A quality evaluation method of bletilla striata

Abstract

The application discloses a quality evaluation method of Bletilla striata, and belongs to the technical field of traditional Chinese medicinal materials. The quality evaluation method of Bletilla striata comprises the following steps: preparing a test sample solution and a control sample solution of Bletilla striata to be evaluated; determining the contents of bletiliaside, rutin, dioscin III and gastrodin in Bletilla striata by an HPLC method; obtaining the comprehensive quality of Bletilla striata to be evaluated by principal component analysis; and obtaining the regional classification result of Bletilla striata by clustering analysis on the component contents. By the method, the quality of Bletilla striata can be quickly evaluated under the condition of multiple evaluation factors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicinal materials, and particularly relates to a quality evaluation method of Bletilla striata. BACKGROUND

[0002] Bletilla striata is the dried tuber of Bletilla striata (Thunb.) Reichb. f. in the Orchidaceae family, which has the effects of astringency, hemostasis, swelling and tissue regeneration, and is mainly used for treating hemoptysis, hematemesis, external trauma, hemorrhage, sore, and skin chapping. Studies have shown that Bletilla striata has the effects of antibacterial, antiviral, antitumor, antioxidant, promoting wound healing, and promoting gastrointestinal mucosal injury, and is currently used in the treatment of internal and external bleeding, digestive tract ulcers, ulcerative colitis, anorectal diseases, tuberculosis, and chloasma. Bletilla striata is mainly distributed in Sichuan, Guizhou, Yunnan, and Anhui, and studies have shown that different growth environments, planting years, and other conditions of Bletilla striata affect the quality of the medicinal material. Bletilla striata is widely planted, and the quality of Bletilla striata in different producing areas is uneven, and there is no clear specification.

[0003] At present, the quality evaluation method of Bletilla striata only evaluates the quality of Bletilla striata under a single evaluation factor, and the speed of obtaining the quality result of Bletilla striata is slow. SUMMARY

[0004] The present application aims to solve the problem that the quality evaluation method of Bletilla striata only evaluates the quality of Bletilla striata under a single evaluation factor, and the speed of obtaining the quality result of Bletilla striata is slow. The present application provides a quality evaluation method of Bletilla striata, which can quickly evaluate the quality of traditional Chinese medicinal materials under multiple evaluation factors by HPLC combined with cluster analysis and principal component analysis, and can be used as a means of medicinal material quality evaluation. The present application simultaneously applies cluster analysis and principal component analysis to Bletilla striata, evaluates the correlation between various factors and the quality of Bletilla striata, and provides a certain theoretical basis for the further cultivation and utilization of Bletilla striata.

[0005] The pharmacological effect of the plant medicine is related to the secondary metabolites contained therein, and the quality of the plant medicine can be measured by the content of medicinal ingredients. In Bletilla striata, bletilins have a nervous system protection function, can promote intelligence and delay aging, prevent and treat senile dementia, are the pharmacologically active ingredients with a higher content in Bletilla striata, rutin has pharmacological activities such as anti-myocardial injury, anti-inflammatory, anti-oxidation, anti-apoptosis, antibacterial, and antiviral, as a flavonoid substance, rutin also has the effects of protecting plants from ultraviolet rays, pests and diseases, and Dioscorea saponin III has been proved to have certain anticancer activity, gastrodin is widely used for treating vascular and nervous system diseases, in addition, it also has the effects of anti-oxidation, anti-inflammatory and anti-apoptosis. In the 2020 edition of Chinese Pharmacopoeia, 1,4-di[-(glucosyl oxygen)benzyl]-2 isobutyl malonate (bletilin) is taken as a single index, and gastrodin, rutin and Dioscorea saponin III have important medicinal values, so the four components can be used to evaluate the quality of Bletilla striata medicinal materials.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] A quality evaluation method of Bletilla striata, comprising the following steps:

[0008] Preparation of a test sample solution and a control sample solution of the Bletilla striata to be evaluated for quality;

[0009] Determination of the contents of bletilins, rutin, Dioscorea saponin III and gastrodin in Bletilla striata by HPLC method;

[0010] Obtaining the comprehensive quality of the Bletilla striata to be evaluated for quality by principal component analysis;

[0011] Obtaining the regional classification results of Bletilla striata by cluster analysis of the component contents.

[0012] Preferably, the method for obtaining the comprehensive quality of the Bletilla striata to be evaluated for quality by principal component analysis comprises determining the comprehensive quality score y of Bletilla striata by calculating the principal component 1 characteristic vector weight y1 and the principal component 2 characteristic vector weight y2 determined according to the content size, wherein the principal component 1 characteristic vector weight value y1 is obtained by the following formula: y1=0.278*x1+0.296*x2+0.197*x3+0.286*x4, and the principal component 2 characteristic vector weight value y2 is obtained by the following formula: y2=-0.430*x1+0.048*x2+1.031*x3-0.343*x4;

[0013] Wherein, x1, x2, x3 and x4 represent the results of rutin, gastrodin, bletilins and Dioscorea saponin III, respectively.

[0014] Preferably, the bletilla striata is classified according to the comprehensive quality score y, wherein the bletilla striata with a quality score greater than 1.5 is classified into a first category, the bletilla striata with a quality score of 0-1.5 is classified into a second category, and the bletilla striata with a quality score less than 0 is classified into a third category.

[0015] Preferably, the step of obtaining the regional classification result of the bletilla striata comprises: sorting the bletilla striata according to the comprehensive quality of the bletilla striata, using the content of the ingredients of bletilla striata glycoside, rutin, gastrodin and dioscin III as variables to perform cluster analysis on the bletilla striata, and performing regional classification on the bletilla striata through the determination of Euclidean distance.

[0016] Preferably, the selected Euclidean distance is 4.

[0017] Preferably, the test sample solution is prepared according to the following method:

[0018] The bletilla striata tuber is crushed and passed through an 80-mesh sieve. 0.250 g of bletilla striata tuber powder is accurately weighed into a stoppered conical flask, 25 mL of 70% ethanol is added, and the flask is placed at room temperature for 30 min for soaking. Then, ultrasonic extraction is performed at 40°C for 30 min. The extract is filtered through a 0.22 μm microporous filter membrane. The filtrate is taken to obtain a sample solution with a mass concentration of 0.1 g / mL of bletilla striata tuber dry powder.

[0019] Preferably, the control sample solution is prepared according to the following method:

[0020] 2 mg of each of the bletilla striata glycoside, rutin, dioscin III and gastrodin control samples is weighed, 1 mL of anhydrous ethanol is added to prepare a standard solution with a mass concentration of 2 mg / mL. Then, gradient dilution is performed to obtain standard solutions with mass concentrations of 1 mg / mL, 0.5 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.02 mg / mL and 0.01 mg / mL.

[0021] Preferably, the HPLC conditions are as follows: the mobile phase is acetonitrile-0.1% phosphoric acid using gradient elution, the column temperature is 30°C, the detection wavelength is 270 nm, and the injection volume is 10 μL.

[0022] The present application has the following beneficial effects:

[0023] The present application takes bletilla striata samples from multiple production areas and different growth years in Guizhou and Anhui, the main production areas of bletilla striata, determines the contents of four ingredients of bletilla striata glycoside, rutin, dioscin III and gastrodin in bletilla striata from different production areas and different growth years through HPLC, and combines principal component analysis and cluster analysis methods to obtain the quality of bletilla striata from different production areas and different growth years and determine the cultivation conditions of bletilla striata. The quality of traditional Chinese medicinal materials can be quickly evaluated in the case of multiple evaluation factors. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 A chromatogram of a test solution of the invention is shown (abscissa: time, ordinate: response value);

[0025] Figure 2 A chromatogram of a test solution of the invention is shown (abscissa: time, ordinate: response value)

[0026] Figure 3 A chromatogram of a test solution of the invention is shown (abscissa: time, ordinate: response value);

[0027] Figure 4 A chromatogram of a test solution of the invention is shown (abscissa: time, ordinate: response value);

[0028] Figure 5 A chromatogram of a test solution of the invention is shown (abscissa: time, ordinate: response value);

[0029] Figure 6 A chromatogram of a test solution of the invention is shown (abscissa: time, ordinate: response value); DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application, i.e., the described embodiments are only a part of the embodiments of the present application, but not all the embodiments.

[0031] Example 1

[0032] The present embodiment provides a quality evaluation method for Bletilla striata, comprising the following steps:

[0033] 1. Instrument preparation

[0034] Agilent 1260 high performance liquid chromatograph (Agilent, USA);

[0035] BT125D electronic analytical balance (Sartorius Scientific Instruments Co., Ltd.);

[0036] DHG-9070A air drying oven (Shanghai Hengyi Scientific Instrument Co., Ltd.);

[0037] 2500C multifunctional pulverizer (Dongguan Fangtai Electrical Appliance Co., Ltd.);

[0038] SG8200HBT ultrasonic cleaner (Shanghai Guantai Ultrasonic Instrument Co., Ltd.);

[0039] UPR-11-15TW type ultrapure water machine (Sichuan UPR Ultrapure Technology Co., Ltd.).

[0040] 2. Reagent preparation

[0041] Anhydrous ethanol (AR, Chuan Dong Chemical Co., Ltd. in Chongqing);

[0042] Acetonitrile (AR, Beijing Mai Rui Da Technology Co., Ltd.);

[0043] Phosphoric acid (AR, Chengdu Jinshan Chemical Reagent Co., Ltd.);

[0044] Baiji Shuang (purity ≥98%, batch number CFS202102);

[0045] Rutin (purity ≥98%, batch number CFS2022101);

[0046] Dioscin III (purity ≥98%, batch number CFS202102);

[0047] Gastrodin (purity ≥98%, batch number CFS202102) reference substance (Wuhan Tianzhi Biological Technology Co., Ltd.).

[0048] 3. Preparation of medicinal materials

[0049] Baiji medicinal materials (number: S1-S22) from Guizhou Province and Anhui Province for 15 Baiji producing areas, stored in the Chinese Herbal Medicine Laboratory of Guizhou Academy of Agricultural Sciences.

[0050] Table 1 Information of Baiji medicinal material samples

[0051]

[0052] Note: In Table 1, Baiji medicinal materials of S18 and S19 are from different producing areas in Dejiang County.

[0053] 4. Test sample solution was prepared as follows:

[0054] The Baiji tuber was crushed and passed through an 80-mesh sieve. 0.250 g of Baiji tuber powder was accurately weighed into a stoppered conical flask, 25 mL of 70% ethanol was added, and it was immersed at room temperature for 30 min. Then, it was extracted at 40°C for 30 min under ultrasonic extraction. The extract was filtered through a 0.22 μm microporous filter membrane. The filtrate was taken to obtain a sample solution with a Baiji tuber dry powder mass concentration of 0.1 g / mL.

[0055] 5. Reference substance solution was prepared as follows:

[0056] Accurately weigh 2 mg of each of the reference substances of bletilavoside, rutin, dioscin III and gastrodin, add 1 mL of absolute ethanol to prepare a standard solution with a mass concentration of 2 mg / mL, and then dilute it to a mass concentration of 1 mg / mL, 0.5 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.02 mg / mL and 0.01 mg / mL by gradient dilution.

[0057] 6. The peak retention time and peak area of bletilavoside, rutin, dioscin III and gastrodin are determined by HPLC method for the reference substance solutions of bletilavoside, rutin, dioscin III and gastrodin respectively, and the peak area of each specific peak time of the test sample solution is recorded by HPLC method.

[0058] The chromatographic conditions are as follows:

[0059] Chromatographic column: Agilent HC-C 18 (2) Chromatographic column (250 x 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-0.1% phosphoric acid (B), gradient elution (0-10 min, 5% A→20% A; 10-30 min, 20% A→60% A; 30-40 min, 60% A-90% A, 40-50 min, 90% A-90% A); collection time: 50 min; flow rate: 1 mL / min; column temperature: 30°C; detection wavelength: 270 nm; injection volume: 10 μL.

[0060] The chromatograms of the reference substance solutions of gastrodin, rutin, bletilavoside and dioscin III are shown in Figure 2 , Figure 3 , Figure 4 and Figure 5 respectively.

[0061] The chromatogram of the test sample solution is shown in Figure 1 .

[0062] The contents of bletilavoside, rutin, dioscin III and gastrodin in No. S1-S22 bletilavoside are calculated by referring to the peak retention time and peak area of the reference substances bletilavoside, rutin, dioscin III and gastrodin, and the results are shown in Table 2.

[0063] Table 2 Determination results of the content of each component (mg / mL)

[0064]

[0065] 7. Principal component analysis:

[0066] 7.1 KMO test and Bartlett's test of sphericity were performed on the data in Table 2, the KMO value was 0.688, and the Bartlett's test of sphericity resulted in a concomitant probability of 0.000, which was less than the significance level 0.05, so the null hypothesis of Bartlett's test of sphericity was rejected, and it was considered that there was strong correlation between the variables, and it was suitable for principal component analysis.

[0067] 7.2 According to step 7.1, principal component analysis was performed on 4 components of 22 batches of different origin and different age of Bletilla striata samples, and the variance characteristic values and cumulative contribution rates of the four component indicators were obtained as shown in Table 3; and the component matrix of the component content of the 22 batches of Bletilla striata samples was shown in Table 4. The principal components in the variables were determined by the contribution rate.

[0068] Table 3 Variance characteristic values and cumulative contribution rates of four component indicators

[0069]

[0070] From Table 3, the variance characteristic value of component 1 was 3.136, the variance characteristic value of component 2 was 0.731, and the cumulative contribution rate of the two components reached 96.682%, which was greater than 85%, so the above two principal components could represent the information of the four components of the 22 samples, and therefore the two principal components could be selected as the comprehensive evaluation index of the component content of Bletilla striata.

[0071] Table 4 Component content component matrix of 22 batches of Bletilla striata samples

[0072]

[0073] From the data in Table 3 and Table 4, the contribution rate of principal component 1 was the largest, which was 78.404%, and the characteristic value was 3.316, indicating that principal component 1 played a dominant role in the analysis, and the information of principal component 1 mainly came from rutin, gastrodin and bletilavoside, and the three indicators had positive influence on principal component 1. The contribution rate of principal component 2 was 18.278%, and the characteristic value was 0.731, and the information mainly came from dioscin III.

[0074] 7.3 According to the calculation formula of the principal component characteristic vector coefficient, the principal component characteristic vector coefficient was calculated, and the characteristic vector weight value of principal component 1 and the characteristic vector weight value of principal component 2 were obtained, and the calculation formula was as follows:

[0075] y1 = 0.278 * x1 + 0.296 * x2 + 0.197 * x3 + 0.286 * x4

[0076] y2 = -0.430 * x1 + 0.048 * x2 + 1.031 * x3 - 0.343 * x4

[0077] Wherein, x1, x2, x3, x4 represent the results of rutin, gastrodin, bletilla striata glycoside and dioscorea opposita Ⅲ respectively.

[0078] The calculation formula of the comprehensive quality score of bletilla striata is as follows:

[0079] y = 0.7840 * y1 + 0.828 * y2

[0080] y1 represents the characteristic vector weight value of principal component 1, and y2 represents the characteristic vector weight value of principal component 2.

[0081] 7.4 The variable calculation is carried out by SPSS 25.0, and the data of each component content after normalization is respectively substituted into y1 and y2 formula, so that the y1 and y2 principal component factor scores can be obtained. Then, the scores are substituted into the calculation formula of the comprehensive quality score of bletilla striata, so that the comprehensive quality scores of 22 bletilla striata samples can be obtained, and the ordering is carried out. The ordering results are shown in Table 5.

[0082] Table 5 Principal component scores and comprehensive scores of bletilla striata quality of different producing areas and different years

[0083]

[0084]

[0085] According to the related research, the higher the comprehensive score of the bletilla striata sample is, the better the overall quality of the bletilla striata is. Therefore, according to Table 5, the bletilla striata samples with the comprehensive scores of the quality of different producing areas and different years greater than 0 are in turn three-year-old (S21) from Pingtang County of Guizhou Province, three-year-old (S22) from Tongling City of Anhui Province, three-year-old (S17) from Huaxi District of Guiyang City of Guizhou Province, one-year-old (S8) from Pu'an County of Guizhou Province, two-year-old (S19) from Dejiang County of Tongren City of Guizhou Province, three-year-old (S16) from Zongyang County of Anhui Province, and one-year-old (S18) from Dejiang County of Tongren City of Guizhou Province. It is preliminarily considered that the comprehensive quality of these batches of bletilla striata samples is better.

[0086] 7.5 The bletilla striata samples with the comprehensive quality scores greater than 1.5 are classified into the first type, the bletilla striata samples with the comprehensive quality scores of 0-1.5 are classified into the second type, and the bletilla striata samples with the comprehensive quality scores less than 0 are classified into the third type. The numbers S21 and S22 are in the first type, the numbers S8 and S16-S19 are in the second type, and the numbers S1-S7, S9-S15 and S20 are in the third type. The comprehensive quality of the numbers S21 and S22 is the best, the comprehensive quality of the numbers S16-S19 is the second, and the comprehensive quality of the numbers S1-S15 and S20 is the lowest.

[0087] 8. Cluster analysis:

[0088] The three-year-old samples of 22 Bletilla samples from different producing areas were sorted, and the sorting results are shown in Table 6. The three-year-old Bletilla samples from 15 producing areas in the sorted 22 Bletilla samples were used as variables with four components of bletilline, rutin, gastrodin and dioscin III, and cluster analysis was performed by using SPSS 25.0 software, and the clustering results obtained by analysis are shown in Figure 6

[0089] Table 6 Different producing areas of three-year-old Bletilla

[0090]

[0091] From Figure 2 It can be seen that when the Euclidean distance is 4, the Bletilla samples from the 15 producing areas can be divided into 3 categories, among which the three-year-old Bletilla from the producing areas of S2, S4, S6, S11, S13, S15, S16, S17 and S20 can be divided into one category, the three-year-old Bletilla from the producing areas of S8, S10, S18 and S19 can be divided into one category, and S21 and S22 can be divided into one category.

[0092] Results: According to the results of principal component analysis, the quality of the 22 batches of Bletilla samples is the best in Pingtang County, Guizhou Province (S21) three-year-old Bletilla, and in the case of the same producing area, the quality of three-year-old Bletilla is generally better than that of one-year-old Bletilla. The three-year-old Bletilla samples from 15 producing areas are divided into three categories by cluster analysis, and combined with the results of principal component analysis, it can be known that the quality of the 22 batches of Bletilla samples is the best in Pingtang County, Guizhou Province (S21) three-year-old Bletilla and Tongling City, Anhui Province (S22) three-year-old Bletilla.

[0093] From the results of principal component analysis and cluster analysis, it can be known that the environmental conditions in Pingtang County, Guizhou Province and Tongling City, Anhui Province are very suitable for the growth of Bletilla, and the environmental conditions in the two producing areas of Pingtang County, Guizhou Province and Tongling City, Anhui Province can be used as the basis for cultivating Bletilla.

[0094] From the quality classification in principal component analysis and the classification of different producing areas in cluster analysis, for example: in principal component analysis, S8 and S16-S19 are in the same category, and in cluster analysis, S8 and S10, S18 and S19 are in the same category, it shows that even under the same growing conditions, the quality of Bletilla will be different, which may be related to the personnel cultivating Bletilla, which is only a guess, and also provides research value for the growth factors of Bletilla.

[0095] ​Conclusion: In this example, HPLC method was used to determine the content of 15 production areas, one-year-old and three-year-old Bletilla striata of four components of Bletilla striata, rutin, dioscin III, gastrodin. Combined with principal component analysis and cluster analysis, the best conditions for the growth of Bletilla striata were determined. According to the results of principal component analysis, it can be seen that the comprehensive quality of Bletilla striata in different production areas and different growth years has certain differences. In the same production area, the quality of Bletilla striata generally increases with the growth of the growth year.

[0096] Cluster analysis of 15 production areas of three-year-old Bletilla striata is roughly divided into three categories, indicating that the production area has a certain degree of influence on the content of various components of Bletilla striata. This influence may come from different climate conditions, soil conditions, light conditions, etc. in different production areas. Bletilla striata with similar geographical location or similar conditions is classified into a category.

[0097] In this example, in the case of the same production area, the comprehensive quality of three-year-old Bletilla striata is generally better than that of one-year-old Bletilla striata, and the quality of Bletilla striata with long growth years is better than that of Bletilla striata with short growth years, which may be related to the accumulation of metabolic products with the growth of time. The secondary metabolites in Bletilla striata accumulate continuously with the growth of planting time. Although the content may not always show an upward trend due to the synthesis of other substances, the content of Bletilla striata, rutin, dioscin III, and gastrodin as the basis of effective substances in Bletilla striata is evaluated, and the overall quality is still on the rise. This is of great significance to determine the quality of medicinal plants and the rule of cultivation years, and to cultivate and rationally develop and utilize medicinal materials.

[0098] According to relevant research, the content of metabolites of traditional Chinese medicinal materials varies with the production area. In this example, the comprehensive quality of Bletilla striata in different production areas is significantly different, which is closely related to various environmental factors in the production area. According to relevant research, different environmental conditions, such as light, CO2 concentration, air temperature, water, altitude, and biological factors, can affect the synthesis of plant secondary metabolites by regulating plant gene expression and the activity of synthesis pathways, thereby affecting the accumulation of plant metabolites. And this influence is the result of the synergistic effect of various environmental factors. It can be seen that the interaction between various environmental factors in the production area affects the accumulation of Bletilla striata metabolites, thereby affecting the quality of Bletilla striata. This influence mechanism is complex and needs further research.

[0099] The present application can quickly evaluate the quality of Chinese medicinal materials under various evaluation factors by determining the contents of four effective medicinal ingredients, namely, bletilloside, rutin, dioscin III and gastrodin, by HPLC method, combining with cluster analysis and principal component analysis, and can be used as a means for evaluating the quality of medicinal materials. In the present embodiment, the cluster analysis and the principal component analysis are simultaneously applied to the Chinese medicinal material bletilla, the correlation between various factors and the quality of bletilla is evaluated, the suitable growth conditions of bletilla are determined, and certain research value is provided for the further cultivation and utilization of bletilla medicinal materials.

[0100] Test Example

[0101] 1. Linear relationship investigation:

[0102] The control sample solutions of bletilloside, rutin, dioscin III and gastrodin with different concentrations in step 5 of Example 1 were injected and determined for 3 times under the chromatographic conditions of step 6 of Example 1. The regression was performed with the mass concentration of the control sample as the abscissa (X) and the peak area as the ordinate (Y), and the results are shown in Table 7:

[0103] Table 7 Linear relationship of each component

[0104]

[0105] As shown in Table 7, each component has good linear relationship within its own range.

[0106] 2. Precision test:

[0107] The control sample solutions in step 5 of Example 1 were respectively taken and injected for 6 times under the chromatographic conditions of step 6 of Example 1, and the peak area RSDs of bletilloside, rutin, dioscin III and gastrodin were 0.53%, 0.21%, 0.34% and 1.29% respectively, indicating that the precision of the instrument is good.

[0108] 3. Reproducibility test:

[0109] Precisely weighed bletilla tuber powder was prepared into 6 test sample solutions according to the method in step 4 of Example 1, and was continuously injected under the chromatographic conditions of step 6 of Example 1, and the peak area RSDs of the contents of bletilloside, rutin, dioscin III and gastrodin were 0.89%, 1.28%, 1.21% and 1.50% respectively, indicating that the method has good reproducibility.

[0110] 4. Stability test:

[0111] The Bletilla striata tuber powder was precisely weighed, and one sample solution was prepared according to the method in Step 4 of Example 1. Under the chromatographic conditions of Example 1, 10 μL of the sample solution was injected at 0, 2, 4, 6, 8, 12, and 24 h after preparation, respectively. The RSDs of the peak areas of Bletilla striata glycoside, rutin, dioscin III, and Gastrodia elata glycoside were 0.238%, 0.35%, 1.18%, and 1.35%, respectively, indicating that the sample was stable within 24 h.

[0112] 5. Sample injection recovery test:

[0113] The same batch of sample with known content was precisely weighed, and 1 mL of the standard solution of Bletilla striata glycoside, rutin, dioscin III, and Gastrodia elata glycoside (with mass concentrations of 1.5616, 0.0147, 0.0220, and 0.2534 mg / mL, respectively) was added. The sample solution was prepared according to the method in Step 4 of Example 1, and was injected under the chromatographic conditions of Step 6 of Example 1. The average sample injection recovery rates of Bletilla striata glycoside, rutin, dioscin III, and Gastrodia elata glycoside were 100.73%, 94.10%, 98.02%, and 101.43%, respectively, and the RSDs were 0.55%, 1.75%, 0.56%, and 1.38%, respectively, indicating that the method had high accuracy.

[0114] The above examples only express the specific embodiments of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the protection scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the technical concept of the present application, a number of modifications and improvements can be made, which are within the protection scope of the present application.

Claims

1. A method for evaluating the quality of Paeonia lactiflora, characterized by, The method comprises the following steps: Preparation of the sample solution and the control solution of the bletilla striata to be evaluated; Determination of the contents of bletilla striate glycoside, rutin, dioscin III and gastrodin in the bletilla striata by HPLC; Obtaining the comprehensive quality of the bletilla striata to be evaluated by principal component analysis; Obtaining the regional classification results of the bletilla striata by cluster analysis of the component contents; The method for obtaining the comprehensive quality of the bletilla striata to be evaluated by principal component analysis comprises determining the comprehensive quality score y of the bletilla striata by calculating the feature vector weight y1 of the principal component 1 and the feature vector weight y2 of the principal component 2 determined according to the contents, wherein the feature vector weight value y1 of the principal component 1 is obtained by the following formula: y1=0.278*x1+0.296*x2+0.197*x3+0.286*x4, and the feature vector weight value y2 of the principal component 2 is obtained by the following formula: y2=-0.430*x1+0.048*x2+1.031*x3-0.343*x4; Wherein, x1, x2, x3 and x4 represent the results of rutin, gastrodin, bletilla striate glycoside and dioscin III respectively; The step of obtaining the regional classification results of the bletilla striata comprises: sorting the bletilla striata according to the comprehensive quality, using the component contents of bletilla striate glycoside, rutin, gastrodin and dioscin III as variables, performing cluster analysis on the bletilla striata, and dividing the bletilla striata into regions according to the Euclidean distance; The sample solution is prepared by crushing the bletilla striata tubers, and extracting the bletilla striata tuber dry powder by ultrasonic extraction at 40℃ with 70% ethanol; The conditions of the HPLC are: HC-C 18 Column 250 x 4.6 mm, 5 μm; mobile phase: acetonitrile A - 0.1% phosphoric acid B, gradient elution 0-10 min, 5% A→20% A; 10-30 min, 20% A→60% A; 30-40 min, 60% A-90% A, 40-50 min, 90% A-90% A; detection wavelength 270 nm.

2. The quality evaluation method of the Bletilla striata according to claim 1, characterized in that, The bletilla striata is divided into three categories according to the quality score, wherein the bletilla striata with a quality score greater than 1.5 is classified into the first category, the bletilla striata with a quality score of 0-1.5 is classified into the second category, and the bletilla striata with a quality score less than 0 is classified into the third category, and the results of bletilla striate glycoside, rutin, gastrodin and dioscin III are expressed in mg / mL.

3. The quality evaluation method of the Bletilla striata according to claim 1, characterized in that, The selected Euclidean distance is 4.

4. The quality evaluation method of the Bletilla striata according to claim 1, characterized in that, The sample solution is prepared by crushing the bletilla striata tubers, and extracting the bletilla striata tuber dry powder by ultrasonic extraction at 40℃ with 70% ethanol; The sample solution is prepared by crushing the bletilla striata tubers, and extracting the bletilla striata tuber dry powder by ultrasonic extraction at 40℃ with 70% ethanol; 5. The quality evaluation method of the Bletilla striata according to claim 1, characterized in that, The control solution is prepared by weighing 2 mg of bletilla striate glycoside, rutin, dioscin III and gastrodin, respectively, and adding 1 mL of anhydrous ethanol to prepare a standard solution with a mass concentration of 2 mg / mL, and then diluting it into standard solutions with mass concentrations of 1 mg / mL, 0.5 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.02 mg / mL and 0.01 mg / mL by gradient dilution. The HPLC conditions are as follows: column temperature 30℃, injection volume 10μL.

6. The quality evaluation method of the Bletilla striata according to Claim 1, wherein, ​

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