Gastrodin grading method based on q-marker and application thereof

Abstract

The application provides a Gastrodia elata grading method based on Q-marker and application thereof, and belongs to the technical field of traditional Chinese medicine native medicinal material grading evaluation. The method firstly determines six candidate compounds as gastrodin, p-hydroxybenzyl alcohol, balesenoside A, balesenoside B, balesenoside C and balesenoside E, and then verifies the six candidate compounds as Q-markers of the antioxidant activity of Gastrodia elata by integrating multi-dimensional data such as the content of the six candidate compounds, in-vitro anti-free radical (ABTS) and in-vivo antioxidant (anti-nematode H2O2 damage) biological activity. The Q-marker coefficient of the content of the candidate compounds, in-vitro chemical activity and in-vivo biological activity is constructed by using a radar chart area, an integrated quantity-effect model for rapidly predicting the antioxidant activity of Gastrodia elata by the content of the Q-marker is established, and the results show that the model prediction value is consistent with the experiment, and the high-quality Gastrodia elata with good antioxidant and anti-aging activity has a score of greater than or equal to 8.79.

Description

Technical Field

[0001] This invention relates to the field of grading and evaluation technology for traditional Chinese medicinal materials, and in particular to a grading method for Gastrodia elata based on Q-marker and its application. Background Technology

[0002] Traditional methods for quality control of Chinese medicinal materials mostly rely on their appearance, which is not accurate enough. Modern methods focus on chemical component control, which is precise but fails to directly reflect the fundamental requirements of the efficacy of processed Chinese medicinal materials. Evaluation of the potency of Chinese medicinal materials based on bioactivity can be correlated with the clinical efficacy of drugs, but this evaluation suffers from high experimental requirements, long cycles, and poor precision and reproducibility. Therefore, establishing a chemical-biological potency-correlation evaluation system is crucial. Academician Liu Changxiao proposed the concept of quality markers (Q-Markers) for Chinese medicinal materials, closely linking efficacy and chemical components, which has gained nationwide attention. However, there are few reports on how to integrate multiple Q-marker components into a complete quality evaluation system for the grading and evaluation of authentic Chinese medicinal materials.

[0003] Gastrodia elata is a traditional and precious Chinese medicinal herb, traditionally used for its effects of calming wind and relieving spasms, suppressing liver yang, and dispelling wind and unblocking meridians. Modern research has found that Gastrodia elata has good anti-inflammatory, antioxidant, anti-aging, anticonvulsant, and sedative effects. The activity of Gastrodia elata mainly comes from its abundant phenolic acid compounds, among which gastrodin (Gas) has a hypnotic effect, and baicalin A can alleviate the cardiotoxicity caused by DOX. Traditional methods for quality control of Gastrodia elata rely on appearance evaluation. Modern methods often use chemical fingerprinting and multi-component content determination of gastrodin and baicalin A to conduct quality control research on the effective components. These methods can accurately measure these components, but neither can directly reflect the biological activity of Gastrodia elata.

[0004] How to quickly predict the antioxidant activity of Gastrodia elata in order to achieve a comprehensive evaluation of its chemical composition and biological activity, and to determine the high-quality grading limit of Gastrodia elata through a scoring system, is a worthy research task, and it also has certain reference value for the research of other Chinese medicinal materials. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing a Q-marker-based grading method for Gastrodia elata and its application. Taking the antioxidant properties of Gastrodia elata as an example, a comprehensive scoring system of Q-marker coefficients is established. This system enables quantitative scoring and prediction of the antioxidant activity of Gastrodia elata by measuring the content of Q-marker components, and the accuracy of the model has been verified in real samples.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: 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 candidate compound added; 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 trend, 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. Based on the obtained Q-Marker coefficient, calculate the antioxidant score of the Gastrodia elata sample according to formula (9), and then classify the Gastrodia elata according to the antioxidant score. (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; S8. Using a support vector machine model, the Gastrodia elata samples were divided into two categories: high-quality and ordinary, and the grading threshold QS was calculated and determined. 界限 The antioxidant score of high-quality Gastrodia elata should be no less than the QS score. 界限 .

[0007] Furthermore, the aforementioned The calculation formula is: (3), In the formula, Normalized values ​​for the content of different candidate compounds in Gastrodia elata; Six different candidate compounds; The average content of a single candidate compound in the Gastrodia elata sample; The maximum value of the average content among the 6 candidate compounds.

[0008] Furthermore, the aforementioned The calculation formula is: (4), In the formula, Normalized scores were assigned to the contributions of different candidate compounds to the ABTS· scavenging ability; The contribution of individual candidate compounds to the ABTS· scavenging ability in Gastrodia elata samples. The maximum contribution of ABTS· scavenging ability among the six candidate compounds is represented by this value.

[0009] Furthermore, the aforementioned The calculation formula is: (5), In the formula, Normalized scores were assigned to the contributions of different candidate compounds to the survival rate of Caenorhabditis elegans. The contribution of individual candidate compounds in the Gastrodia elata sample to the protective capacity against oxidative stress in Caenorhabditis elegans. The highest contribution of the six candidate compounds to the protective ability of Caenorhabditis elegans against oxidative stress was observed.

[0010] Furthermore, the area of ​​the radar map The calculation is performed using equation (6): (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.

[0011] Furthermore, the method for normalizing the area of ​​the radar image is as follows: (8) In the formula, 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.

[0012] Furthermore, the method for determining the content of six candidate compounds in the Gastrodia elata sample was as follows: high performance liquid chromatography was used for determination. An octadecylsilane-bonded silica gel column was used as the packing material, and 0.1% formic acid aqueous solution and acetonitrile were used as the mobile phase for gradient elution. The elution program was: 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 was 30℃, and the detection wavelength was 220 nm.

[0013] Furthermore, in the ABTS free radical scavenging assay, vitamin C was used as a positive control. Absorbance was measured at 734 nm using a microplate reader, and nonlinear regression analysis was employed to calculate the IC50 of the test sample. 50 value.

[0014] Furthermore, the antioxidant effect of Gastrodia elata extract was evaluated in the oxidative stress experiment of Caenorhabditis elegans using the H2O2 oxidative damage model.

[0015] The second objective of this invention is to provide the application of the above-described method in the quality control of Gastrodia elata.

[0016] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention provides a Q-marker-based grading method for Gastrodia elata, integrating multidimensional data such as Q-marker content, in vitro anti-free radical (ABTS) activity, and in vivo antioxidant (anti-nematode H2O2 damage) bioactivity, to create a quantitative model for predicting antioxidant activity by measuring Q-marker content. Briefly, the contents of six major blood-entering chemical components in 27 batches of GE were measured: gastrodin (Gas), p-hydroxybenzyl alcohol (HBA), Parishin A (PA), Parishin B (PB), Parishin C (PC), and Parishin E (PE), as well as the IC50 of ABTS scavenging. 50 Principal component analysis (PCA) was used to classify Gastrodia elata into "high-quality" and "qualified" grades. The correlation between six components and the antioxidant activity of Gastrodia elata was calculated. The ABTS scavenging assay and the H2O2-induced aging model of Caenorhabditis elegans were then used to verify that the six components were Q-markers for the antioxidant activity of Gastrodia elata. Finally, Q-marker coefficients integrating chemical component content, in vitro chemical activity, and in vivo biological activity were constructed using radar chart area. An integrated dose-response model for rapidly predicting the antioxidant activity of Gastrodia elata using Q-marker content was established, and experimental verification was performed on real samples. The results showed that the model predictions were consistent with the experimental results (R0). 2 =0.8062), and it is believed that the score of high-quality Gastrodia elata with good antioxidant and anti-aging activity is greater than or equal to 8.79.

[0017] (2) This invention provides a Q-marker-based grading method for Gastrodia elata. Compared with the 5 days required for the aging experiment of Caenorhabditis elegans with H2O2, the content of Q-marker can be measured within 1 hour to quickly predict the antioxidant activity of Gastrodia elata. This achieves a comprehensive evaluation of chemical components and biological activities. Furthermore, the scoring system determines the activity-based high-quality grading limit of Gastrodia elata, providing a new method for the quality control of Gastrodia elata. It also has certain reference value for the research of other Chinese medicinal materials. Attached Figure Description

[0018] Figure 1 Standard fingerprints of six candidate compounds from Gastrodia elata are shown: 1: Gas; 2: HBA; 3: PE; 4: PB; 5: PC; 6: PA. Figure 2 The structural formulas of six candidate compounds from Gastrodia elata are shown. Figure 3 This is a typical dose-effect curve for the ABTS· scavenging experiment of Gastrodia elata extract; Figure 4The graph shows the results of PCA correlation calculations between the ABTS scavenging ability of multiple batches of Gastrodia elata extract and the contents of six components. Figure 5 The correlation coefficients between the contents of six candidate compounds and the ABTS· scavenging ability of Gastrodia elata extract were calculated. Figure 6 The graph shows the changes in ABTS· scavenging ability of Gastrodia elata samples after adding six candidate compounds to the monomer. In the figure, (A) is for the addition of Gas; (B) is for the addition of HBA; (C) is for the addition of PA; (D) is for the addition of PB; (E) is for the addition of PC; and (F) is for the addition of PE. Figure 7 The graph shows the changes in the protective ability of Gastrodia elata samples against the CEH2O2 damage model after adding six candidate compounds to the monomer. In the graph, (A) is for the addition of Gas; (B) is for the addition of HBA; (C) is for the addition of PA; (D) is for the addition of PB; (E) is for the addition of PC; and (F) is for the addition of PE. Figure 8 The content distribution trend of 6 candidate compounds in 60 batches of Gastrodia elata; Figure 9 To calculate the contribution of six candidate compounds to the ABTS· scavenging ability of Gastrodia elata extract; Figure 10 To calculate the contribution of six candidate compounds in Gastrodia elata extract to the protective CE against H2O2 stimulation; Figure 11 To establish a multidimensional evaluation system for six candidate compounds by using a three-dimensional approach of content, in vivo activity, and in vitro activity to create a visual radar chart; Figure 12 The Q-Marker scoring system is obtained after calculating the area of ​​the visualized radar chart of 6 candidate compounds; Figure 13 To determine the survival rate of CEs after 1 h of stimulation with H2O2 following protection of CEs with 7 different batches of Gastrodia elata extract for 24 h; Figure 14 To be Figure 13 A linear fitting plot of CE survival rate and antioxidant score was performed, where Y is the CE survival rate and X is the antioxidant score of Gastrodia elata. Figure 15 A visualization of the results of SVM classifying Gastrodia elata into high-quality and ordinary categories. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0020] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0021] The fresh Gastrodia elata samples collected in this invention came from five provinces: Yunnan, Guizhou, Hubei, Anhui, and Shaanxi, and were identified as the rhizomes of the orchid Gastrodia elata. After steaming, the fresh Gastrodia elata samples were sliced ​​into 2 mm thin slices, dried in an oven at 50 ℃ for 36 h, then pulverized and sieved to obtain 87 batches of Gastrodia elata powder samples.

[0022] Example 1 This embodiment provides a Q-marker-based method for grading Gastrodia elata, with the following specific steps: 1. Chemical composition analysis For each batch of Gastrodia elata sample, 0.2 g of powder was transferred to a 10 mL volumetric flask and extracted with 50% methanol aqueous solution at 25 °C using ultrasonic extraction for 30 min. After cooling to room temperature, the extract was diluted to the mark with 50% methanol aqueous solution. Subsequently, the sample solution was centrifuged at 3500 rpm for 15 min at 4 °C. The supernatant was collected after centrifugation, filtered, and then used for HPLC-DAD analysis.

[0023] Chromatographic analysis was performed on an Agilent 1290 HPLC-DAD system. Fingerprint chromatogram and content determination conditions: Gradient elution was performed on a ZORBAX SB-C18 reversed-phase column (4.6 × 250 mm, 5 μm) at 30 °C. The mobile phase consisted of 0.1% phosphoric acid aqueous solution (A) and acetonitrile (B), with a flow rate of 0.800 mL / min. The following gradient elution program was used: 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 was set to 220 nm. The injection volume was 10 μL.

[0024] The fingerprint chromatograms were analyzed using Agilent Data Analyse and the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System software. The similarity range for 87 batches was 0.835-0.997.

[0025] The analysis of blood components followed the experimental method of Lee et al., but with some modifications (Lee et al., 2023). Twelve male mice were randomly divided into a blank control group and a drug-treated group (n=6). After fasting for 12 h, Gastrodia elata extract was administered by gavage. The preparation method of Gastrodia elata extract was the same as that in the chemical component analysis, and the dosage was 1.7 g / kg (crude drug / body weight). Plasma was collected before administration and at 0, 1, 1.5, 2, 2.5, and 3 hours after administration and stored at –80°C. For plasma sample preparation, plasma from the blank control group and the drug-treated group at different time points was mixed (approximately 1.0 ml), and three times the volume of ice-cold methanol was added. The mixture was vortexed, centrifuged at 15,000 rpm for 30 min at 4°C, and the supernatant was collected. The supernatant was concentrated to dryness by vacuum centrifugation, then reconstituted with 100 ml of 50% methanol-water solution, filtered through a 0.22 mm filter membrane, and analyzed by HPLC-UV under the following conditions: Gradient elution was performed on a ZORBAX SB-C18 reversed-phase column (4.6 × 250 mm, 5 μm) at 30 °C. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B), with a flow rate of 0.800 mL / min. The gradient elution program was as follows: 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 elata extract was performed. Typical chromatograms of the sample solutions are shown below. Figure 1 As shown. Further analysis of mouse serum samples identified six common candidate compounds that enter the bloodstream from Gastrodia elata (such as...). Figure 2 The following are listed as gastrodin (Gas), p-hydroxybenzyl alcohol (HBA), parishin A (PA), parishin B (PB), parishin C (PC), and parishin E (PE), respectively.

[0027] Based on the established HPLC-DAD method, six candidate compounds were detected and quantified in 60 batches of Gastrodia elata. The contents of these candidate compounds ranged from 0.0226% to 1.4725%. Specifically, the contents were as follows: Gas (0.0537-0.4687%), HBA (0.0319-0.3656%), PA (0.0957%-0.8700%), PB (0.1555-0.6280%), PC (0.0226-0.1859%), and PE (0.1727-1.4725%). The contents of GAS and HBA were similar to those reported by Du et al. (Du et al., 2022), indicating that the Gastrodia elata samples in this study are representative. 2. ABTS·Free Radical Scavenging Experiment From 87 batches of samples, 27 samples were selected stratified and randomly assigned to each reagent for ABTS testing. The methodology followed that of Re et al. (Re et al., 1999). Absorbance was measured at 734 nm using a microplate reader, and nonlinear regression analysis (GraphPadPrism 9 software) was used to calculate the IC50 of the tested samples. 50 Value. The detection method was examined using VC as a positive control, and the results showed R... 2 =0.99. Typical ABTS· scavenging ability of Gastrodia elata samples is as follows: Figure 3 As shown.

[0028] The ABTS scavenging rate IC of 27 batches of Gastrodia elata samples was analyzed. 50 The values ​​and contents of the six candidate compounds were classified unsupervised using principal component analysis (PCA). Figure 4 (As shown). 27 batches of Gastrodia elata were clearly divided into two categories, suggesting that the quality grading of Gastrodia elata may be divided into two levels: high-quality and ordinary. Furthermore, during the classification process, correlation coefficients were obtained between six candidate compounds and the ABTS· free radical scavenging ability of Gastrodia elata, revealing the degree of influence of these six candidate compounds on the ABTS· free radical scavenging ability of Gastrodia elata extract. In the results analysis, HBA, PA, and Gas showed strong correlations with ABTS· scavenging ability, with correlation coefficients of -1, 0.57644, and 0.30702, respectively. HBA showed a strong negative correlation with ABTS· scavenging ability, while PA and Gas showed a strong positive correlation. The correlation coefficients of the remaining three candidate compounds, PB, PB, and PC, were 0.29449, 0.21805, and 0.08521, respectively (as shown). Figure 5 (As shown).

[0029] The contribution of six candidate compounds to antioxidant activity was predicted based on the Pearson correlation coefficients of their PCA classification. Based on the correlation coefficients of candidate compounds in *Gastrodia elata* obtained from PCA, an ABTS· scavenging assay was used to verify whether they were Q-markers for antioxidant activity in *Gastrodia elata*. From 87 batches of samples, six NO37 samples with the lowest content of each component were selected as matrices (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). Using the highest content as the endpoint (the contents of the six components were 0.4687%, 0.3656%, 0.8700%, 0.6280%, 0.1859%, and 1.4725%, respectively), monomeric compounds were gradually added, and the ABTS· scavenging rate of samples with different monomer additions was measured.

[0030] The results are as follows Figure 6 As shown, the results predicted by HBA and PCA are consistent and negatively correlated with ABTS·scavenging rate. Gas and PB are positively correlated with ABTS·scavenging rate. PC and PE have almost no effect on ABTS·scavenging rate. PA shows an interesting phenomenon: as the concentration of PA in the sample increases, the ABTS·scavenging rate of the sample first increases to a certain limit and then decreases.

[0031] 3. Oxidative damage experiment of Caenorhabditis elegans The *C. elegans* used in the experiment were wild-type and provided by the Yunnan University laboratory. Prior to the experiment, the *C. elegans* underwent synchronization treatment. The antioxidant effect of *Gastrodia elata* extract was evaluated using an H2O2 oxidative damage model. Briefly, synchronized *C. elegans* to the L4 stage were washed into centrifuge tubes with M9 buffer, rinsed three times, and the worm solution was adjusted to 15-20 nematodes per 10 μL. Then, 10 μL of worm solution, 80 μL of the test sample solution, 1.5 μL of 5-FuDR (5 mg / mL), and 2 μL of ampicillin (5 mg / mL) were added to each well of a 96-well plate. Finally, the total volume of each well was diluted to 90 μL with M9 buffer. After stable incubation at 20 ℃ for 24 h, 10 μL of hydrogen peroxide (200 mM) was added to each well. The survival rate of *C. elegans* was observed after 1 h, and statistical analysis was performed using one-way ANOVA (GraphPad Prism 9).

[0032] The sample selection scheme for the *C. elegans* oxidative stress assay was consistent with that of the ABTS-scavenging assay. High concentration represented the highest concentration of monomeric compound added, low concentration represented no monomeric compound added, and medium concentration represented the median of the low and high concentrations. Monomeric compounds were added stepwise, and the nematode survival rate was measured for samples with different monomeric compound additions. Results are as follows: Figure 7As shown, the protective effects of the six candidate compounds on H2O2-stimulated *C. elegans* are largely consistent with the results of the ABTS experiment. HBA showed a negative correlation with antioxidant capacity, while the survival rate of *C. elegans* increased significantly with increasing concentration of Gas. With increasing PA concentration, the survival rate of *C. elegans* first increased to a certain limit and then decreased. With increasing PB and PC concentrations, the survival rate of *C. elegans* was slightly increased at high concentrations, but not significantly. PE had virtually no effect on the survival rate of *C. elegans*.

[0033] 4. Establish an antioxidant quality evaluation system for Gastrodia elata through multidimensional data analysis. Because Chinese medicinal herbs contain a wide variety of active ingredients and have a broad range of applications, we chose to comprehensively predict the antioxidant quality markers of Gastrodia elata from three aspects: content, ABTS scavenging ability, and oxidative stress protection ability of Caenorhabditis elegans. Analyzing the quality markers in Gastrodia elata from these three dimensions can avoid analytical biases caused by high content but weak activity or strong activity but low content.

[0034] First, in order to more clearly and intuitively show the ABTS· scavenging ability of the six candidate compounds and the oxidative stress protection ability of Caenorhabditis elegans, the contribution 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).

[0035] (1); (2), in Scoring the contribution of different candidate compounds to ABTS· scavenging ability; Six different candidate compounds; To determine the concentration of the candidate compound added; 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.

[0036] 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; The survival rate of *C. elegans* without the addition of candidate compounds.

[0037] Meanwhile, in order to eliminate data bias caused by big data fluctuations, the data normalization method was used to process the content distribution trend of the six candidate compounds, the contribution of ABTS· scavenging ability and the contribution of Caenorhabditis elegans oxidative stress protection ability, and the calculation was performed using equations (3), (4) and (5).

[0038] (3), (4), (5), Normalized values ​​for the content of different candidate compounds in Gastrodia elata; The six candidate compounds are Gas, HBA, PA, PB, PC, and PE. The average content of a single candidate compound in 87 batches of Gastrodia elata samples; The maximum average content among the six candidate compounds is shown in the following figure. Figure 8 As shown, Gas, HBA, PA, PB, PC, and PE The values ​​are 0.414, 0.297, 0.803, 0.879, 0.195 and 1.000, respectively.

[0039] Normalized scores were assigned to the contributions of different candidate compounds to the ABTS· scavenging ability, such as... Figure 9 As shown, Gas, HBA, PA, PB, PC, and PE The values ​​were 0.531, -0.449, 0.194, 1.000, 0.614 and 0.092, respectively.

[0040] The normalized scores for the contributions of different candidate compounds to the survival rate of *C. elegans* are shown below. Figure 10 As shown, HBA, PA, PB, PC, and PE The values ​​are 1.000, -0.180, 0.201, 0.044, 0.280 and 0.012, respectively.

[0041] Then , and The three-dimensional radar image was plotted as a three-dimensional representation, and the result is as follows: Figure 11 As shown, after normalizing the area of ​​the radar chart, the Q-Marker coefficients of candidate compounds were obtained under multidimensional data analysis. A higher Q-Marker coefficient indicates greater importance of the compound in the antioxidant activity of Gastrodia elata, and a greater likelihood of it becoming a Q-Marker. (Radar chart area...) The calculation is performed using the following formula: (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 specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A Q-marker-based grading method for Gastrodia elata, characterized in that, Includes 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 Six different candidate compounds; Scoring the contribution of different candidate compounds to ABTS· scavenging ability; To determine the concentration of the added candidate compound; To add ABTS clearance rate at various 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. Based on the obtained Q-Marker coefficient, calculate the antioxidant score of the Gastrodia elata sample according to formula (9), and then classify the Gastrodia elata according to the antioxidant score. （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; S8. Using a support vector machine model, the Gastrodia elata samples were divided into two categories: high-quality and ordinary. The QS limit was calculated and determined, and it was found that the antioxidant score of high-quality Gastrodia elata should not be less than the QS limit.

2. The method according to claim 1, characterized in that, The The calculation formula is: （3）， In the formula, Normalized values ​​for the content of different candidate compounds in Gastrodia elata; Six different candidate compounds; The average content of a single candidate compound in the Gastrodia elata sample; The maximum value of the average content among the 6 candidate compounds.

3. The method according to claim 2, characterized in that, The The calculation formula is: （4）， In the formula, Normalized scores were assigned to the contributions of different candidate compounds to the ABTS· scavenging ability; The contribution of individual candidate compounds to the ABTS· scavenging ability in Gastrodia elata samples. The maximum contribution of ABTS· scavenging ability among the six candidate compounds is represented by this value.

4. The method according to claim 3, characterized in that, The The calculation formula is: （5）， In the formula, Normalized scores were assigned to the contributions of different candidate compounds to the survival rate of Caenorhabditis elegans. The contribution of individual candidate compounds in the Gastrodia elata sample to the protective capacity against oxidative stress in Caenorhabditis elegans. The highest contribution of the six candidate compounds to the protective ability of Caenorhabditis elegans against oxidative stress was observed.

5. The method according to claim 4, characterized in that, The area of ​​the radar image The calculation is performed using equation (6): （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.

6. The method according to claim 5, characterized in that, The method for normalizing the area of ​​a radar image is as follows: （8） In the formula, 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.

7. The method according to claim 1, characterized in that, The method for determining the content of six candidate compounds in the Gastrodia elata sample was as follows: high performance liquid chromatography (HPLC) was used. An octadecylsilane-bonded silica gel column was used as the packing material, and 0.1% formic acid aqueous solution and acetonitrile were used as the mobile phase for gradient elution. The elution program was: 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 was 30℃, and the detection wavelength was 220 nm.

8. The method according to claim 1, characterized in that, In the ABTS free radical scavenging assay, vitamin C was used as a positive control. Absorbance was measured at 734 nm using a microplate reader, and nonlinear regression analysis was employed to calculate the IC50 of the test samples. 50 value.

9. The method according to claim 1, characterized in that, The antioxidant effect of Gastrodia elata extract was evaluated in an oxidative stress experiment on Caenorhabditis elegans using an H2O2 oxidative damage model.

10. The application of the method as described in any one of claims 1-9 in the quality control of Gastrodia elata.

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