A method for constructing HPLC fingerprint of Pandanus oleraceus and a method for quality detection of Pandanus oleraceus based on spectral efficiency relationship.

Abstract

This invention discloses a method for constructing an HPLC fingerprint of *Pandanus tiglium* and a method for quality detection of *Pandanus tiglium* based on spectral-efficacy relationships, belonging to the technical field of quality evaluation and detection of traditional Chinese medicine. This invention utilizes an online liquid microextraction-high performance liquid chromatography (HPLC-HPLC) system to construct the fingerprint of *Pandanus tiglium*. This method eliminates the need for sample extraction and preparation steps, thereby determining whether the material basis of the sample has changed, evaluating the stability and quality uniformity of different *Pandanus tiglium* samples, and enabling automated detection through instrument settings. It is simple, safe, and economical to operate, making it suitable for the quality detection of *Pandanus tiglium*. Furthermore, the fingerprint established by this invention can be correlated with antioxidant activity data through spectral-efficacy analysis, thereby screening out key active ingredients such as chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid B, achieving a correlation evaluation from "chemical composition" to "pharmacological function," providing more comprehensive technical support for the quality grading and product development of *Pandanus tiglium*.

Description

Technical Field

[0001] This invention relates to the technical field of quality evaluation and testing of traditional Chinese medicine, specifically to a method for constructing an HPLC fingerprint of Panax notoginseng and a method for quality detection of Panax notoginseng based on the spectrum-effect relationship. Background Technology

[0002] Pandanus tectorii radix et rhizoma is the dried root and rhizome of Pandanus tectorius Sol., a plant in the Pandanaceae family, and related species. It is a perennial evergreen shrub or small tree, widely distributed in tropical and subtropical coastal areas of my country, and also found growing naturally in Southeast Asian countries. It is a characteristic medicinal and edible plant in southern my country. Its roots, leaves, fruits, and flowers are rich in flavonoids, phenols, terpenes, and other bioactive components. Modern pharmacology has confirmed that its extracts possess multiple effects, including antibacterial, anti-inflammatory, antioxidant, and hypoglycemic properties, and show broad application prospects in traditional Chinese medicine clinical practice and functional food development.

[0003] With the expanding market demand for Panax notoginseng-related products, stable control of raw material quality has become a core requirement for ensuring product efficacy, safety, and market competitiveness. Establishing a scientific and precise quality evaluation technology system is a key support for achieving this goal and is crucial for promoting its industrialization.

[0004] The quality of Pandanus is directly related to the types and contents of its active ingredients. The synthesis and accumulation of these active ingredients are easily affected by various factors, resulting in significant fluctuations in the content of core active ingredients (such as total polyphenols and flavonoids) in Pandanus raw materials from different sources. These ingredients are the material basis for its core pharmacological effects, such as antioxidant activity. Fluctuations in these ingredients directly lead to difficulties in ensuring the efficacy stability of downstream products. Therefore, accurately characterizing the components of Pandanus and establishing scientific quality evaluation methods are of great significance for standardizing the Pandanus raw material market and promoting the rational and efficient utilization of its resources.

[0005] Fingerprint spectroscopy can comprehensively reflect the types and relative contents of chemical components in natural products, and is a core technology for the quality evaluation of traditional Chinese medicine and natural products. Relevant quality control standards are continuously being added to each edition of the Chinese Pharmacopoeia. Currently, high-performance liquid chromatography (HPLC) technology, due to its advantages such as high separation efficiency and accurate detection, has been widely used in the establishment of fingerprint spectra for natural products.

[0006] However, the complex matrix of Panax notoginseng, the large fluctuations in the content of its active ingredients, and the presence of numerous impurities make direct HPLC analysis susceptible to interference from these impurities. This results in problems such as overlapping chromatographic peaks, poor separation, and insufficient sensitivity, making it difficult to obtain clear and stable fingerprint spectra and accurately characterize its components, thus affecting the accuracy of quality evaluation. Furthermore, traditional fingerprinting techniques suffer from qualitative deficiencies and quantitative blind spots, only able to identify some known components and lacking a systematic correlation with pharmacodynamic activity. This makes it difficult to explain quality differences from an efficacy perspective, which does not align with the trend of combining spectrum and efficacy evaluation in traditional Chinese medicine.

[0007] Online liquid chromatography-microextraction (LC-MS / MS) technology boasts advantages such as high enrichment efficiency and strong interference removal capabilities, demonstrating promising results in the detection of plants like Astragalus membranaceus and tea. For instance, Chinese invention patent CN120468324A discloses a method and application for establishing fingerprint spectra of Astragalus membranaceus cell-wall broken slices based on LC-MS / MS. However, it has not yet been applied to the establishment of fingerprint spectra for Pandanus oleraceus. Existing technologies still cannot overcome bottlenecks such as matrix interference and difficulty in characterizing component differences in Pandanus oleraceus, hindering accurate identification and quality evaluation. Furthermore, Pandanus oleraceus and Astragalus membranaceus exhibit significant differences in medicinal properties, components, and matrix, making it impossible to directly apply the LC-MS / MS detection conditions for Astragalus membranaceus; specific optimization is required. In addition, antioxidant activity is a core pharmacological effect of Pandanus oleraceus, but a correlation analysis system between its fingerprint spectra and antioxidant activity has not yet been established, making it impossible to elucidate efficacy differences through fingerprint spectra.

[0008] Therefore, developing a method for establishing the fingerprint spectrum of Panax notoginseng based on online liquid-phase microextraction technology, obtaining clear and characteristic fingerprint spectra, and establishing a spectrum-effect correlation between fingerprint spectra and antioxidant activity, as well as screening marker components, will enable precise characterization of the components of Panax notoginseng raw materials. This will not only standardize the raw material market and ensure product quality, but also fill the technological gap in this field, promote the innovative development of natural product quality evaluation technology towards spectrum-effect combination, and provide technical support for the industrialization development of Panax notoginseng. Summary of the Invention

[0009] To overcome the aforementioned defects and shortcomings in the existing technology, this invention provides a method for constructing an HPLC fingerprint of Panax notoginseng and a method for quality detection of Panax notoginseng based on the spectral efficiency relationship.

[0010] The first objective of this invention is to provide a method for constructing an HPLC fingerprint of Panax notoginseng.

[0011] A second objective of this invention is to provide a method for quality testing of Pandanus arvense.

[0012] The third objective of this invention is to provide a method for screening quality markers of Panax notoginseng based on the relationship between antioxidant spectrum efficacy and antioxidant activity.

[0013] This invention claims protection for the following: A method for constructing an HPLC fingerprint of Panax notoginseng is provided, which is obtained by using an online liquid microextraction-high performance liquid chromatography (HPLC-HPLC) system. The HPLC system includes a mobile phase, a pump, an online liquid microextraction device, a liquid chromatography column, and a UV detector connected in sequence by tubing. The construction method is as follows: the mobile phase is pumped into the online liquid-phase microextraction device to perform liquid-phase microextraction on Panax notoginseng, and the solution after liquid-phase microextraction is introduced into the liquid chromatography column and UV detector for separation and detection. The chromatogram is recorded to obtain the fingerprint spectrum. When detecting Pandanus, the online liquid-phase microextraction device includes a protective column filled with Pandanus and diatomaceous earth, and the core of the protective column is sealed on both sides with a microporous filter membrane. The mobile phase includes mobile phase A and mobile phase B; The mobile phase A is obtained by mixing formic acid aqueous solution and acetic acid in a volume ratio of (0.8-1.2):(0.8-1.2), wherein the volume fraction of formic acid aqueous solution is 0.3-0.7%; The mobile phase B is acetonitrile; The pump has a pre-stored elution gradient program, in which the volume percentage change of mobile phase A is: 0 min, mobile phase A is 95%; After 10 minutes, mobile phase A reached 90%. After 20 minutes, mobile phase A was 90%. After 30 minutes, the mobile phase A was 88.5%; After 40 minutes, the mobile phase A was 79%. At 65 min, mobile phase A was 77%; At 66 min, mobile phase A was 95%; 70 min, mobile phase A is 95%; The UV detector has a detection wavelength of 326 nm.

[0014] Preferably, during the detection of Pandanus, the online liquid-phase microextraction device includes an empty protective column core, Pandanus, diatomaceous earth, a microporous filter membrane, and a protective column sleeve. The Pandanus and diatomaceous earth are filled in the empty protective column core, and the two sides of the filled protective column core are sealed with the microporous filter membrane. The sealed protective column core is then sealed in the protective column sleeve.

[0015] Preferably, the amount of Pandanus is 2.0 to 8.0 mg.

[0016] More preferably, the amount of pandanus is 6.0 mg.

[0017] Preferably, the mobile phase A is obtained by mixing formic acid aqueous solution and acetic acid in a volume ratio of 1:1, wherein the volume fraction of formic acid aqueous solution is 0.5%.

[0018] Preferably, the method further includes the detection of the reference solution: the mobile phase is pumped into the online liquid microextraction device, and then the reference solution is injected into the liquid chromatography column. The solution after liquid microextraction and the reference solution are separated and detected in the liquid chromatography column and UV detector, and the chromatogram of the reference solution is recorded. Compare the chromatograms of the test sample and the reference sample to identify the characteristic chromatographic peaks in the chromatogram of the test sample. When detecting the reference solution, the online liquid phase microextraction device includes a protective column filled with diatomaceous earth, and the core of the protective column is sealed with microporous filter membranes on both sides.

[0019] Preferably, when detecting the reference solution, the online liquid phase microextraction device includes an empty guard column core, diatomaceous earth, a microporous filter membrane, and a guard column sleeve. The diatomaceous earth is filled in the empty guard column core, and the two sides of the filled guard column core are sealed with the microporous filter membrane. The sealed guard column core is then sealed in the guard column sleeve.

[0020] Preferably, the injection volume of the reference solution is 8–12 μL.

[0021] More preferably, the injection volume of the reference solution is 10 μL.

[0022] Preferably, the reference solution includes neochlorogenic acid solution, chlorogenic acid solution, cryptochlorogenic acid solution, isochlorogenic acid B solution, isochlorogenic acid A solution and isochlorogenic acid C solution.

[0023] More preferably, the reference solution is a methanol solution mixture of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C.

[0024] The reference solution is used for the qualitative localization and peak identification of characteristic chromatographic peaks in fingerprint chromatograms, and also provides a reference standard for method validation and the accuracy, repeatability and comparability of the chromatograms.

[0025] Preferably, the method for preparing the neochlorogenic acid solution is as follows: neochlorogenic acid and methanol are thoroughly mixed, wherein the mass-to-volume ratio of neochlorogenic acid to methanol is (0.04-0.045) mg: 1 mL; The chlorogenic acid solution is prepared by mixing chlorogenic acid and methanol thoroughly, wherein the mass-to-volume ratio of chlorogenic acid to methanol is (0.04-0.045) mg: 1 mL; The method for preparing the cryptochlorogenic acid solution is as follows: cryptochlorogenic acid and methanol are thoroughly mixed, and the mass-volume ratio of cryptochlorogenic acid to methanol is (0.035~0.04) mg: 1 mL; The isochlorogenic acid B solution is prepared by mixing isochlorogenic acid B with methanol thoroughly, wherein the mass-to-volume ratio of isochlorogenic acid B to methanol is (0.035-0.04) mg: 1 mL; The isochlorogenic acid A solution is prepared by mixing isochlorogenic acid A with methanol thoroughly, wherein the mass-to-volume ratio of isochlorogenic acid A to methanol is (0.035-0.04) mg: 1 mL; The isochlorogenic acid C solution is prepared by mixing isochlorogenic acid C with methanol thoroughly, wherein the mass-to-volume ratio of isochlorogenic acid C to methanol is (0.035-0.04) mg: 1 mL.

[0026] More preferably, the method for preparing the neochlorogenic acid solution is as follows: neochlorogenic acid and methanol are thoroughly mixed, wherein the mass-to-volume ratio of neochlorogenic acid to methanol is 0.04365 mg: 1 mL; The chlorogenic acid solution is prepared by mixing chlorogenic acid and methanol thoroughly, wherein the mass-to-volume ratio of chlorogenic acid to methanol is 0.04216 mg: 1 mL. The method for preparing the cryptochlorogenic acid solution is as follows: cryptochlorogenic acid and methanol are thoroughly mixed, and the mass-volume ratio of cryptochlorogenic acid to methanol is 0.03713 mg: 1 mL; The isochlorogenic acid B solution is prepared by mixing isochlorogenic acid B with methanol thoroughly, wherein the mass-to-volume ratio of isochlorogenic acid B to methanol is 0.03818 mg: 1 mL. The isochlorogenic acid A solution is prepared by mixing isochlorogenic acid A with methanol thoroughly, wherein the mass-to-volume ratio of isochlorogenic acid A to methanol is 0.03869 mg: 1 mL. The isochlorogenic acid C solution is prepared by mixing isochlorogenic acid C with methanol thoroughly, wherein the mass-to-volume ratio of isochlorogenic acid C to methanol is 0.03322 mg: 1 mL.

[0027] Preferably, the flow rate of the mobile phase is 0.6 to 1.2 mL / min.

[0028] More preferably, the flow rate of the mobile phase is 0.8 mL / min.

[0029] Preferably, the diameter of the pipes on both sides of the online liquid phase microextraction device is 0.5 to 1.00 mm.

[0030] More preferably, the diameter of the pipes on both sides of the online liquid phase microextraction device is 1.00 mm.

[0031] Preferably, the liquid chromatography column is a C 18 reverse-phase chromatography column; More preferably, the liquid chromatography column is a Capcell Pak MG-C 18 chromatography column.

[0032] Preferably, the column temperature of the liquid chromatography column is 23 to 27 °C.

[0033] More preferably, the column temperature of the liquid chromatography column is 25 °C.

[0034] A method for quality detection of Pandanus tectorius: Using the above construction method to detect several batches of Pandanus tectorius samples, record the chromatogram, and generate a reference fingerprint of Pandanus tectorius after data processing of the chromatogram; Using the above construction method to detect the待测 Pandanus tectorius sample, record the chromatogram, and obtain the chromatogram of the待测 Pandanus tectorius sample; Perform similarity analysis on the chromatogram of the待测 Pandanus tectorius sample and the reference fingerprint of Pandanus tectorius.

[0035] The several batches are not less than 15 batches.

[0036] Preferably, the method of data processing is: Import the chromatogram into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints, screen and confirm the common peaks, and generate the reference fingerprint of Pandanus tectorius by the average method after S-type multi-point calibration and full-spectrum automatic peak matching.

[0037] Preferably, the method further includes the detection of the reference solution: Pump the mobile phase into the on-line liquid phase microextraction device, then inject the reference solution into the liquid chromatography column, and the solution after liquid phase microextraction and the reference solution enter the liquid chromatography column and the UV detector for separation and detection, and record the reference chromatogram; Compare the chromatogram of the待测 Pandanus tectorius sample with the reference chromatogram to confirm the characteristic chromatographic peaks in the chromatogram of the待测 Pandanus tectorius sample.

[0038] The reference fingerprint of Pandanus tectorius contains 6 characteristic chromatographic peaks: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C. The specified values of the retention times of each characteristic chromatographic peak are: neochlorogenic acid 17.321 min, chlorogenic acid 28.041 min, cryptochlorogenic acid 29.011 min, isochlorogenic acid B 52.478 min, isochlorogenic acid A 55.090 min, isochlorogenic acid C 59.087 min; Import the chromatogram of the待测 Pandanus tectorius sample into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints, and perform similarity analysis with the reference fingerprint of Pandanus tectorius. It can be judged as qualified only when the following two conditions are met: The retention time of the characteristic chromatographic peak in the chromatogram of the Pandanus sample to be tested shall be within ±10% of the specified value of the retention time of the corresponding characteristic chromatographic peak in the Pandanus reference fingerprint chromatogram; The similarity between the chromatogram of the test Panax notoginseng sample and the fingerprint chromatogram of the Panax notoginseng control is not less than 0.9.

[0039] A method for screening quality markers of Panax notoginseng based on its antioxidant spectrum relationship includes the following steps: S1. Construct a fingerprint spectrum of Pandanus using the above construction method. The fingerprint spectrum of Pandanus contains 6 characteristic chromatographic peaks: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C. S2. Determine the antioxidant activity of Panax notoginseng; S3. Perform grey relational analysis or partial least squares regression analysis on the peak areas of the characteristic chromatographic peaks in the fingerprint spectrum of Pandanus obtained in step S1 and the antioxidant activity obtained in step S2, and calculate the correlation between each characteristic chromatographic peak and the antioxidant activity to determine the quality markers of Pandanus.

[0040] Preferably, in step S1, the retention time of each characteristic chromatographic peak in the Pandanus fingerprint spectrum is within ±10% of a specified value; The specified retention times for each characteristic chromatographic peak are as follows: neochlorogenic acid 17.321 min, chlorogenic acid 28.041 min, cryptochlorogenic acid 29.011 min, isochlorogenic acid B 52.478 min, isochlorogenic acid A 55.090 min, and isochlorogenic acid C 59.087 min.

[0041] Preferably, in step S2, the indicators of antioxidant activity include DPPH free radical scavenging rate, ABTS free radical scavenging rate and / or comprehensive antioxidant capacity composite index.

[0042] Preferably, in step S3, the quality markers of Panax notoginseng are chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A.

[0043] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a method for constructing an HPLC fingerprint of Pandanus oleraceus and a method for quality detection of Pandanus oleraceus based on spectral efficacy correlation. This invention utilizes an online liquid microextraction-high performance liquid chromatography (HPLC-HPLC) system to construct the fingerprint of Pandanus oleraceus. This method eliminates the need for sample extraction and preparation steps, thereby determining whether the material basis of the sample has changed, evaluating the stability and quality uniformity of different Pandanus oleraceus samples, and enabling automated detection through instrument settings. It is simple, safe, and economical to operate, making it suitable for quality detection of Pandanus oleraceus. Furthermore, the fingerprint established by this invention can be correlated with antioxidant activity data through spectral efficacy correlation analysis, thereby screening out key active ingredients such as chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid B, achieving a correlation evaluation from "chemical composition" to "pharmacological function," providing more comprehensive technical support for the quality grading and product development of Pandanus oleraceus. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of an online liquid microextraction (OLMM-HPLC) analysis system.

[0045] Figure 2 The images show the HPLC chromatograms and control fingerprint chromatograms of 21 Pandanus samples from different origins in Example 1.

[0046] Figure 3 The HPLC chromatogram of the mixed reference standard in Example 1 is shown below; Peak 1: Neochlorogenic acid; Peak 2: Chlorogenic acid; Peak 3: Cryptochlorogenic acid; Peak 4: Isochlorogenic acid B; Peak 5: Isochlorogenic acid A; Peak 6: Isochlorogenic acid C.

[0047] Figure 4 The HPLC chromatograms of Panax notoginseng samples using different mobile phase flow rates are shown in Example 2.

[0048] Figure 5 The HPLC chromatograms of Pandanus samples with different loading amounts are shown in Example 3.

[0049] Figure 6 The HPLC chromatograms of Panax notoginseng samples using PEK tubes of different diameters in Example 4 are shown.

[0050] Figure 7 This is the HPLC chromatogram result of the online liquid phase microextraction repeatability experiment in Example 5.

[0051] Figure 8 The graph shows the partial regression coefficients between the characteristic chromatographic peaks of Panax notoginseng HPLC and the antioxidant indicators; Peak 1: Neochlorogenic acid; Peak 2: Chlorogenic acid; Peak 3: Cryptochlorogenic acid; Peak 4: Isochlorogenic acid B; Peak 5: Isochlorogenic acid A; Peak 6: Isochlorogenic acid C.

[0052] Figure 9The VIP values ​​of characteristic chromatographic peaks and antioxidant indicators in the HPLC chromatogram of Panax notoginseng are shown in the figure. Peak 1: Neochlorogenic acid; Peak 2: Chlorogenic acid; Peak 3: Cryptochlorogenic acid; Peak 4: Isochlorogenic acid B; Peak 5: Isochlorogenic acid A; Peak 6: Isochlorogenic acid C. Detailed Implementation

[0053] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0054] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0055] In this embodiment of the invention, 21 batches of Pandanus truncata medicinal materials were collected from 21 different producing areas across the country. They were identified as Pandanus truncata from the Pandanaceae family and the Pandanus genus. The materials were crushed and passed through a No. 3 sieve. The source of the medicinal materials is shown in the table below.

[0056]

[0057] New chlorogenic acid standard reference was purchased from the National Institutes for Food and Drug Control, batch number 112110-202401; The chlorogenic acid standard reference was purchased from the National Institutes for Food and Drug Control, batch number 110753-202119; Cryptochlorogenic acid standard reference was purchased from the National Institutes for Food and Drug Control, batch number 112111-202401; Isochlorogenic acid A standard reference was purchased from the National Institutes for Food and Drug Control, batch number 111782-202208; Isochlorogenic acid B standard reference was purchased from the National Institutes for Food and Drug Control, batch number 112107-202301; Isochlorogenic acid C standard reference was purchased from the National Institutes for Food and Drug Control, batch number 111894-202205.

[0058] Example 1: A Method for Detecting and Analyzing the Fingerprint Spectrum of Pandanus Based on Online Liquid-Phase Microextraction Technology 1. Preparation of the online liquid-phase microextraction device for the test sample Remove the guard column (Phenomenex KJ0-4282) core and disassemble it. Remove the original packing material from the core, wash and dry it. Accurately weigh 6.0 mg of Pandanus (S1-S21) powder from different origins and place them in the cores respectively. Fill the gaps with diatomaceous earth until it is flush with the edge of the core. Seal both sides of the core with a 0.22 μm organic microporous membrane and trim off any excess filter paper. Place the core with the sample packing into the column sleeve, tighten it, and connect it to the front end of the HPLC column. The mobile phase should flow in from the flat end of the guard column and out from the tip.

[0059] 2. Online liquid microextraction (OLMM-HPLC) analysis A schematic diagram of the OLMM-HPLC analysis system is shown below. Figure 1 As shown.

[0060] The mobile phase was pumped into the pipeline at a flow rate of 0.8 mL / min and introduced into the online liquid-phase microextraction device for the test sample constructed in step 1 through a 1.0 mm diameter PEE tube. The sample was extracted online. The extracted solution flowed out through a 1.0 mm diameter PEE tube on the other side of the online liquid-phase microextraction device and was separated by a high-performance liquid chromatography column. The separated substances flowed out through a UV detector and were then introduced into the waste liquid. The HPLC chromatogram was obtained by the UV detector.

[0061] Capcell Pak MG-C reversed-phase column was used. 18 The mobile phase A was a mixture of 0.5% formic acid aqueous solution (v / v) and acetic acid in a volume ratio of 1:1, and the mobile phase B was acetonitrile. The HPLC chromatograms of 21 Pandanus samples from different origins were obtained by using the mobile phase gradient elution program shown in Table 1. The mobile phase flow rate was 0.8 mL / min, the UV detector wavelength was 326 nm, the injection volume was 10 μL, and the column temperature was 25℃.

[0062] The HPLC chromatograms of 21 Pandanus samples from different origins were imported into the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system. After S-type multi-point correction and full-spectrum automatic peak matching, the reference fingerprint chromatograms of the 21 batches of Pandanus samples were generated by the mean method.

[0063] HPLC chromatograms and control fingerprint chromatograms of 21 Pandanus samples from different origins are shown below. Figure 2 As shown.

[0064] Table 1. Gradient elution conditions of the mobile phase

[0065] 3. Preparation of reference solution and identification of characteristic chromatographic peaks in fingerprint chromatogram Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C reference standards, dissolve them separately in methanol, and dilute to 10 mL volumetric flasks to prepare single reference standard stock solutions with concentrations of 0.4365 mg / mL, 0.4216 mg / mL, 0.3713 mg / mL, 0.3818 mg / mL, 0.3869 mg / mL, and 0.3322 mg / mL, respectively. Then, accurately measure 1 mL of each of the above single reference standard stock solutions, place them in the same 10 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain the mixed reference standard working solution.

[0066] The column core of the guard column was filled with diatomaceous earth, and both sides of the column core were sealed with a 0.22 μm organic microporous filter membrane. Excess filter paper was trimmed. The column core was sealed in the guard column sleeve to obtain the online liquid-phase microextraction device for the reference standard, which was then connected to the front end of the liquid chromatography column. 10 μL of the mixed reference standard working solution was accurately pipetted and injected into the liquid chromatograph connected to the online liquid-phase microextraction device using an HPLC injector. Elution was performed according to the procedure in Table 1 to obtain the HPLC chromatogram of the mixed reference standard, and the retention times of the characteristic chromatographic peaks were recorded. The characteristic chromatographic peaks of the neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C reference standards are shown below. Figure 3 As shown.

[0067] 4. Identification of characteristic chromatographic peaks in the HPLC chromatogram of Pandanus arvense samples The retention times of the six target components in the HPLC chromatogram of the Panax notoginseng sample were compared with the retention times of the characteristic chromatographic peaks of the mixed reference standard HPLC chromatogram and the characteristic chromatographic peaks of the reference fingerprint chromatogram. The comparison results are shown in Table 2.

[0068] The results showed that the retention times of the characteristic chromatographic peaks of the HPLC chromatogram of the Panax notoginseng sample, the characteristic chromatographic peaks of the control fingerprint chromatogram, and the characteristic chromatographic peaks of the mixed control HPLC chromatogram were basically the same, confirming that the retention times of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C did not change significantly in the OLMM-HPLC method.

[0069] Table 2. Identification of characteristic chromatographic peaks in the HPLC chromatograms of Pandanus arvense samples.

[0070] 5. Similarity calculation of HPLC chromatograms of Pandanus samples The HPLC chromatograms of the 21 different Panax notoginseng samples from different origins obtained in step 2 were compared with the reference fingerprint chromatograms and the similarity values ​​were calculated using the Chinese herbal medicine chromatographic fingerprint chromatogram similarity evaluation system. The results are shown in Table 3.

[0071] As shown in Table 3, the similarity between the HPLC chromatograms of *Pandanus tectorius* samples from different origins and the control fingerprint chromatograms ranged from (83.5±1.0)% to (99.9±0.1)%, indicating a relatively high level overall. This suggests that while the chemical composition of *Pandanus tectorius* samples from different origins is highly consistent, there are also some differences. Sample S19 showed the highest similarity at (99.9±0.1)%, indicating that the characteristics of samples from this origin were effectively preserved. Sample S13 showed the lowest similarity at (83.5±1.0)%, suggesting that its chemical composition may have changed due to factors such as climate, soil, and harvesting time. This difference in similarity reflects the inherent quality fluctuations of *Pandanus tectorius* from different origins.

[0072] In summary, this online liquid-phase microextraction fingerprinting technology can serve as an efficient and stable quality evaluation method for the identification and quality consistency evaluation of Panax notoginseng.

[0073] Table 3. Similarity results of HPLC chromatograms of Pandanus samples from different origins (n=4)

[0074] Example 2: Effect of mobile phase flow rate on HPLC chromatogram of Pandanus arvense sample I. Experimental Methods The procedure was carried out in accordance with Example 1, except that the mobile phase flow rates were set to 0.4 mL / min, 0.6 mL / min, 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, respectively.

[0075] II. Experimental Results HPLC chromatograms of *Panax notoginseng* samples obtained using different mobile phase flow rates are as follows: Figure 4 As shown, the results indicate that when the mobile phase flow rate is 0.4 mL / min, the chromatographic peaks are incomplete. When the mobile phase flow rate is 0.6–1.2 mL / min, the chromatographic peak order remains unchanged, and the peak shapes are complete, all of which meet the separation requirements. However, when the mobile phase flow rate is 0.6 mL / min, the peak elution time is significantly delayed, failing to meet the requirements for rapid detection. Specifically, when the mobile phase flow rate is 0.8 mL / min, all common peaks in the HPLC chromatogram of the Pandanus sample appear completely, with complete peak shapes and a suitable elution time, meeting the requirements for rapid detection. Therefore, 0.8 mL / min is set as the optimal mobile phase flow rate.

[0076] Example 3: Effect of sample loading on the HPLC chromatogram of Pandanus arvense sample I. Experimental Methods The procedure was carried out in accordance with Example 1, except that the sample loading amount of Panax notoginseng powder in the online liquid-phase microextraction device was set to 2.0 mg, 2.5 mg, 3.0 mg, 4.0 mg, 5.0 mg, 6.0 mg, 7.0 mg, and 8.0 mg, respectively.

[0077] II. Experimental Results HPLC chromatograms of *Panax notoginseng* samples obtained with different sample loading amounts are as follows: Figure 5 As shown in Table 4, the similarity between each chromatogram and the reference fingerprint chromatogram was calculated using the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine. The results indicate that when the sample loading in the online liquid microextraction device is within the range of 2.0–8.0 mg, the similarity between the HPLC chromatogram of the *Plantaina scabra* sample and the reference fingerprint chromatogram is higher than 99.5%, fully meeting the consistency technical requirements for fingerprint chromatograms of traditional Chinese medicine. The overall retention behavior and peak shape characteristics of the characteristic chromatographic peaks under different loading amounts are basically consistent.

[0078] from Figure 5 The HPLC chromatograms of *Pandanus* samples show that as the sample loading increased from 2.0 mg to 6.0 mg, the signal intensity of the characteristic chromatographic peaks increased significantly, and the resolution and peak shape symmetry of each characteristic peak were also optimized, indicating that increasing the sample loading can effectively improve the extraction efficiency and detection sensitivity of the target components. When the sample loading exceeded 6.0 mg (e.g., 7.0 mg, 8.0 mg), although the overall similarity remained above 99.5%, some high-concentration characteristic chromatographic peaks showed slight overload broadening, and the similarity showed a slight decreasing trend. This suggests that excessively high sample loading may lead to enhanced matrix effects or column overload, which may affect the fine characteristics and quantitative accuracy of the *Pandanus* sample HPLC chromatograms.

[0079] Based on the similarity data in Table 4, a sample loading of 6.0 mg resulted in a similarity of 100.0%, the highest among all levels. Simultaneously, the HPLC chromatogram of the Pandanus sample at this loading level exhibited moderate peak intensity, sharp peak shape, and good separation, balancing detection sensitivity and chromatographic stability. Considering both chromatographic characteristics and similarity evaluation, 6.0 mg was selected as the optimal loading level to ensure the representativeness and reliability of the online liquid chromatography-microextraction fingerprint, providing stable technical support for qualitative analysis and quality control.

[0080] Table 4. Similarity results of HPLC chromatograms of Pandanus samples with different sample loading amounts

[0081] Example 4: Effect of connecting pipe diameter on HPLC chromatogram of Pandanus oleraceus sample I. Experimental Methods The procedure was carried out according to Example 1, except that the diameter of the PEK tube was set to 0.5 mm, 0.75 mm, and 1.0 mm respectively.

[0082] II. Experimental Results HPLC chromatograms of *Panax notoginseng* samples obtained using different tube diameters are as follows: Figure 6 As shown in Table 5, the HPLC chromatograms of each Panax notoginseng sample and the control fingerprint chromatogram were calculated using the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine.

[0083] The results showed that when the diameter of the connecting pipe of the online liquid phase microextraction device was in the range of 0.50 to 1.00 mm, the similarity between the HPLC chromatograms of each Panax notoginseng sample and the control fingerprint chromatogram was higher than 97.9%, indicating that the method has good stability and durability within this pipe diameter range.

[0084] from Figure 6 The HPLC chromatograms of the *Panax notoginseng* sample show that as the tube diameter increases from 0.50 mm to 1.00 mm, the signal intensity of the characteristic chromatographic peaks increases significantly, and the response values ​​of the characteristic chromatographic peaks increase significantly. This indicates that increasing the tube diameter can effectively increase sample throughput and improve the extraction efficiency and detection sensitivity of the target components. At a tube diameter of 1.00 mm, the characteristic chromatographic peaks not only have the strongest signals but also exhibit sharp peak shapes, good separation, and no obvious peak broadening or tailing, demonstrating the best chromatographic separation effect and signal response.

[0085] Based on the similarity data in Table 5, the similarity at a tube diameter of 1.00 mm reached 99.5%, the highest value among all tube diameter levels, indicating that the HPLC chromatogram of the Pandanus sample at this tube diameter best matches the control fingerprint chromatogram. Considering the signal intensity, peak shape quality, and similarity results, 1.00 mm is the optimal tube diameter for connection, providing the highest detection sensitivity while ensuring chromatographic consistency, thus providing reliable support for qualitative and quantitative analysis and quality control.

[0086] Table 5. Similarity results of HPLC chromatograms of Pandanus samples with different connecting pathway diameters.

[0087] Example 5 Repeatability Experiment I. Experimental Methods The procedure was carried out in accordance with Example 1, except that the S5 Pandanus sample was tested 6 times.

[0088] II. Experimental Results The fingerprint similarity test results of six online liquid chromatography-microextraction experiments on Pandanus oleraceus sample S5 are as follows: Figure 7As shown in Table 6, the similarity between each chromatogram and the control fingerprint chromatogram was calculated using the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system.

[0089] The results showed that, under the experimental conditions of Example 1, the similarity between the HPLC chromatograms of the Pandanus samples obtained from six consecutive repeated experiments and the control fingerprint chromatograms was between 99.3% and 99.9%, which was at an extremely high level overall. This indicates that the online liquid-phase microextraction method has excellent repeatability and stability.

[0090] from Figure 7 The HPLC chromatograms of the Panax notoginseng samples showed that the characteristic chromatographic peaks in the six parallel experiments were highly consistent in terms of retention time, peak shape, and relative peak height, with no obvious retention time drift or peak shape distortion. This indicates that the method has good reproducibility during operation and can stably capture the characteristic components in the sample.

[0091] The similarity data in Table 6 further confirms this point. The similarity of all parallel experiments is higher than 99.3%, with the highest reaching 99.9%. This indicates that the method can obtain highly consistent HPLC chromatograms of Pandanus samples under the same experimental conditions, providing a reliable technical guarantee for qualitative identification and quality evaluation.

[0092] Table 6 Results of Repeatability Experiments

[0093] Example 6: A method for constructing a fingerprint map of Paniclea spp. 1. Preparation of the online liquid-phase microextraction device for the test sample Remove the guard column (Phenomenex KJ0-4282) core and disassemble it. Remove the original packing material from the core, wash and dry it. Accurately weigh 6.0 mg of Panax notoginseng powder and place it in the core. Fill the gaps with diatomaceous earth until it is flush with the edge of the core. Seal both sides of the core with a 0.22 μm organic microporous membrane and trim off any excess filter paper. Place the sample-packed core into the column sleeve, tighten it, and connect it to the front end of the HPLC column. The mobile phase should flow in from the flat end of the guard column and out from the tip.

[0094] 2. Preparation of an online liquid-phase microextraction device for reference standards The preparation of the online liquid chromatography-microextraction device for the reference standard was carried out according to step 1, "Preparation of the online liquid chromatography-microextraction device for the test sample," except that only the core of the guard column was filled with diatomaceous earth, and both sides of the core were sealed with a 0.22 μm organic microporous filter membrane, with excess filter paper trimmed off. The core was then sealed in the guard column sleeve to obtain the online liquid chromatography-microextraction device for the reference standard, which was then connected to the front end of the liquid chromatography column.

[0095] 3. Construction of fingerprint map The mobile phase system, pump, online liquid microextraction device (online liquid microextraction device for test sample or online liquid microextraction device for reference), liquid chromatography column and UV detector are connected in sequence through tubing. The diameter of the PEK tubes on both sides of the online liquid microextraction device is 1.0 mm.

[0096] A mobile phase A was prepared by mixing 0.5% formic acid aqueous solution and acetic acid in a volume ratio of 1:1, and a mobile phase B was prepared by mixing acetonitrile. The mobile phases were pumped into the pipeline at a flow rate of 0.8 mL / min, and gradient elution was performed according to the procedure shown in Table 1 of Example 1.

[0097] The mobile phase is introduced into the online liquid-phase microextraction device for the test sample constructed in step 1 through a 1.0 mm diameter PEE tube to extract the Panax notoginseng powder online. The extracted solution flows out through a 1.0 mm diameter PEE tube on the other side of the online liquid-phase microextraction device and is separated by a high-performance liquid chromatography column. The separated substances flow out through a UV detector and are introduced into the waste liquid. The liquid chromatogram is obtained by the UV detector and recorded.

[0098] The HPLC chromatograms of each Panax notoginseng sample were imported into the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system. After S-type multi-point correction and full-spectrum automatic peak matching, the reference fingerprint chromatogram of Panax notoginseng was generated by the mean method.

[0099] 4. Characteristic chromatographic peak identification Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C reference standards, dissolve them separately in methanol, and dilute to 10 mL volumetric flasks to prepare single reference standard stock solutions with concentrations of 0.4365 mg / mL, 0.4216 mg / mL, 0.3713 mg / mL, 0.3818 mg / mL, 0.3869 mg / mL, and 0.3322 mg / mL, respectively. Then, accurately measure 1 mL of each of the above single reference standard stock solutions, place them in the same 10 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain the mixed reference standard working solution.

[0100] The mobile phase was pumped into the tubing at a flow rate of 0.8 mL / min, and gradient elution was performed according to the procedure shown in Table 1 of Example 1. The mobile phase was then passed through a 1.0 mm PEEK tube into the online liquid-phase microextraction device containing the reference standard prepared in step 2 for liquid-phase microextraction.

[0101] Accurately pipette 10 μL of the mixed reference standard working solution and inject it into the liquid chromatography column using an HPLC injector. The solution after liquid microextraction and the mixed reference standard working solution are separated by high performance liquid chromatography. The separated substances are eluted by a UV detector and passed into the waste liquid. The liquid chromatogram is obtained by the UV detector and recorded. This chromatogram is the HPLC chromatogram of the mixed reference standard.

[0102] The characteristic chromatographic peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C in the reference fingerprint obtained in step 3 were confirmed based on the HPLC chromatogram of the mixed reference standard.

[0103] Example 7: Screening of key antioxidant active ingredients and analysis of their spectral efficacy based on fingerprinting of Panax notoginseng I. Experimental Methods 1. Preparation of sample solution The *Pandanus* sample was pulverized, passed through a No. 3 sieve (50 mesh), and the sieved *Pandanus* sample powder was collected. 3 g of the powder was placed in a stoppered conical flask, and 20 mL of methanol was added. The flask was weighed, and the mixture was sonicated at 40 kHz and 500 W for 45 min. The volume was then adjusted to the final weight using methanol. The solution was filtered, concentrated, and dissolved in methanol to a final volume of 20 mL to obtain the sample solution.

[0104] 2. ABTS free radical scavenging rate experiment Weigh 192.04 mg ABTS and 67.58 mg potassium persulfate into a reagent bottle, add 100 mL of ultrapure water, and shake well to prepare the ABTS stock solution (3.5 mM ABTS and 2.5 mM potassium persulfate). Store at 4°C in the dark for at least 12 h to prepare the ABTS stock solution. Dilute with anhydrous ethanol solution to an absorbance of 1.0 ± 0.05 at 734 nm before use to obtain the ABTS working solution.

[0105] The experiment was set up with a blank group and a sample group, with 4 replicates in each group. The reactants were added to the 96-well plate according to the dosage shown in Table 7. After standing at room temperature in the dark for 5 min, the plate was shaken for 1 min in a microplate reader and the absorbance at 734 nm was measured. The ABTS radical scavenging rate of the sample solution was calculated according to the following formula (1).

[0106]

[0107] In the formula, A1 is the absorbance of the sample group at 734 nm, and A0 is the absorbance of the blank group at 734 nm.

[0108] Table 7. Dosage and order of reactants added in the ABTS free radical experiment (unit: μL)

[0109] 3. DPPH free radical scavenging rate experiment Weigh 39.43 mg of DPPH, add anhydrous ethanol to make up to 500 mL, and shake well to prepare DPPH working solution (0.2 mM DPPH solution).

[0110] The experiment included a blank group and a sample group, with four replicates in each group. Reactants were added sequentially to the 96-well plate according to the dosages shown in Table 8. After standing at room temperature in the dark for 30 min, the plate was shaken for 1 min in a microplate reader, and the absorbance at 517 nm was measured. The DPPH radical scavenging rate of the sample solution was calculated using formula (2):

[0111] In the formula, B1 is the absorbance of the sample group at 517 nm, and B0 is the absorbance of the blank group at 517 nm.

[0112] Table 8. Dosage and order of reactants added in the DPPH free radical experiment (unit: μL)

[0113] 4. APC Composite Index Analysis The ability of Panax notoginseng to scavenge ABTS and DPPH free radicals was systematically evaluated using the Antioxidant Potency Composite (APC) index. The APC index of the two antioxidant methods was calculated according to formula (3), and the APC composite index was the average of the APC index scores of the two antioxidant methods.

[0114]

[0115] 5. Grey Relational Analysis (GRA) The grey relational degree was calculated directly using the SPSSPRO online analysis platform (https: / / www.spsspro.com). The analysis steps are as follows: (1) Perform dimensionless processing (meaning) on ​​the data: Divide the data of this sequence by the mean to normalize it to around the order of 1.

[0116] (2) Establish reference and comparison sequences: APC index (pharmacological activity index) of different Panax notoginseng medicinal materials was selected as the reference sequence, and peak area data of each characteristic chromatographic peak in the HPLC chromatogram of Panax notoginseng sample was used as the comparison sequence.

[0117] (3) Solve for the reference sequence and compare the gray relation values ​​between the sequences.

[0118] (4) Solve for the grey relational degree value and sort the grey relational degree value.

[0119] 6. Partial Least Squares Regression Analysis In the partial least squares regression (PLSR) model, the correlation between the independent variables of the X matrix and the dependent variables of the Y vector is established. Using the APC composite index as the dependent variable and the peak areas of six common characteristic chromatographic peaks as the independent variables, SIMCA 14.1 software was used to establish spectral-efficacy models between the peak areas of each characteristic chromatographic peak in the HPLC chromatogram of Pandanus oleraceus samples and their antioxidant activity (pharmacological activity index), thus screening for pharmacodynamic active ingredients highly correlated with these two factors.

[0120] II. Experimental Results 1. ABTS free radical scavenging rate of Pandanus officinalis from different origins The ABTS free radical scavenging test mainly reflects the sample's ability to scavenge water-soluble cationic free radicals. It can complement the DPPH method and comprehensively evaluate the in vitro antioxidant activity of plants. Table 8 shows that the ABTS free radical scavenging rate of Pandanus oleraceus from different origins differed by more than 51 percentage points, further confirming that the origin has a universal and significant impact on the antioxidant activity of Pandanus oleraceus.

[0121] In terms of activity, samples S5, S16, and S8 all showed ABTS scavenging rates higher than 76%, which was highly consistent with the DPPH free radical scavenging results. This indicates that the Pandanus from this region is rich in both fat-soluble and water-soluble antioxidants, exhibiting highly efficient scavenging effects against different types of free radicals and demonstrating outstanding comprehensive antioxidant advantages. While samples S6, S7, S9, and S11 showed higher DPPH scavenging rates, their ABTS scavenging rates were only at a moderate level. This suggests that the antioxidant components in these samples are mainly fat-soluble, with relatively insufficient accumulation of water-soluble antioxidants, indicating a polar bias in the composition of antioxidant components.

[0122] Table 8. Free radical scavenging rate (%) of Pandanus samples from different origins

[0123] 2. DPPH free radical scavenging rate of Pandanus officinalis from different origins DPPH free radical scavenging rate is a classic indicator for evaluating the in vitro lipophilic free radical scavenging activity of plant materials. It can directly reflect the content and antioxidant potential of reducing active substances such as phenols and flavonoids in the sample. As shown in Table 8 above, the DPPH scavenging rate of Pandanus tectorius samples from 21 production areas varied by more than 54 percentage points, indicating that the ecological environment of the production area is a key factor affecting the DPPH free radical scavenging activity of Pandanus tectorius.

[0124] The DPPH scavenging rates of samples S5, S8, S6, S16, and S18 were all above 84%, significantly higher than the other samples. This indicates that the growth conditions in these areas are more conducive to the synthesis and accumulation of antioxidant active substances in *Pandanus tectorius*, which may be closely related to ecological factors such as light, temperature, soil fertility, and altitude in these areas. Such areas can be considered preferred production areas for the development of *Pandanus tectorius* antioxidant raw materials. The scavenging rates of samples S20 and S2 were significantly lower, presumably due to unfavorable environmental conditions for the accumulation of secondary metabolites or the genetic characteristics of the germplasm itself. These samples should be avoided in raw material screening.

[0125] Overall, the DPPH scavenging rate of Pandanus in most producing areas was higher than 45%, and half of the samples were higher than 70%, indicating that Pandanus has high development value as a natural antioxidant plant resource, and there are rich active variations within the species, which has room for further breeding and quality improvement.

[0126] 3. APC Composite Index Analysis To more scientifically and comprehensively evaluate the in vitro antioxidant capacity of Pandanus oleraceus from different origins, this invention uses the APC comprehensive index to evaluate the antioxidant capacity of Pandanus oleraceus, obtaining the DPPH-APC index, ABTS-APC index (Table 9) and APC comprehensive index (Table 10), and ranking the antioxidant activity of 21 samples according to the APC comprehensive index.

[0127] Regarding the DPPH-APC index, the range of each sample was 36.62% to 100.00%. Sample S5 had the highest DPPH-APC index, which was set at 100.00% as the reference benchmark. Sample S20 had the lowest DPPH-APC index (36.62%), followed by Sample S2 (41.40%). The DPPH-APC indices of the remaining samples ranged from 47.06% to 99.28%, showing a clear hierarchical differentiation.

[0128] Regarding the ABTS-APC index, the values ​​for each sample ranged from 36.23% to 100.00%, with sample S5 having the highest value (100.00%) and sample S2 having the lowest ABTS-APC index (36.23%). High values ​​were concentrated in samples S16 and S8, at 96.41% and 94.21% respectively, while low values ​​were concentrated in samples S2, S17, and S4. The overall distribution was similar to the trend of the DPPH-APC index, but the magnitudes differed.

[0129] Table 9. DPPH-APC and ABTS-APC indices of Pandanus tectorius from different origins

[0130] The APC composite index, obtained by weighted integration of the DPPH-APC and ABTS-APC indices, comprehensively reflects the overall antioxidant level of the samples. The APC composite index of the 21 samples from different origins ranged from 38.62% to 100.00%. The ranking results showed that S5 (100.00%, 1st), S16 (97.12%, 2nd), S8 (96.74%, 3rd), S6 (86.64%, 4th), and S10 (86.17%, 5th) were the top five samples in terms of overall antioxidant activity. Samples with lower composite indices were S21 (47.89%), S1 (48.98%), S4 (49.30%), S3 (51.94%), and S13 (54.16%). Among these, S20 (38.62%) and S2 (38.82%) had the lowest composite indices, exhibiting the worst overall antioxidant performance.

[0131] Table 10. APC Composite Index and Ranking of Pandanus arvense from Different Origins

[0132] The APC comprehensive index and ranking results show that the comprehensive antioxidant activity of the 21 samples exhibits a continuous gradient distribution, indicating that the place of origin is the core influencing factor determining the comprehensive antioxidant quality of Pandanus tectorius. Differences in ecological environment, soil conditions, cultivation mode, and germplasm genetic background are ultimately reflected in the comprehensive antioxidant activity through differences in the accumulation of secondary metabolites. The top 5 samples, S5, S16, S8, S6, and S10, all have comprehensive indices above 86%, demonstrating stable and outstanding antioxidant efficacy. They can serve as core raw material sources for the development of Pandanus tectorius antioxidant functional products, breeding of superior varieties, and screening of standardized planting bases. In contrast, S2 and S20 have comprehensive indices below 40%, with multiple indicators ranking last. Their comprehensive antioxidant quality is inferior to that of other origins, making them unsuitable as preferred materials for industrial development.

[0133] Furthermore, the APC comprehensive index ranking results are highly consistent with the trend of the original clearance rate, proving that the antioxidant assay system and comprehensive evaluation method used in this study are stable and reliable, and the evaluation results can truly reflect the differences in antioxidant activity of Pandanus tectorius from different producing areas. Combining the aforementioned original clearance rate analysis and APC comprehensive evaluation results, this study can provide systematic data support and theoretical basis for the quality evaluation, regional classification, and efficient utilization of Pandanus tectorius medicinal resources. It also lays an experimental foundation for the isolation of antioxidant active ingredients, research on their mechanisms of action, and the construction of quality standards.

[0134] 4. Grey Relational Analysis Using the APC composite index as the parent sequence (reference sequence) and the peak areas of the characteristic chromatographic peaks in the HPLC chromatograms of 21 Pandanus samples from different origins as the sub-sequences (comparison sequences), the grey relational degree between the two was calculated, which can directly reflect the degree of influence of the parent sequence on the sub-sequence. The peak areas of the characteristic chromatographic peaks in the HPLC chromatograms of Pandanus from 21 origins are shown in Table 11, and the grey relational degree results are shown in Table 12.

[0135] Table 11 Peak areas of six characteristic chromatographic peaks in the HPLC chromatogram of Pandanus arvense sample.

[0136] Table 12 Correlation between characteristic peak areas and APC composite index of HPLC chromatograms of Pandanus arvense samples

[0137] To clarify the core material basis of the antioxidant activity of *Pandanus tectorius*, grey relational analysis was used to calculate and rank the correlation between the peak areas of six main characteristic chromatographic peaks (isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid) in its HPLC chromatogram and the APC comprehensive index. The correlation between the six characteristic components and the APC comprehensive index ranged from 0.747 to 0.798, with all correlations greater than 0.7, indicating a strong positive correlation between each component and the comprehensive antioxidant activity, and that all are important material bases affecting the antioxidant efficacy of *Pandanus tectorius*. The correlations, from highest to lowest, are as follows: isochlorogenic acid A (0.798, ranked 1st) > isochlorogenic acid B (0.794, ranked 2nd) > isochlorogenic acid C (0.792, ranked 3rd) > chlorogenic acid (0.763, ranked 4th) > cryptochlorogenic acid (0.749, ranked 5th) > neochlorogenic acid (0.747, ranked 6th). Among them, isochlorogenic acid A has the highest correlation with the APC comprehensive index, making it the characteristic component most strongly correlated with the comprehensive antioxidant activity of Panax notoginseng; isochlorogenic acid B and isochlorogenic acid C follow closely behind, with all three having correlations higher than 0.79, forming a core component group highly correlated with antioxidant activity.

[0138] The results of this study indicate that chlorogenic acid and its isomers (isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, cryptochlorogenic acid, and neochlorogenic acid) are key components in Pandanus oleracea that scavenge DPPH and ABTS free radicals and contribute to its comprehensive antioxidant activity. These components are rich in phenolic hydroxyl groups and can exert antioxidant effects by providing hydrogen protons, blocking free radical chain reactions, and chelating metal ions. This is consistent with the antioxidant mechanism of natural phenolic acids and also indicates that the antioxidant activity of Pandanus oleracea is not caused by a single component acting independently, but rather by the synergistic effect of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C as the core components.

[0139] 5. Analysis of characteristic chromatographic peak VIP values ​​and partial regression coefficients based on partial least squares method To further screen key marker components affecting the antioxidant activity of Panax notoginseng and clarify the contribution and direction of each characteristic component to the antioxidant effect, partial least squares (PLS) regression analysis was performed on the peak areas of six characteristic chromatographic peaks in the HPLC fingerprint and the antioxidant APC comprehensive index. The variable importance projection value (VIP) and partial regression coefficient of each characteristic chromatographic peak were calculated. The results are shown in [Figure number missing]. Figure 8 , Figure 9 Peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is isochlorogenic acid B, peak 5 isochlorogenic acid A, and peak 6 isochlorogenic acid C.

[0140] Partial regression coefficient results ( Figure 8 The results showed that the partial regression coefficients of the six characteristic chromatographic peaks were all positive, indicating that the peak areas of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C were all positively correlated with the APC comprehensive index. This means that the increase in the content of each component can have a positive promoting effect on the comprehensive antioxidant activity of Panax notoginseng. The partial regression coefficients of each peak were similar and the overall distribution was stable, with no negative regulatory components, suggesting that the six phenolic acid components are all positive contributing factors to antioxidant activity.

[0141] VIP value results ( Figure 9 The results showed a clear stratification of VIP values ​​for each characteristic chromatographic peak. Chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A all had VIP values ​​greater than 1.0, making them key variables significantly affecting antioxidant activity. Neochlorogenic acid, cryptochlorogenic acid, and isochlorogenic acid C had VIP values ​​between 0.8 and 1.0, contributing to antioxidant activity, but less significantly than the former three. Ranked by VIP value, the contribution of each component to antioxidant activity was as follows: peak 2 > peak 4 > peak 5 > peak 1 > peak 3 > peak 6. A higher VIP value indicates a greater explanatory power for the variation in the component's content on the differences in overall antioxidant activity among samples, making it a core variable driving the differentiation of antioxidant quality between different production areas.

[0142] The correlation between the antioxidant active components of *Pandanus tectorius* was analyzed using a comprehensive GRA correlation coefficient > 0.7, a comprehensive regression coefficient > 0, and a VIP value > 1. The analysis revealed that the main components influencing the differences in antioxidant activity among *Pandanus tectorius* from different origins were chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A. All three components showed correlation coefficients higher than 0.76 in the grey relational analysis, exhibiting a strong positive correlation with the APC comprehensive index. Their partial regression coefficients were all positive in the partial least squares regression analysis, indicating that increased content significantly enhances the free radical scavenging capacity and overall antioxidant activity of *Pandanus tectorius*. Furthermore, the VIP values ​​were all greater than 1.0, making them core variables with significant explanatory power for differences in antioxidant effects and key material basis for the hierarchical differentiation of antioxidant activity among samples from different origins. Statistical analysis of fingerprint peak areas and activity indicators reveals that these components are all chlorogenic acid isomers, rich in catechol-type phenolic hydroxyl groups in their molecular structure. They exert antioxidant effects through pathways such as donating hydrogen atoms, scavenging reactive oxygen free radicals, and blocking oxidation chain reactions. Their accumulation in samples from high-activity origins is significantly higher than in low-activity origins, which is the fundamental material reason for the differentiation in antioxidant quality of Pandanus officinalis from different origins. While other phenolic acid components show a correlation greater than 0.7 and a positive partial regression coefficient, indicating a good positive synergistic contribution, their VIP values ​​are all below 1.0, meaning they only participate in the antioxidant effect as auxiliary active ingredients, and their contribution to the activity differences between origins is weaker than that of the aforementioned core components.

[0143] The results of the joint analysis of multiple statistical models corroborate each other and are highly consistent, confirming that chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A can serve as marker components for evaluating the antioxidant quality of Panax notoginseng, providing a stable and reliable target basis for the formulation of its quality control standards, the selection of high-quality production areas, and the breeding of superior varieties.

[0144] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for constructing an HPLC fingerprint of Panax notoginseng, comprising an online liquid microextraction-high performance liquid chromatography (HPLC-HPLC) system, wherein the system comprises a mobile phase, a pump, an online liquid microextraction device, a liquid chromatography column, and a UV detector connected sequentially via tubing, characterized in that, The mobile phase includes mobile phase A and mobile phase B; The mobile phase A is obtained by mixing formic acid aqueous solution and acetic acid in a volume ratio of (0.8-1.2):(0.8-1.2), wherein the volume fraction of formic acid aqueous solution is 0.3-0.7%; The mobile phase B is acetonitrile; The pump has a pre-stored elution gradient program, in which the volume percentage change of mobile phase A is: 0 min, mobile phase A is 95%; After 10 minutes, mobile phase A reached 90%. After 20 minutes, mobile phase A was 90%. After 30 minutes, the mobile phase A was 88.5%; After 40 minutes, the mobile phase A was 79%. At 65 min, mobile phase A was 77%; At 66 min, mobile phase A was 95%; 70 min, mobile phase A is 95%; The UV detector has a detection wavelength of 326 nm.

2. The construction method according to claim 1, characterized in that, The method further includes the detection of a reference solution, which includes neochlorogenic acid solution, chlorogenic acid solution, cryptochlorogenic acid solution, isochlorogenic acid B solution, isochlorogenic acid A solution and isochlorogenic acid C solution.

3. The construction method according to claim 2, characterized in that, The method for preparing the neochlorogenic acid solution is as follows: neochlorogenic acid and methanol are thoroughly mixed, wherein the mass-to-volume ratio of neochlorogenic acid to methanol is (0.04-0.045) mg: 1 mL; The chlorogenic acid solution is prepared by thoroughly mixing chlorogenic acid and methanol, wherein the mass-to-volume ratio of chlorogenic acid to methanol is (0.04-0.045) mg: 1 mL; The method for preparing the cryptochlorogenic acid solution is as follows: cryptochlorogenic acid and methanol are thoroughly mixed, and the mass-volume ratio of cryptochlorogenic acid to methanol is (0.035~0.04) mg: 1 mL; The isochlorogenic acid B solution is prepared by mixing isochlorogenic acid B with methanol thoroughly, wherein the mass-to-volume ratio of isochlorogenic acid B to methanol is (0.035-0.04) mg: 1 mL; The isochlorogenic acid A solution is prepared by mixing isochlorogenic acid A with methanol thoroughly, wherein the mass-to-volume ratio of isochlorogenic acid A to methanol is (0.035-0.04) mg: 1 mL; The isochlorogenic acid C solution is prepared by mixing isochlorogenic acid C with methanol thoroughly, wherein the mass-to-volume ratio of isochlorogenic acid C to methanol is (0.035-0.04) mg: 1 mL.

4. The construction method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.6–1.2 mL / min.

5. The construction method according to claim 1, characterized in that, The diameter of the pipes on both sides of the online liquid phase microextraction device is 0.5 to 1.00 mm.

6. A method for quality testing of Pandanus, characterized in that, The method described in any one of claims 1 to 5 was used to test several batches of Pandanus samples, and chromatograms were recorded. The chromatograms were then processed to generate a Pandanus reference fingerprint spectrum. The sample of Panax notoginseng to be tested was tested using the construction method described in any one of claims 1 to 5, and the chromatogram was recorded to obtain the chromatogram of the sample of Panax notoginseng to be tested. The chromatograms of the samples of Pandanus to be tested were compared with the fingerprint chromatograms of the Pandanus control.

7. The method according to claim 6, characterized in that, The Pandanus tectorius control fingerprint contains 6 characteristic chromatographic peaks: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C. The specified values of the retention times of each characteristic chromatographic peak are: neochlorogenic acid 17.321 min, chlorogenic acid 28.041 min, cryptochlorogenic acid 29.011 min, isochlorogenic acid B 52.478 min, isochlorogenic acid A 55.090 min, and isochlorogenic acid C 59.087 min; The results of the similarity analysis can be judged as qualified only when the following two conditions are met: The retention time of the characteristic chromatographic peak in the chromatogram of the待测 Pandanus tectorius sample is within the range of ±10% of the specified value of the retention time of the corresponding characteristic chromatographic peak in the Pandanus tectorius control fingerprint; The similarity between the chromatogram of the待测 Pandanus tectorius sample and the Pandanus tectorius control fingerprint is not less than 0.

9.

8. A method for screening quality markers of Panax notoginseng based on its antioxidant spectrum relationship, characterized in that, It includes the following steps: S1. Construct a Pandanus tectorius fingerprint using the construction method described in any one of claims 1 to 5. The Pandanus tectorius fingerprint contains 6 characteristic chromatographic peaks: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C; S2. Determine the antioxidant activity of Pandanus tectorius; S3. Perform grey relational analysis or partial least squares regression analysis on the peak areas of the characteristic chromatographic peaks in the Pandanus tectorius fingerprint obtained in step S1 and the antioxidant activity obtained in step S2, calculate the correlation degree between each characteristic chromatographic peak and the antioxidant activity, and determine the quality markers of Pandanus tectorius.

9. The screening method according to claim 8, characterized in that, In step S1, the retention time of each characteristic chromatographic peak in the Pandanus tectorius fingerprint is within ±10% of the specified value; The specified values of the retention times of each characteristic chromatographic peak are: neochlorogenic acid 17.321 min, chlorogenic acid 28.041 min, cryptochlorogenic acid 29.011 min, isochlorogenic acid B 52.478 min, isochlorogenic acid A 55.090 min, and isochlorogenic acid C 59.087 min.

10. The screening method according to claim 8, characterized in that, In step S2, the indexes of the antioxidant activity include DPPH free radical scavenging rate, ABTS free radical scavenging rate, and / or comprehensive antioxidant capacity composite index.

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