Construction of chinaroot greenbrier medicinal material fingerprint and application of chinaroot greenbrier medicinal material fingerprint in medicinal material quality control

Abstract

The invention relates to construction of a chinaroot greenbrier medicinal material fingerprint spectrum and application of the chinaroot greenbrier medicinal material fingerprint spectrum in medicinal material quality control. The HPLC fingerprint spectrum of the chinaroot greenbrier medicinal material has 23 characteristic fingerprint chromatographic peaks, the peak 6 is chlorogenic acid, the peak 7 is epicatechin, the peak 9 is 5-O-caffeoylshikimic acid, the peak 10 is polydatin, the peak 11 is oxyresveratrol, the peak 12 is neoastilbin, the peak 13 is astilbin, the peak 14 is neoiso-astilbin, the peak 15 is resveratrol, the peak 16 is engeletin, and the peak 17 is quercitrin. The peak 20 is kaempferol.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine analysis, and particularly relates to the construction of a fingerprint of Smilax china L. and its application in the quality control of medicinal materials. Background Art

[0002] Smilax china L. is the dried rhizome of the plant Smilax china L. of the Liliaceae family. It tastes sweet, slightly bitter and astringent, and is of mild nature. It belongs to the liver and kidney meridians. Also known as Jin Gang Ci, Jin Gang Teng, Red Tuckahoe, etc., it is mainly distributed in mountain slopes and river valleys below 2000m above sea level. It has the effects of promoting diuresis and removing turbidity, expelling wind and relieving arthralgia, and detoxifying and dispersing stasis. It is used for symptoms such as turbid urine, excessive leukorrhea, rheumatic arthralgia, furuncles and carbuncles. The main components of Smilax china L. include flavonoids, glycosides, steroidal saponins, organic acids, etc. Modern pharmacological studies have shown that Smilax china L. has various pharmacological effects such as hypoglycemic, anti-inflammatory, antioxidant, anti-rheumatic, and anti-tumor effects.

[0003] Fingerprint combined with multi-index analysis methods have been widely used in the overall quality evaluation of traditional Chinese medicine and compound preparations. Spectrum-effect analysis is based on the research of traditional Chinese medicine fingerprints to explore the effective ingredient groups of traditional Chinese medicine and the correlation between the components and efficacy of traditional Chinese medicine. At present, there are studies on the antioxidant activities of different extraction parts such as ethyl acetate and petroleum ether in Smilax china L., but there are no reports on the spectrum-effect relationship of Smilax china L. Therefore, the present invention establishes an HPLC fingerprint of Smilax china L. and measures its in vitro antioxidant activity. For the first time, starting from the fingerprint and antioxidant activity, multiple spectrum-effect analysis methods are combined to screen the antioxidant active ingredient groups of Smilax china L., providing a reference basis for improving the quality evaluation system and development and utilization of Smilax china L. medicinal materials. Summary of the Invention

[0004] The present invention provides an HPLC fingerprint (or its construction method) of Smilax china L. medicinal materials, characterized in that when the HPLC chromatographic conditions are as follows, the HPLC fingerprint of the Smilax china L. medicinal materials is basically consistent with Figure 2 the following;

[0005] The HPLC chromatographic conditions are as follows:

[0006] The chromatographic column is an Agilent 5TC-C 18 chromatographic column, specification: 250mm×4.6mm, 5μm; mobile phase: methanol as phase A, 0.05% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0 - 20min, 25% - 35% A; 20 - 30min, 35% A; 30 - 40min, 35% - 38% A; 40 - 45min, 38% A; 45 - 55min, 38% - 45% A; 55 - 70min, 45% - 63% A; 70 - 75min, 63% - 70% A; 75 - 80min, 70% A; column temperature 30°C, injection volume 10μL, flow rate 0.8mL·min-1 , the detection wavelength is 295 nm.

[0007] Another embodiment of the present invention provides the HPLC fingerprint of the above-mentioned Smilax china L. medicinal materials (or its construction method), which is characterized in that the HPLC fingerprint of the Smilax china L. medicinal materials is basically the same as Figure 2 consistent, with 23 characteristic fingerprint chromatographic peaks, among which peak 6 is chlorogenic acid, peak 7 is epicatechin, peak 9 is 5-O-caffeoylshikimic acid, peak 10 is piceid, peak 11 is oxyresveratrol, peak 12 is neoponcirin, peak 13 is poncirin, peak 14 is neoheteroponcirin, peak 15 is resveratrol, peak 16 is engelitin, peak 17 is quercitrin, and peak 20 is kaempferol.

[0008] Another embodiment of the present invention provides the application of the HPLC fingerprint of the above-mentioned Smilax china L. medicinal materials (or its construction method) in the quality control and component analysis of Smilax china L. medicinal materials.

[0009] Another embodiment of the present invention provides the application of the HPLC fingerprint of the above-mentioned Smilax china L. medicinal materials (or its construction method) in the quality control of Smilax china L. medicinal materials, which is characterized in that the application includes the following steps:

[0010] (1) Crush the Smilax china L. medicinal materials, add methanol solution, perform ultrasonic treatment, and after filtering through a microporous filter membrane, obtain a test solution;

[0011] (2) Take the test solution obtained in step (1), perform HPLC detection to obtain the HPLC chromatogram of the test solution, and the chromatographic conditions are as follows:

[0012] The chromatographic column is an Agilent 5TC-C 18 chromatographic column, specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: methanol is phase A, 0.05% phosphoric acid aqueous solution is phase B; gradient elution conditions: 0 - 20 min, 25% - 35% A; 20 - 30 min, 35% A; 30 - 40 min, 35% - 38% A; 40 - 45 min, 38% A; 45 - 55 min, 38% - 45% A; 55 - 70 min, 45% - 63% A; 70 - 75 min, 63% - 70% A; 75 - 80 min, 70% A; column temperature 30 °C, injection volume 10 μL, flow rate 0.8 mL·min -1 , the detection wavelength is 295 nm;

[0013] (3) Compare the HPLC chromatogram of the test solution obtained in step (2) with the HPLC fingerprint of the Smilax china L. medicinal materials described in the present invention, and the Smilax china L. medicinal materials with a similarity above 0.80 are qualified products.

[0014] Among them, in step (1), after pulverization, it is sieved through a 40-mesh sieve. The methanol solution is a methanol solution with a volume fraction of 60%-70%. The ultrasonic time is 20-40 min. The microporous filter membrane is selected from a 0.22-μm filter membrane or a 0.45-μm filter membrane.

[0015] Another embodiment of the present invention provides a method for exploring the relationship between the common peaks of the fingerprint of Smilax china L. and its antioxidant activity, which is characterized by optionally including the following steps (1) and / or (2):

[0016] (1) Grey relational analysis (GRA): Taking the reciprocal of the antioxidant IC 50 value as the mother sequence, and the peak areas of each common peak as the sub-sequences to form a data matrix. The initial value method is used to perform dimensionless quantization processing on the data matrix, and the resolution coefficient is taken as 0.5 for GRA analysis to calculate the correlation degree between the common peaks of Smilax china L. and the antioxidant activity. The greater the correlation degree, the greater the influence of the comparison sequence on the reference sequence. The results show that the correlation degrees between 23 common peaks and the antioxidant activity results are all greater than 0.7, indicating that the antioxidant effect of Smilax china L. is affected by the combined action of multiple components;

[0017] (2) Partial least squares regression (PLSR) analysis: After standardizing the peak areas of 23 common peaks and the antioxidant activity data in the fingerprint spectra of 29 batches of Smilax china L., they are imported into the SIMCA14.1 software to calculate the standardized regression coefficients and variable importance in projection VIP values. A positive regression coefficient represents a positive correlation between the peak area of the common peak and the antioxidant activity, and vice versa. The VIP value represents the contribution degree of the variable. When VIP>1, it indicates that the variable has a significant impact on the model; The chromatographic peaks 6, 11-13, and 15 have VIP>1 and are positively correlated with the DPPH radical scavenging ability; The chromatographic peaks 6, 10, 12, 15, and 18 have VIP>1 and are positively correlated with the ABTS radical scavenging ability; It shows that the compounds represented by these characteristic peaks play an important role in the antioxidant effect of Smilax china L.

[0018] Another embodiment of the present invention provides the above method, which is characterized in that by comprehensively analyzing the results of GRA and PLSR, the characteristic peaks showing differences in antioxidant effects among different batches of Smilax china L. are peak 6: chlorogenic acid, peak 10: polydatin, peak 11: oxyresveratrol, peak 12: neoponierin, peak 13: ponierin, peak 15: resveratrol, and peak 18.

[0019] Unless otherwise specified, the ratio of the mobile phase in the chromatographic conditions of the present invention is a volume ratio; 0.05% phosphoric acid aqueous solution refers to an aqueous solution of phosphoric acid with a mass fraction of 0.05%. The Smilax china L. or Smilax china L. medicinal materials mentioned in the present invention generally refer to Smilax china L. decoction pieces.

[0020] Compared with the prior art, the advantages of the present invention are as follows: (1) The present invention makes up for the deficiencies of the prior art, establishes the HPLC fingerprint of 29 batches of Smilax china samples from different sources, and identifies 23 common peaks. The Smilax china samples were preliminarily comprehensively evaluated by hierarchical cluster analysis (HCA) and principal component analysis (PCA). The antioxidant activities of the samples were evaluated by different test methods, and the spectrum-effect relationship of Smilax china was established by grey relational analysis (GRA) and partial least squares regression (PLSR) analysis. It was found that components such as chlorogenic acid, resveratrol glucoside, oxyresveratrol, neoastilbin, astilbin, and peak resveratrol have relatively large contribution values to the antioxidant activity of Smilax china, and may be the key components for the antioxidant effect of Smilax china. The present invention provides a scientific and effective method for the study of the pharmacodynamic material basis and quality control of Smilax china, and also provides a reference for the development and utilization of Smilax china. Description of the Drawings

[0021] Figure 1 is the HPLC fingerprint and reference fingerprint (R) of 29 batches of Smilax china medicinal materials.

[0022] Figure 2 is the reference fingerprint generated by fitting the fingerprint of 29 batches of Smilax china medicinal materials of the present invention.

[0023] Figure 3 is the HPLC chromatogram of the test sample (A) and mixed reference substances (B) of Smilax china medicinal materials; peak 6 is chlorogenic acid; peak 7 is epicatechin; peak 9 is 5-O-caffeoylshikimic acid; peak 10 is resveratrol glucoside; peak 11 is oxyresveratrol; peak 12 is neoastilbin; peak 13 is astilbin; peak 14 is neoisoastilbin; peak 15 is resveratrol; peak 16 is engelitin; peak 17 is quercitrin; peak 20 is kaempferol.

[0024] Figure 4 is the hierarchical cluster analysis chart of 29 batches of Smilax china medicinal materials.

[0025] Figure 5 is the PCA score chart of 29 batches of Smilax china medicinal materials.

[0026] Figure 6 is the partial least squares regression coefficient chart of each common peak of Smilax china medicinal materials.

[0027] Figure 7 is the VIP value of each common peak of Smilax china medicinal materials. Detailed Embodiments

[0028] 1 Materials

[0029] 1.1 Instruments

[0030] Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA); KQ5200DE numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); DFT-200A portable high-speed grinder (Wenling Lindaj Machinery Co., Ltd.); BT25S electronic analytical balance (d = 0.01 mg), BS210S electronic analytical balance (d = 0.1 mg) (Sartorius Beijing Co., Ltd.); MULTISKAN Sky microplate reader (Thermo Fisher Scientific, USA).

[0031] 1.2 Reagents and drugs

[0032] Reference substances astilbin (batch number: CHB201131), 5-O-caffeoylshikimic acid (batch number: CHB230906), resveratrol (batch number: CHB201107), polydatin (batch number: CHB201129) were all purchased from Chengdu Cromar Biotechnology Co., Ltd. (purity ≥ 98%); engeletin (batch number: 111906 - 202303), chlorogenic acid (batch number: 110753 - 201314), kaempferol (batch number: 110861 - 201310), quercitrin (batch number: 111538 - 202308) were all purchased from National Institutes for Food and Drug Control (purity ≥ 93.6%); neoastilbin (batch number: HR4164W2), astillbin (batch number: HR4163W4), epicatechin (batch number: HR15126S1), oxyresveratrol (batch number: HS052957) were all purchased from Baoji Chenguang Biotechnology Co., Ltd. (purity ≥ 98%); methanol and acetonitrile were chromatographically pure (Thermo Fisher Scientific, USA); 2,2-diphenyl-1-picrylhydrazyl (DPPH, batch number: C16266521) was purchased from Shanghai Macklin Biochemical Co., Ltd.; vitamin C (VC, batch number: D01GB169601) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (purity ≥ 98%); 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) scavenging ability kit (batch number: 20241011) was purchased from Nanjing Jiancheng Bioengineering Institute. Water was ultrapure water, and other reagents were all of analytical grade.

[0033] Twenty-nine batches of Smilax china L. rhizome pieces were identified by Researcher Zhang Hong of Shaanxi Academy of Traditional Chinese Medicine as the dried rhizomes of the Liliaceae plant Smilax china L. The sample source information is shown in Table 1.

[0034] Table 1 Information of Smilax china L. samples

[0035]

[0036] 2 Methods and results

[0037] 2.1 Chromatographic conditions

[0038] Use an Agilent 5TC-C 18 (2)(250 mm×4.6 mm, 5 μm) chromatographic column; Use methanol (A) - 0.05% phosphoric acid aqueous solution (B) as the mobile phase, elution gradient (0 - 20 min, 25% - 35% A; 20 - 30 min, 35% A; 30 - 40 min, 35% - 38% A; 40 - 45 min, 38% A; 45 - 55 min, 38% - 45% A; 55 - 70 min, 45% - 63% A; 70 - 75 min, 63% - 70% A; 75 - 80 min, 70% A), column temperature 30 °C, injection volume 10 μL, flow rate 0.8 mL·min -1 , and the detection wavelength is 295 nm.

[0039] 2.2 Preparation of reference substance solution

[0040] Accurately weigh appropriate amounts of reference substances epicatechin, 5-O-caffeoylshikimic acid, neofasciculin, fasciculin, neo-isofasciculin, engelitin, resveratrol, chlorogenic acid, piceid, oxyresveratrol, quercitrin, and kaempferol respectively, dissolve and make up the volume with 70% methanol to prepare a mixed reference substance solution with mass concentrations of 37.5, 25, 33, 33, 37.5, 37.5, 17, 25, 50, 42, 83, 83 μg·mL -1 respectively.

[0041] 2.3 Preparation of test solution

[0042] Accurately weigh 0.7 g of Smilax glabra Roxb. powder (passing through a 40-mesh sieve), place it in a conical flask, accurately add 10 mL of 70% methanol solution by volume, weigh it, ultrasonically extract for 30 min, let it stand at room temperature, weigh it again, make up the weight loss with 70% methanol solution, shake well, filter, and take the continuous filtrate through a 0.45 μm microporous filter membrane to obtain the solution.

[0043] 2.4 Methodology investigation

[0044] 2.4.1 Precision test

[0045] Take an appropriate amount of sample No. S24, prepare the test solution according to the method under "2.3", inject samples continuously for 6 times according to the chromatographic conditions under "2.1", and record the chromatogram. Using the 13th peak fasciculin as the reference peak (S), calculate the relative retention time and relative peak area RSD of each common peak. The results show that the RSD of the relative retention time of each common peak is 0.03% - 0.48%, and the RSD of the relative peak area is 0.12% - 4.37%, indicating good instrument precision.

[0046] 2.4.2 Repeatability Test

[0047] Take 6 portions of Sample No. S24, prepare the test solution in parallel according to the method under "2.3", inject and determine under the chromatographic conditions under "2.1", and record the chromatogram. Using the astilbin peak at peak No. 13 as the reference peak (S), calculate the relative retention time and relative peak area RSD of each common peak. The results show that the RSD of the relative retention time of each common peak is 0.02% - 0.32%, and the RSD of the relative peak area is 0.22% - 4.95%, indicating that this method has good repeatability.

[0048] 2.4.3 Stability Test

[0049] Take an appropriate amount of Sample No. S24, prepare the test solution according to the method under "2.3", inject and determine under the chromatographic conditions under "2.1" at 0, 2, 4, 6, 8, 12, and 24 h respectively, and record the chromatogram. Using the astilbin peak at peak No. 13 as the reference peak (S), calculate the relative retention time and relative peak area RSD of each common peak. The results show that the RSD of the relative retention time of each common peak is 0.31% - 3.63%, and the RSD of the relative peak area is 0.18% - 4.53%, indicating that the test solution is stable within 24 h.

[0050] 2.5 Establishment and Similarity Evaluation of HPLC Fingerprint of Smilax china

[0051] 2.5.1 Similarity Evaluation

[0052] Take 29 batches of Smilax china samples, accurately weigh them, prepare the test solution according to the method under "2.3", then inject and determine under the chromatographic conditions under "2.1", and record the chromatogram. Import the data into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (2012 Edition) for analysis. Using the chromatogram of Sample No. S1 as the reference chromatogram, with a time window width of 0.1, select the average method, and after multi-point calibration, use Mark peak matching to generate the HPLC superimposed fingerprint of 29 batches of Smilax china samples, and a total of 23 common peaks are calibrated ( Figure 1 ). By comparing the retention times of the test samples and reference substances, a total of 12 common peaks are identified. Among them, peak No. 6 is chlorogenic acid, peak No. 7 is epicatechin, peak No. 9 is 5-O-caffeoylshikimic acid, peak No. 10 is piceid, peak No. 11 is oxyresveratrol, peak No. 12 is neoastilbin, peak No. 13 is astilbin, peak No. 14 is neoisoastilbin, peak No. 15 is resveratrol, peak No. 16 is engelitin, peak No. 17 is quercitrin, and peak No. 20 is kaempferol ( Figure 3 ). Using the reference fingerprint ( Figure 1 R, Figure 2)Taking it as a reference, the similarity between 29 batches of Smilax china and the control fingerprint was calculated using software. The results showed that the similarity range between the 29 batches of samples and the control fingerprint was 0.784 - 0.996, as shown in Table 2, indicating that there were certain differences in the quality of Smilax china from different origins. Samples with a similarity above 0.80 to the control fingerprint were qualified products.

[0053] Table 2 Similarity of HPLC fingerprint of 29 batches of Smilax china

[0054]

[0055] 2.5.2 Hierarchical cluster analysis (HCA)

[0056] Taking the peak areas of the common peaks of 29 batches of Smilax china as variables, after Z-standardization, they were imported into SPSS 27.0 software. The between-group linkage system clustering method was used, and clustering analysis was carried out with the squared Euclidean distance as the interval. The results are shown in Figure 4 . When the squared Euclidean distance was 10, the 29 batches of Smilax china samples were clustered into 3 categories: S24 was in category I, S9 - S13, S19 - S23 were clustered into category II, and the remaining samples were clustered into category III.

[0057] 2.5.3 Principal component analysis (PCA)

[0058] Using SIMCA-P 14.1 software, unsupervised PCA was carried out with the peak areas of 23 common peaks of 29 batches of Smilax china samples as the X variable and the sample batches as the Y variable. The eigenvalues and variance contribution rates of the correlation coefficients were calculated. The eigenvalues of the first 5 principal components (PC1 - PC5) were all >1, indicating that the first 5 principal components played a dominant role in the PCA analysis. The cumulative variance contribution rate was 83.20%, which could represent most of the chemical information in the Smilax china medicinal materials. The results are shown in Table 3. Graphing and analysis were carried out with the information represented by the first 5 principal components obtained from PCA. As Figure 5 can be seen, there was a certain separation trend for Smilax china from different origins, indicating that there were certain differences in the quality of Smilax china from different sources. The spatial distribution of the 29 batches of Smilax china was roughly divided into 3 groups. Among them, S9 - S13, S19 - S23 were relatively close in distribution, S24 was far from other samples, and the remaining batches were relatively close in distribution, which was basically consistent with the HCA results.

[0059] Table 3 Eigenvalues and variance contribution rates of principal components

[0060]

[0061] 2.6 Study on the in vitro antioxidant activity of Smilax china

[0062] 2.6.1 Determination of DPPH Free Radical Scavenging Ability

[0063] Preparation of sample solution: Prepare the sample solution according to the method under "2.3", and successively dilute it with 70% methanol into sample solutions with different mass concentrations for testing. Take an appropriate amount of VC reference substance, accurately weigh it, dissolve it with 70% methanol, and dilute it into solutions with concentrations of 1, 2, 5, 10, 15, and 20 μg·mL -1 respectively as positive controls.

[0064] Preparation of DPPH solution: Take an appropriate amount of DPPH, dissolve it with absolute ethanol and prepare a DPPH solution with a mass concentration of 0.08 mg·mL -1 .

[0065] Take 100 μL of sample solutions and reference substance solutions with different mass concentrations, place them in a 96-well plate, add 150 μL of DPPH solution to each well, mix well, incubate in a water bath at 37 °C in the dark for 30 min, and measure the absorbance A1 at 517 nm using a microplate reader; for the blank group, use 70% methanol instead of the sample solution and measure its absorbance A0; for the control group, use 70% methanol instead of the DPPH solution and measure its absorbance A2. Set 3 replicates for each group and take the average value. Calculate the scavenging rate according to the following formula: DPPH free radical scavenging rate = [1 - (A1 - A2) / A0] × 100%. Use GraphPad Prism 8.3.0 software with the sample concentration (mg·mL -1 ) as the abscissa and the scavenging rate as the ordinate to perform logarithmic curve fitting to obtain the fitting formula, and calculate its half maximal inhibitory concentration (IC 50 ), as shown in Table 4. The magnitude of the IC 50 value is inversely proportional to the antioxidant ability, and the larger the value, the weaker its antioxidant ability. The results show that Smilax china from different sources all have a certain DPPH free radical scavenging effect, and the scavenging ability of free radicals increases with the increase of concentration. The IC 50 for DPPH free radicals is 0.1003 - 0.2539 mg·mL -1 . Among them, S26 (Hubei), S28 (Jiangxi), and S20 (Anqing, Anhui) have stronger abilities to scavenge DPPH free radicals.

[0066] Table 4 IC 50 of the antioxidant activity of Smilax china (mg·mL -1 , n = 3)

[0067]

[0068]

[0069] 2.6.2 Determination of ABTS free radical scavenging ability

[0070] Preparation of sample solution: Prepare sample solution according to the method under "2.3", and dilute it with 70% methanol to different mass concentrations of sample solution. Take an appropriate amount of VC reference substance, accurately weigh it, dissolve it with 70% methanol, and dilute it to concentrations of 0.05, 0.1, 0.15, 0.20, 0.22, and 0.25 mg·mL -1 solution as a positive control.

[0071] According to the instructions of the ABTS kit, the sample solution was added to the 96-well plate in sequence, and then the ABTS working solution was added. The reaction was carried out at room temperature in the dark for 6 minutes, and the absorbance A1 was measured at 405nm; the blank group used 70% methanol instead of the sample solution, and its absorbance A0 was measured; the control group used 70% methanol instead of the ABTS free radical working solution, and its absorbance A2 was measured. Three replicate wells were set for each group, and the average value was taken. The clearance rate was calculated according to the following formula: ABTS free radical clearance rate = [1-(A1-A2) / A0] × 100%. IC 50 The calculation method of the value is the same as that of "2.6.1", and the results are shown in Table 4. The scavenging effect of 29 batches of Smilax china samples on ABTS free radicals increased with the increase of concentration. 50 0.3338~0.9441mg·mL -1 Among them, S7 (Jiangsu), S4 (Henan), and S1 (Jiangxi) have stronger abilities to scavenge ABTS free radicals.

[0072] 2.7 Spectrum-effect relationship analysis

[0073] 2.7.1 Grey relational analysis (GRA)

[0074] In order to further explore the relationship between the common peaks of the fingerprint spectrum of Smilax china and its antioxidant activity, this study used GRA to analyze its spectrum-activity relationship. 50 The reciprocal of the value was used as the parent sequence, and the peak area of each common peak was used as the subsequence to form a data matrix. The data matrix was dimensionlessly quantized using the initial value method, and the resolution coefficient was taken as 0.5. GRA analysis was performed to calculate the correlation between the common peaks of Smilax china and the antioxidant activity (see Table 5). The greater the correlation, the greater the influence of the comparison sequence on the reference sequence. The results showed that the correlation between the 23 common peaks and the antioxidant activity results was greater than 0.7, indicating that the antioxidant effect of Smilax china was affected by the combined action of multiple components.

[0075] Table 5 Correlation between common peaks of Smilax china and antioxidant activity

[0076]

[0077]

[0078] 2.7.2 Partial Least Squares Regression (PLSR) Analysis

[0079] In order to further clarify the spectral-effect relationship between the components of Smilax china L. and its antioxidant activity, the peak areas of 23 common peaks in the fingerprint spectra of 29 batches of Smilax china L. and the antioxidant activity data were imported into SIMCA 14.1 software after standardization, and the standardized regression coefficients and variable importance in projection (VIP) values were calculated. A positive regression coefficient indicates a positive correlation between the peak area of the common peak and the antioxidant activity, and vice versa for a negative correlation. The regression coefficients are shown in Figure 6 . The VIP value represents the contribution degree of the variable. When VIP > 1, it can be considered that the variable has a significant impact on the model. The results are shown in Figure 7 . The chromatographic peaks 6, 11 - 13, and 15 have VIP > 1 and are positively correlated with the DPPH radical scavenging ability; the chromatographic peaks 6, 10, 12, 15, and 18 have VIP > 1 and are positively correlated with the ABTS radical scavenging ability. This indicates that the compounds represented by these characteristic peaks play an important role in the antioxidant effect of Smilax china L.

[0080] Based on the comprehensive results of GRA and PLSR analyses, the characteristic peaks that may account for the differences in antioxidant effects among different batches of Smilax china L. are likely to be peak 6 (chlorogenic acid), peak 10 (piceid), peak 11 (oxyresveratrol), peak 12 (neofilipenduloside), peak 13 (filipenduloside), peak 15 (resveratrol), and peak 18.

Claims

1. The HPLC fingerprint of Smilax china L. medicinal materials is characterized in that When the HPLC chromatographic conditions are as follows, the HPLC fingerprint of the Smilax china L. medicinal material is basically consistent with Figure 2; The HPLC chromatographic conditions are as follows: The chromatographic column is an Agilent 5TC-C 18 chromatographic column, specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: methanol as phase A, 0.05% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0 - 20 min, 25% - 35% A; 20 - 30 min, 35% A; 30 - 40 min, 35% - 38% A; 40 - 45 min, 38% A; 45 - 55 min, 38% - 45% A; 55 - 70 min, 45% - 63% A; 70 - 75 min, 63% - 70% A; 75 - 80 min, 70% A; Column temperature: 30 °C, injection volume: 10 μL, flow rate: 0.8 mL·min -1 , detection wavelength: 295 nm 2. The HPLC fingerprint spectrum according to claim 1, characterized in that The HPLC fingerprint of the Smilax china L. medicinal material has 23 characteristic fingerprint chromatographic peaks, among which peak 6 is chlorogenic acid, peak 7 is epicatechin, peak 9 is 5-O-caffeoylshikimic acid, peak 10 is piceid, peak 11 is oxyresveratrol, peak 12 is neoponidic acid, peak 13 is ponidic acid, peak 14 is neoheteroponidic acid, peak 15 is resveratrol, peak 16 is engeletin, peak 17 is quercitrin, and peak 20 is kaempferol.

3. The method for constructing the HPLC fingerprint spectrum according to any one of claims 1-2, characterized in that When the HPLC chromatographic conditions are as follows, the HPLC fingerprint of the Smilax china L. medicinal material has 23 characteristic fingerprint chromatographic peaks, among which peak 6 is chlorogenic acid, peak 7 is epicatechin, peak 9 is 5-O-caffeoylshikimic acid, peak 10 is piceid, peak 11 is oxyresveratrol, peak 12 is neoponidic acid, peak 13 is ponidic acid, peak 14 is neoheteroponidic acid, peak 15 is resveratrol, peak 16 is engeletin, peak 17 is quercitrin, and peak 20 is kaempferol.

4. Application of the HPLC fingerprint according to any one of claims 1-3 or the method for constructing the same in quality control and component analysis of Smilax china L. medicinal materials.

5. Use of the HPLC fingerprint spectrum according to any one of claims 1-3 or a method for constructing the same in quality control of Smilax china L. medicinal materials, characterized in that The application includes the following steps: (1) Crush the Smilax china L. medicinal material, add a methanol solution, perform ultrasonic treatment, and filter through a microporous filter membrane to obtain a test solution; (2) Take the test solution obtained in step (1), perform HPLC detection to obtain the HPLC chromatogram of the test sample, and the chromatographic conditions are as follows: The chromatographic column was Agilent 5TC-C 18 chromatographic column, specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: methanol as phase A, 0.05% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0 - 20 min, 25% - 35% A; 20 - 30 min, 35% A; 30 - 40 min, 35% - 38% A; 40 - 45 min, 38% A; 45 - 55 min, 38% - 45% A; 55 - 70 min, 45% - 63% A; 70 - 75 min, 63% - 70% A; 75 - 80 min, 70% A; column temperature 30°C, injection volume 10 μL, flow rate 0.8 mL·min -1 , and the detection wavelength was 295 nm; (3) Compare the HPLC chromatogram of the test sample obtained in step (2) with the HPLC fingerprint of the Smilax china L. medicinal material described in the present invention. The Smilax china L. medicinal material with a similarity above 0.80 is a qualified product.

6. The application according to claim 5, characterized in that In step (1), after crushing, it is sieved through a 40-mesh sieve. The methanol solution is a methanol solution with a volume fraction of 60%-70%, the ultrasonic time is 20-40 min, and the microporous filter membrane is selected from a 0.22-μm filter membrane or a 0.45-μm filter membrane.

7. A method for exploring the relationship between the common peaks of the fingerprint of Smilax china L. medicinal materials and its antioxidant activity, characterized in that Optionally, it includes the following steps (1) and / or (2): (1)Grey relational analysis (GRA): The reciprocal of the antioxidant IC 50 value was taken as the mother sequence, and the peak areas of each common peak were used as the sub-sequences to form a data matrix. The initial value method was used to perform dimensionless quantization processing on the data matrix, with the resolution coefficient taken as 0.

5. GRA analysis was carried out to calculate the correlation degree between the common peaks of Smilax china L. herbs and antioxidant activity. The greater the correlation degree, the greater the influence of the comparison sequence on the reference sequence. The results showed that the correlation degrees between the 23 common peaks and the antioxidant activity results were all greater than 0.7, indicating that the antioxidant effect of Smilax china L. herbs was affected by the combined action of multiple components; (2) Partial least squares regression (PLSR) analysis: After standardizing the peak areas of 23 common peaks and the antioxidant activity data in the fingerprint chromatograms of 29 batches of Smilax china L. medicinal materials, import them into SIMCA 14.1 software, calculate the standardized regression coefficients and variable importance in projection VIP values. A positive regression coefficient indicates a positive correlation between the peak area of the common peak and the antioxidant activity, and vice versa. The VIP value represents the contribution degree of the variable. When VIP>1, it indicates that the variable has a significant impact on the model; the chromatographic peaks 6, 11-13, 15 are those with VIP>1 and a positive correlation with the DPPH free radical scavenging ability; the chromatographic peaks 6, 10, 12, 15, 18 are those with VIP>1 and a positive correlation with the ABTS free radical scavenging ability; it shows that the compounds represented by these characteristic peaks play an important role in the antioxidant effect of Smilax china L.

8. The method according to claim 7, wherein Based on the comprehensive analysis results of GRA and PLSR, the characteristic peaks showing differences in antioxidant effects among different batches of Smilax china L. medicinal materials were identified as Peak 6: chlorogenic acid, Peak 10: polydatin, Peak 11: resveratrol oxide, Peak 12: neoponidic acid, Peak 13: ponidic acid, Peak 15: resveratrol, and Peak 18.

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