is also the Q-Marker coefficient of the six different candidate compounds; Among the 6 candidate compounds The highest value. Results are visualized as follows: Figure 12 As shown, Gas and PB performed the best. The values ​​were 1.000 and 0.826, respectively; HBA was negatively correlated with the antioxidant activity of Gastrodia elata. The value is -0.229; for PA, PC, and PE. The values ​​were 0.306, 0.297 and 0.090, respectively.

[0043] Based on the obtained Q-Marker coefficient, the antioxidant capacity score of different batches of Gastrodia elata is calculated using the following formula. By comparing the antioxidant capacity scores, the actual antioxidant capacity of different batches of Gastrodia elata can be predicted.

[0044] (9) In the formula, The antioxidant score of the Gastrodia elata sample. The Q-Marker coefficients of the candidate compounds are... The content of a single candidate compound in the Gastrodia elata sample; 5. Validation of the Gastrodia elata antioxidant scoring system This invention selected seven batches of Gastrodia elata for experiments. The contents of six candidate compounds in the seven batches of Gastrodia elata were determined by HPLC-DAD method, and the antioxidant capacity score QS was calculated by formula (9), which were 2.87; 3.52; 6.34; 7.90; 8.13; 9.55; 12.04, respectively. Oxidative stress experiments were conducted using Caenorhabditis elegans (e.g.,...). Figure 13As shown in the figure, it was found that the antioxidant capacity of Gastrodia elata increased with the increase of the antioxidant capacity score, which was consistent with the prediction results, indicating that the model for predicting the antioxidant capacity of Gastrodia elata through the antioxidant coefficient is consistent with the actual results. Furthermore, linear regression analysis revealed a good correlation between the antioxidant score of Gastrodia elata and its activity (e.g., ...). Figure 14 As shown), R 2 =0.8062.

[0045] 6. Determination of grading boundaries for high-quality antioxidant Gastrodia elata Gastrodia elata was graded as high-quality based on its antioxidant rating. Figure 4 It is evident that Gastrodia elata was divided into two categories. Therefore, this invention uses the Support Vector Machine (SVM) method, which is more suitable for binary classification problems, to classify Gastrodia elata into two categories: high-quality and ordinary. 20 batches (80%) of the 27 batches of Gastrodia elata were designated as the learning set, and 7 batches were designated as the test set (20%). The Gastrodia elata were then classified into two categories: high-quality and ordinary (e.g.,...). Figure 15 As shown in the figure, the antioxidant score (QS) of Gastrodia elata at the boundary was calculated to be 8.79. Therefore, the boundary for high-quality Gastrodia elata is determined to be an antioxidant score ≥ 8.79.

[0046] The chemical composition of traditional Chinese medicine (TCM) herbs is extremely complex. It includes active ingredients, auxiliary ingredients, and inactive ingredients. The biological effects of TCM herbs do not originate from any single active ingredient, but from the interaction of many active ingredients, and even with so-called inactive ingredients. A single ingredient cannot represent the full efficacy of TCM; therefore, in treating another disease, the active ingredient may be ineffective. This invention attempted an ABTS·free radical scavenging experiment on gastrodin monomer, and the results showed that a single ingredient cannot represent the actual effects in a multi-component TCM herb. Therefore, the addition of monomeric compounds to actual Gastrodia elata extract was chosen to eliminate matrix effects in the extract. The specific method is as follows: after identifying the target compound, a batch of Gastrodia elata samples with low levels of this compound were screened. After extraction, the target compound was gradually added until the highest level of this compound was found in all Gastrodia elata samples. This method yields a series of samples with only different levels of the target compound. This method takes into account the interaction factors between different chemical components in the actual Gastrodia elata samples, eliminates the influence of different matrix effects between different batches of TCM herbs on the experimental results, and ensures that the content range of the target compound does not exceed the range that Gastrodia elata itself may contain.

[0047] For any points not covered above, existing technologies shall apply.

[0048] Although some specific embodiments of the present application have been described in detail by way of example with reference to the drawings, it is to be understood that the above examples are intended to be illustrative only and are not intended to limit the scope of the present application, and that various modifications and changes can be made by those skilled in the art to the particular embodiments described without departing from the spirit and scope of the present application. It is intended that the scope of the present application be limited only by the broadest interpretation of the appended claims to be accorded under 35 U.S.C. § 112.

Claims

1. A method for grading Gastrodia elata based on Q-marker, characterized in that, The method comprises the following steps: S1, determining the content of 6 candidate compounds in the Gastrodia sample, wherein the candidate compounds are gastrodin, p-hydroxybenzyl alcohol, bartzin A, bartzin B, bartzin C and bartzin E; performing ABTS·radical scavenging test on the Gastrodia sample; performing Caenorhabditis elegans oxidative stress experiment on the Gastrodia sample; S2, calculating the contribution of the 6 candidate compounds to the ABTS·radical scavenging capacity and the Caenorhabditis elegans oxidative stress protection capacity of the Gastrodia sample by "amount-effect" slope change, as shown in formula (1) and formula (2): （1）； （2）， wherein is the score of the contribution of the different candidate compounds to the ABTS· scavenging capacity; is the score of the contribution of the different candidate compounds to the ABTS· scavenging capacity; is the concentration of the added candidate compound; is the ABTS· scavenging rate at the concentration of the added ABTS· scavenging rate; is the ABTS· scavenging rate without the addition of the candidate compound; is the number of monomers added to the sample; score the contribution of different candidate compounds to the survival rate of C. elegans, the survival rate of C. elegans in the presence of the candidate compound at a concentration of the survival rate of C. elegans in the absence of the candidate compound; S3, the content distribution trend, ABTS· scavenging ability contribution degree and Caenorhabditis elegans oxidative stress protection ability contribution degree of the six candidate compounds are processed by using data normalization method, and the normalized values of different candidate compounds in the content dimension of Gastrodia are obtained , the contribution degree scoring normalized value of different candidate compounds to ABTS· scavenging ability , the contribution degree scoring normalized value of different candidate compounds to the survival rate of Caenorhabditis elegans ; S6, will , and The radar chart is drawn in three dimensions as a three-dimensional radar chart. After the area of ​​the radar chart is normalized, the Q-Marker coefficients of different candidate compounds are obtained. S7, calculating the antioxidant score of the Gastrodia sample according to the obtained Q-Marker coefficient according to formula (9), and grading the Gastrodia according to the antioxidant score; （9） wherein, is the antioxidant score of the Gastrodia elata sample, is the Q-Marker coefficient of the candidate compound, is the content of the individual candidate compound in the Gastrodia elata sample; S8, classifying the Gastrodia sample into two categories of high quality and ordinary by using a support vector machine model, and calculating and determining a grading boundary QS boundary, so as to determine that the antioxidant score of high-quality Gastrodia should be not less than the QS boundary.

2. The method of claim 1, wherein, The The calculation formula is: （3）， wherein, is the normalized value of the content dimension of different candidate compounds in Gastrodia; is 6 different candidate compounds; is the average content of a single candidate compound in Gastrodia samples; is the maximum value of the average content of the 6 candidate compounds.

3. The method of claim 2, wherein, The The calculation formula is: （4）， wherein, is the normalized value of the contribution of the different candidate compounds to the ABTS· scavenging capacity; is the contribution of the individual candidate compound in the Gastrodia elata sample to the ABTS· scavenging capacity, is the maximum value of the contribution of the ABTS· scavenging capacity of the 6 candidate compounds.

4. The method of claim 3, wherein, The The calculation formula is: （5）， wherein is the contribution of the different candidate compounds to the survival rate of C. elegans normalized value; is the contribution of the individual candidate compound in the Gastrodia sample to the oxidative stress protection ability of C. elegans; is the maximum contribution of the 6 candidate compounds to the oxidative stress protection ability of C. elegans.

5. The method of claim 4, wherein, the area of the radar chart is calculated by equation (6): （6）； Wherein, （7）， wherein, is the normalized value of the content dimension of different candidate compounds in Gastrodia elata; is the normalized value of the contribution degree score of different candidate compounds to ABTS· scavenging ability; is the normalized value of the contribution degree score of different candidate compounds to the survival rate of Caenorhabditis elegans.

6. The method of claim 5, wherein, The method for normalizing the area of the radar chart is: （8） wherein is the normalized value for a different candidate compound is also the Q-Marker coefficient for the 6 different candidate compounds; is the highest value among the 6 candidate compounds .

7. The method of claim 1, wherein, The method for determining the content of the 6 candidate compounds in the Gastrodia sample is: high performance liquid chromatography is used for determination, an octadecylsilane bonded silica gel column is used as a filler, 0.1% formic acid aqueous solution and acetonitrile are used as mobile phases, gradient elution is performed, the elution program is: 0-8 min, 3-10% acetonitrile; 8-15 min, 10-12% acetonitrile; 15-25 min, 15-18% acetonitrile; 25-35 min, 18% acetonitrile; 35-35.1 min, 18-95% acetonitrile, the column temperature is 30°C, and the detection wavelength is 220 nm.

8. The method of claim 1, wherein, In the ABTS free radical scavenging experiment, vitamin C was used as a positive drug for comparison, the absorbance was detected at 734 nm by a microplate reader, and non-linear regression was used for analysis to calculate the IC 50 value of the sample to be tested.

9. The method of claim 1, wherein, In the Caenorhabditis elegans oxidative stress experiment, the H2O2 oxidative damage model is used to evaluate the antioxidant effect of the Gastrodia extract.

10. The method according to any one of claims 1-9 in the quality control of Gastrodia.

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