Two-dimensional chromatography screening method for lipid-lowering active ingredients of lindera root

Abstract

The application discloses a two-dimensional chromatography screening method of lipid-lowering active ingredients of Linderae Radix, and comprises the following steps: extraction of Linderae Radix terpenoid enrichment, establishment of a two-dimensional HSCCCxUHPLC-MS chromatogram, establishment of a two-dimensional bioactivity atlas and identification of the lipid-lowering active ingredients of Linderae Radix; the method integrates the advantages of two chromatography technologies, can quickly and accurately screen the effective active ingredients in the Linderae Radix terpenoid enrichment, and is used for screening acetyl linderae resinoid from the Linderae Radix terpenoid enrichment; the acetyl linderae resinoid has a lipid-lowering activity, which is reported for the first time; and the application provides a new basis for clarifying the lipid-lowering pharmacodynamic material basis of Linderae Radix and related drug development.

Description

Technical Field

[0001] This invention relates to an efficient screening method for lipid-lowering active ingredients in natural products and a study on the activity of pharmacodynamic substances. Based on HSCCC×UHPLC-MS two-dimensional bioactivity spectroscopy, acetyl lindera ester, an unreported lipid-lowering active ingredient in Lindera terpenoids, was rapidly screened, and the lipid-lowering effect of acetyl lindera ester was studied. Background Technology

[0002] As the role of natural products in drug innovation and functional food development becomes increasingly prominent, their bioactive components exhibit significant advantages in regulating physiological activities. In recent years, through in-depth research on natural products and advancements in chemical separation techniques, an increasing number of novel bioactive components have been discovered. However, natural products are typically complex in composition, potentially containing hundreds to thousands of compounds, leading to significant obstacles to their comprehensive development and utilization. Therefore, there is an urgent need to establish a simple, rapid, and high-throughput screening method to overcome the shortcomings of existing technologies, thereby promoting the systematic and efficient development and application of natural products.

[0003] Lindera is derived from the plant Lindera strychnifolia, which belongs to the Lauraceae family. Lindera aggregata The dried tuberous root of *Sims. Kosterm.* was first recorded in the Tang Dynasty's *Compendium of Materia Medica*, boasting a long history of medicinal use. The *Kaibao Materia Medica* states that it is warm in nature and pungent in taste, entering the liver, spleen, and kidney meridians, possessing the effects of regulating qi, relieving stagnation, dispelling cold, and relieving pain. It can treat symptoms such as qi stagnation, chest and abdominal distension and pain, indigestion, vomiting, cold hernia, beriberi, and frequent urination. Modern research further reveals that terpenoid components, especially those with a lindera alkyl structure, are an important material basis for the lipid-lowering effects of many traditional Chinese medicines. Lindera is rich in structurally diverse sesquiterpenoid components, and pharmacological studies have confirmed that its ethanol extract can effectively alleviate diet-induced hyperlipidemia by regulating intestinal microbiota and bile acid metabolism, exhibiting clear lipid-lowering activity. Given that Lindera is rich in a large amount of lindera alkyl sesquiterpenoid components, and that its extract has been confirmed by modern pharmacological studies to have a clear effect on improving hyperlipidemia by regulating the intestinal microbiota-bile acid axis, the specific components responsible for its lipid-lowering effect remain unclear. Therefore, systematic screening of the lipid-lowering activity of its terpenoid components is a key scientific approach to elucidating the pharmacodynamic material basis of this medicinal material and discovering lead compounds.

[0004] Two-dimensional chromatography (2D chromatography) refers to the online or offline combination of two chromatographic techniques with different separation mechanisms to achieve the separation and preparation of complex chemical components. It offers advantages such as high capacity, high resolution, and high sensitivity, overcoming the problems of peak repetition or low abundance in traditional one-dimensional chromatography when separating complex samples. Currently, bioactivity screening strategies based on 2D chromatography platforms are mainly divided into traditional separation screening, bioactivity-guided screening, and affinity chromatography screening. Traditional separation screening methods are primarily based on column chromatography. This method is advantageous for separating the major components of natural products, but some secondary bioactive components are easily overlooked. Bioactivity-guided screening methods mainly include microfractional activity screening and affinity ultrafiltration bioactivity screening. Microfractional activity screening is a rapid and efficient screening method with the advantage of reducing false positive rates, but its low injection volume limits its application in 2D chromatography to some extent. Affinity ultrafiltration is a technique based on affinity and selectivity to "capture" drug targets and their specific ligands. By coupling with high-performance liquid chromatography-mass spectrometry (HPLC-MS), it can achieve high-throughput screening of active ingredients in natural products; however, this method suffers from false positives and false negatives. Affinity chromatography screening utilizes the specific binding of a stationary phase and potential ligands to achieve selective separation, making it highly suitable for screening bioactive components in complex samples. However, some limitations still need to be overcome.

[0005] To date, high-speed countercurrent chromatography (HSCCC) and HPLC have been very popular in two-dimensional chromatographic screening due to their high loading capacity and efficiency. This invention replaces HPLC with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) on the basis of HSCCC and HPLC, which greatly shortens the two-dimensional chromatographic analysis time and also shows a powerful ability to characterize and identify relevant bioactive components. Summary of the Invention

[0006] To address the issue of unclear lipid-lowering active substances in Lindera strychnifolia terpenoids, this invention provides a method for obtaining lipid-lowering active ingredients by combining UHPLC-MS technology with two-dimensional chromatographic screening, and for the first time discovers that acetyl Lindera strychnifolia terpenoids have significant lipid-lowering activity.

[0007] The technical solution of the present invention is as follows: A two-dimensional chromatographic screening method for lipid-lowering active ingredients of Lindera strychnifolia includes: (1) Extraction of terpenoid-rich compounds from Lindera strychnifolia Lindera powder was extracted by reflux with 90% (volume fraction) ethanol aqueous solution to obtain crude extract of Lindera; the crude extract of Lindera was then redissolved with 20% (volume fraction) ethanol aqueous solution and extracted with petroleum ether to obtain terpene enrichment of Lindera. The specific operation is as follows: Lindera powder is mixed with 90% ethanol aqueous solution at a material-to-liquid ratio of 1:8 (g / mL), refluxed for 2 hours, filtered, and the residue is extracted twice more. The filtrates are combined and evaporated to dryness to obtain crude extract of Lindera; the crude extract of Lindera is redissolved in 20% ethanol aqueous solution at a material-to-liquid ratio of 1:40 (g / mL), extracted with an equal volume of petroleum ether, the petroleum ether layer is collected, the extraction is repeated three times, the petroleum ether layers are combined and evaporated to dryness to obtain terpene-rich Lindera; (2) Establishment of a full two-dimensional HSCCC×UHPLC-MS chromatogram The terpene enrichment of Lindera obtained in step (1) was separated by HSCCC in the first dimension, and each separated component was analyzed by UHPLC-MS in the second dimension. A two-dimensional contour plot was drawn using Matlab R2024b script to establish a full two-dimensional HSCCC×UHPLC-MS chromatogram. HSCCC chromatographic conditions: The solvent system was petroleum ether / ethyl acetate / methanol / water. A head-to-tail elution mode was used, with the upper organic phase as the stationary phase and the lower aqueous phase as the mobile phase. The elution program was as follows: 0-65 min, petroleum ether / ethyl acetate / methanol / water volume ratio 5:5:6:4; 65-200 min, petroleum ether / ethyl acetate / methanol / water volume ratio 5:5:7:3; 200-275 min, petroleum ether / ethyl acetate volume ratio 5:5 (push-extraction); flow rate 2 mL / min, rotation speed 800 rpm, detection wavelength 254 nm. UHPLC chromatographic conditions: Eclipse Plus C18 column (4.6 × 50 mm, 1.8 μm); mobile phase A: 0.1% (v / v) formic acid in water; mobile phase B: acetonitrile; elution program: 0.00–8.00 min, 45%–54% (v / v) B; 8.00–10.00 min, 54%–60% B; 10.00–15.00 min, 60%–75% B; 15.00–25.00 min, 75%–100% B; 25.00–27.00 min, 100% B; 27.50–27.51 min, 100%–45% B; 27.51–30.00 min, 45% B; flow rate: 0.4 mL / min; column temperature: 35℃. MS mass spectrometry conditions: Data acquisition was performed using a Q-Exactive Plus MS device in positive ion mode; parameters were as follows: injection voltage: 3000 V; dry gas (N2) flow rate: 40 L / min; dry gas (N2) temperature: 350℃; data were collected using full MS-ddMS2 scan mode, with MS1 and MS2 scan ranges of 100-1500 m / z, MS1 resolution of 70000, and MS2 resolution of 17500; (3) Establishment of a complete two-dimensional bioactivity map and identification of lipid-lowering active ingredients of Lindera strychnifolia The terpene enrichment obtained in step (1) was prepared into a sample solution and separated under the HSCCC chromatographic conditions in step (2). The free lipase inhibitory activity of each separated component was determined, and an HSCCC activity spectrum was established. The terpene enrichment was preferably dissolved in a solvent system with a volume ratio of petroleum ether / ethyl acetate / methanol / water of 5:5:6:4 to prepare a sample solution with a concentration of 20 mg / mL. The terpene enrichment obtained in step (1) was prepared into a sample solution and eluted under the UHPLC chromatographic conditions in step (2). The eluent was collected in a 96-well plate and the free lipase inhibitory activity was determined to establish a UHPLC activity chromatogram. Preferably, the terpene enrichment was dissolved in methanol to prepare a sample solution with a concentration of 50 mg / mL. The HSCCC activity spectrum, UHPLC activity spectrum and the full two-dimensional HSCCC×UHPLC-MS chromatogram of step (2) were combined to draw a full two-dimensional bioactivity spectrum; the active substance was located by cross-analysis of HSCCC and UHPLC, and the lipid-lowering active ingredient of Lindera strychnifolia was identified as acetyl Lindera strychnifolia ester by comparison of molecular ion peaks and fragment ions of MS mass spectrometry.

[0008] This invention evaluates the activity of screened active ingredients based on HepG2 cell line / zebrafish juveniles using indicators such as Oil Red O staining, total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C).

[0009] The beneficial effects of this invention are: This invention integrates the advantages of two chromatographic techniques, enabling rapid and accurate screening of effective active ingredients in Lindera strychnifolia terpenoid concentrates with high-resolution spectra. Compared to existing two-dimensional chromatographic screening techniques, the UHPLC-MS coupling technology used in this method significantly shortens the analysis time of the second-dimensional chromatography, while demonstrating strong capabilities in the characterization and identification of relevant bioactive components, achieving efficient simultaneous screening and identification.

[0010] The lipid-lowering activity of acetylated lindera ester, screened from terpene-rich extracts of Lindera using this method, is reported for the first time. This invention provides new evidence for elucidating the pharmacodynamic material basis of Lindera and the development of related drugs. Attached Figure Description

[0011] Figure 1 Two-dimensional contour plot of terpenoid-enriched Lindera root HSCCC×UHPLC-MS.

[0012] Figure 2 Spatial coverage map of a two-dimensional contour map.

[0013] Figure 3 Linear plot of a two-dimensional contour map.

[0014] Figure 4 Two-dimensional activity diagram of free lipases enriched in Lindera strychnifolia terpenoids.

[0015] Figure 5 Oil Red O staining results of HepG2 cells treated with acetylgynyl buergerianum.

[0016] Figure 6 : TC, TG, and LDL-C content in HepG2 cells treated with acetylglycine.

[0017] Figure 7 : TC, TG, and LDL-C content in zebrafish juveniles treated with acetylosin. Detailed Implementation

[0018] The present invention is further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0019] The Lindera used in the following examples was purchased from the Bozhou Traditional Chinese Medicine Market.

[0020] Example 1: Extraction of terpenoid-rich extracts from Lindera root: Weigh 100.00 g of Lindera powder into a 2 L round-bottom flask, add 800 mL of 90% ethanol (solid-liquid ratio 1:8), reflux for 2 h, filter, repeat the extraction twice with the residue, combine the filtrates, and dry the filtrate under reduced pressure at 45℃ to obtain 9.04 g of crude Lindera extract, with a yield of 9.04%.

[0021] Weigh 5.00 g of crude extract of Lindera strychnifolia, redissolve it in 200 mL of 20% ethanol, extract it with 200 mL of petroleum ether, collect the petroleum ether layer, repeat the extraction 3 times, and dry the petroleum ether layer under reduced pressure at 40 °C to obtain 488.23 mg of yellow paste-like terpene-rich extract, with a yield of 9.76%.

[0022] Example 2: Screening and optimization of two-dimensional chromatographic conditions

[0023] (1) Establishment of HSCCC analytical conditions for Lindera strychnifolia terpenoids enriched: HSCCC adopted a head-to-tail elution mode with the upper organic phase as the stationary phase and the lower aqueous phase as the mobile phase, and petroleum ether / ethyl acetate / methanol / water (PE / EA / MeOH / H2O) as the solvent system. The stationary phase was the upper organic phase (PE:EA), and the mobile phase was the lower aqueous phase (MeOH:H2O). The elution program was as follows: 0-65 min, PE / EA / MeOH / H2O (5:5:6:4, v / v); 65-200 min, PE / EA / MeOH / H2O (5:5:7:3, v / v); 200-275 min, using the stationary phase (PE:EA=5:5, v / v) for pushing. The flow rate was 2 mL / min, the rotation speed was 800 rpm, and the detection wavelength was 254 nm. When the initial two phases (PE / EA / MeOH / H2O, 5:5:6:4, v / v) reached equilibrium, 55.85 mg of terpene concentrate dissolved in 20 mL of the initial two-phase system was injected into the equilibrium system. The stationary phase retention rate was 57.89%.

[0024] (2) Establishment of UHPLC-MS for enriching terpenoids from Lindera root: ① UHPLC conditions: Two-dimensional analysis was performed using an Eclipse Plus C18 column (4.6 × 50 mm, 1.8 μm). Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile. Elution program: 0.00-8.00 min, 45%-54% B; 8.00-10.00 min, 54%-60% B; 10.00-15.00 min, 60%-75% B; 15.00-25.00 min, 75%-100% B; 25.00-27.00 min, 100% B; 27.50-27.51 min, 100%-45% B; 27.51-30.00 min, 45% B. Injection volume: 5 μL, flow rate: 0.4 mL / min, column temperature: 35℃; ②MS conditions: Data acquisition was performed using a Q-Exactive Plus MS device in positive ion mode. Specific parameters were as follows: injection voltage: 3000 V; drying gas (N2) flow rate: 40 L / min; drying gas (N2) temperature: 350℃. Data were collected using full MS-ddMS2 scan mode. The scan range of MS1 and MS2 was 100-1500 m / z, the resolution of MS1 was 70000, and the resolution of MS2 was 17500.

[0025] (3) Establishment of HSCCC×UHPLC-MS: HSCCC is used for the first dimension of two-dimensional separation, and UHPLC-MS is used for the second dimension of two-dimensional separation. Fractions collected from HSCCC at a rate of 1 tube (2 mL) per minute were desolvated, redissolved in 1 mL of methanol, and then transferred to UHPLC-MS for the second dimension of analysis. UHPLC-MS chromatograms of the corresponding fractions were obtained. Two-dimensional contour plots were drawn using Matlab R2024b scripts to establish the complete two-dimensional HSCCC×UHPLC-MS chromatogram, as shown below. Figure 1 As shown. Origin 2019b was used to calculate the spatial coverage and linearity of the chromatogram, where the spatial coverage was 62.18%, as shown. Figure 2 As shown; the linearity is 0.35, as... Figure 3 As shown.

[0026] Example 3: Establishment of an experimental method for free lipase

[0027] (1) Solution preparation: ① Tris-HCl buffer: Measure 10 mL of 1 M Tri-HCl stock solution, add 144.3 mg CaCl2, 5.84 g NaCl and 1 mL Triton X-100, dilute with purified water to prepare a Tri-HCl buffer with a final concentration of 10 mM; ② Free lipase solution: Weigh 5.00 mg of free lipase, dissolve in 5 mL Tri-HCl buffer, centrifuge at 10000 g for 5 min, and the supernatant is a free lipase solution with a concentration of 1 mg / mL; ③ 4-methyl umbelliferone oleate solution (4-MUO): Prepare a 0.1 mM 4-MUO solution with Tri-HCl buffer containing 1% DMSO; ④ Sample solution: Remove the solution from the collected UHPLC / HSCCC fraction, add Tri-HCl buffer containing 1% DMSO to obtain the sample solution.

[0028] (2) Free lipase inhibitory activity experiment: 25 μL of sample solution, 25 μL of free lipase solution, and 50 μL of 4-MUO were added to each well of a 96-well plate, with a total reaction volume of 100 μL. The plate was incubated at 25℃ for 30 min before detection. The amount of released 4-methylumbelliferone was determined by fluorescence detection at an excitation wavelength of 255 nm and an emission wavelength of 460 nm. The inhibition rate of the fraction in each well on the free lipase activity was calculated using formula (1), where ΔA 样品 The fluorescence value of the reaction system containing distillate, ΔA 空白 The fluorescence value represents the reaction system without distillate.

[0029] Inhibition rate (%) = (1 - ΔA) 样品 / △A 空白)×100% (1)

[0030] Example 4: Establishment of a two-dimensional bioactivity map

[0031] (1) Establishment of HSCCC activity spectrum of Lindera strychnifolia terpenoids: A sample solution with a concentration of 20 mg / mL was prepared using a 20 mL PE / EA / MeOH / H2O (5:5:6:4, v / v) system. The sample was eluted according to the optimized HSCCC conditions in Example 2. The fractions were collected at a rate of 10 mL / tube for 45-275 min to obtain 46 components. Each component was evaporated using a solvent evaporator. The free lipase inhibitory activity of each component was determined according to the conditions in Example 3.

[0032] (2) Establishment of UHPLC activity chromatogram of Lindera strychnifolia terpenoids: A sample solution with a concentration of 50 mg / mL was prepared using 1 mL of methanol and eluted under the optimized UHPLC conditions in Example 2, with an injection volume of 20 μL. The fraction from 0 to 30 min was collected into a 96-well plate with a resolution of 7 s / well, and the free lipase inhibitory activity of each fraction was determined under the conditions in Example 3.

[0033] (3) Establishment of high-resolution two-dimensional activity chromatograms: By drawing and combining the HSCCC activity chromatogram, UHPLC activity chromatogram, and the full two-dimensional HSCCC×UHPLC-MS chromatogram in Example 2, a full two-dimensional activity fingerprint chromatogram of free lipase was obtained, such as... Figure 4 As shown. Cross-analysis using both HSCCC and UHPLC revealed that the compound exhibited certain free lipase inhibitory activity at a liquid phase retention time of 17.46 min and a countercurrent fraction of 42, suggesting that this compound may be the main inhibitory monomeric component among the terpenoids of Lindera root. Mass spectrometry analysis of molecular ion peaks and fragment ions identified the compound as acetyl Lindera ester. No previous reports on the lipid-lowering effects of acetyl Lindera ester were found.

[0034] Example 5: Verification of the lipid-lowering activity of acetyl wuyao ester.

[0035] (1) Culture of HepG2 cells: HepG2 cells were cultured in DMEM high glucose medium containing a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C, 5% CO2, and saturated humidity. The medium was changed every two days. When the cell adhesion area reached 80%-90%, the medium was aspirated, an appropriate amount of trypsin was added for digestion, and the cells were passaged.

[0036] (2) Grouping, modeling, and administration of HepG2 cells: 12 mL of cells with a concentration of 1×10⁻⁶ were administered. 5HepG2 cell suspension at concentrations of 100 cells / mL was evenly seeded into 6-well plates and divided into control, model, positive control, and low-, medium-, and high-acetyl-codone ester treatment groups. Cells were spread evenly using the cross-hatching method and cultured in an incubator for 24 h. The old culture medium was discarded, and except for the control group, 2 mL of 600 μM free fatty acids (oleic acid:palmitic acid = 2:1) was added to each well for 24 h. Significant lipid droplet accumulation was observed under a high-power microscope after 24 h. After 24 h, 2 mL of high-glucose medium containing 100 μM orlistat, 25 μM, 50 μM, and 100 μM acetyl-codone ester were added to the positive control, low-, medium-, and high-treatment groups, respectively, and treatment continued for another 24 h.

[0037] (3) Grouping and culture of zebrafish juveniles: Healthy zebrafish embryos were selected under a microscope and cultured in E3 medium for 5 days. The juveniles were randomly divided into a control group, a high-fat model group, and a drug-treated group, with 80 fish in each group. The control group was fed zebrafish feed (particle size <50 microns) at 1 mg / fish at 9 am and 4 pm daily, with the E3 medium changed 1 hour after feeding. The model group and the drug-treated group were cultured in E3 medium with an egg yolk concentration of 1 mg / mL, with the medium changed twice daily. After 7 days of culture, the drug-treated group was fed egg yolk powder plus acetyl balsaminate to a concentration of 3.125 μM. After 7 days of drug treatment, the zebrafish were collected for subsequent index determination. All zebrafish juveniles were kept in a culture medium at 28℃, with 14 hours of light and 10 hours of darkness daily.

[0038] (4) Oil Red O staining: HepG2 cells were evenly seeded into 12-well plates, with 1 mL of cell suspension added to each well. When the cell adhesion area reached 80%, the original culture medium was discarded, and the cells were washed once with PBS buffer. 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 30 min. The paraformaldehyde fixative was then removed, and the cells were washed twice with PBS. 1 mL of Oil Red O staining solution was added, and the cells were stained at room temperature in the dark for 30-40 min. The Oil Red O staining solution was then removed, and the cells were washed twice with PBS. Finally, the cells were observed and photographed under a microscope. Figure 5 As shown, the presence of distinct deep red spots in the model group indicates significant lipid droplet accumulation in the cells. Treatment with different concentrations of the drug reduced the number of red spots in the cells. Quantitative analysis using ImageJ software showed that the effect of acetylgynyl ester on lipid droplets in HepG2 cells was concentration-dependent; treatment with 40 μM acetylgynyl ester resulted in a relative decrease in lipid droplet area of ​​approximately 71.76%.

[0039] (5) Lipid-related index determination: ① Cell level: Cells were cultured according to the steps in (2) of Example 5. The original culture medium was discarded, and the cells were washed twice with PBS buffer. The cells were scraped off and collected. After centrifugation to remove the supernatant, 200 μL of 1% Trition X-100 solution was added, and the cells were lysed on ice for 30 min. The contents of TC, TG, and LDL-C in each group were determined according to the kit (Nanjing Jiancheng) instructions. The results are as follows: Figure 6 As shown, A, B, and C represent the relative contents of TC, TG, and LDL-C, respectively. The results showed that these indicators were all increased in the model group, and these indicators could be reduced by treatment with orlistat and acetyl balsamin. At the same time, the lipid-lowering effect of acetyl balsamin was concentration-dependent. At 100 μM, acetyl balsamin was better than orlistat in reducing TC and TG, and the LDL-C content was significantly different from that in the model group, indicating that acetyl balsamin had a good lipid-lowering effect on high-lipid HepG2 cells. ② Animal level: Zebrafish were cultured according to the steps in Example 5 (3). After the juvenile fish were treated with the drug, they were fasted for 24 h. After fasting, they were washed with PBS, and the water was fully absorbed by filter paper and weighed. Ethanol was added according to the ratio of body weight (g): volume (mL) = 1:9. The mixture was homogenized at 4°C. The centrifuge was set to 4°C in advance and centrifuged at 2500 rpm for 10 min. The supernatant was collected and stored for later use. The contents of TC, TG, and LDL-C in each group were determined according to the instructions of the kit (Nanjing Jiancheng). The results are as follows. Figure 7 As shown, the contents of TC, TG and LDL-C in zebrafish juveniles were significantly reduced under 3.125 μM treatment, indicating that acetyl wuyu ester has a significant lipid-lowering effect in high-fat zebrafish.

[0040] Based on the results of cell oil red O staining and lipid-related index determination in cells and zebrafish, acetyl lindera ester, a component screened from lindera terpenoid enrichment using a two-dimensional bioactivity mapping method, showed good lipid-lowering effects. This result also verified the accuracy of this screening method.

Claims

1. A two-dimensional chromatographic screening method for lipid-lowering active ingredients of Lindera strychnifolia, characterized in that, The method includes: (1) Extraction of terpenoid-rich compounds from Lindera strychnifolia Lindera powder was extracted by reflux with 90% ethanol aqueous solution to obtain crude extract of Lindera; the crude extract of Lindera was then redissolved with 20% ethanol aqueous solution and extracted with petroleum ether to obtain terpene concentrate of Lindera. (2) Establishment of a full two-dimensional HSCCC×UHPLC-MS chromatogram The terpene enrichment of Lindera obtained in step (1) was separated by HSCCC in the first dimension, and each separated component was analyzed by UHPLC-MS in the second dimension. A two-dimensional contour plot was drawn using Matlab R2024b script to establish a full two-dimensional HSCCC×UHPLC-MS chromatogram. HSCCC chromatographic conditions: The solvent system was petroleum ether / ethyl acetate / methanol / water. A head-to-tail elution mode was used, with the upper organic phase as the stationary phase and the lower aqueous phase as the mobile phase. The elution program was as follows: 0-65 min, petroleum ether / ethyl acetate / methanol / water volume ratio 5:5:6:4; 65-200 min, petroleum ether / ethyl acetate / methanol / water volume ratio 5:5:7:3; 200-275 min, petroleum ether / ethyl acetate volume ratio 5:5 (push-extraction); flow rate 2 mL / min, rotation speed 800 rpm, detection wavelength 254 nm. UHPLC chromatographic conditions: Eclipse Plus C18 column; Mobile phase A: 0.1% formic acid in water, Mobile phase B: acetonitrile; Elution program: 0.00-8.00 min, 45%-54% B; 8.00-10.00 min, 54%-60% B; 10.00-15.00 min, 60%-75% B; 15.00-25.00 min, 75%-100% B; 25.00-27.00 min, 100% B; 27.50-27.51 min, 100%-45% B; 27.51-30.00 min, 45% B; Flow rate: 0.4 mL / min; Column temperature: 35℃; MS mass spectrometry conditions: Data acquisition was performed using a Q-Exactive Plus MS device in positive ion mode; parameters were as follows: injection voltage: 3000 V; dry gas N2 flow rate: 40 L / min; dry gas N2 temperature: 350℃; data were collected using full MS-ddMS2 scan mode, with MS1 and MS2 scan ranges of 100-1500 m / z, MS1 resolution of 70000, and MS2 resolution of 17500; (3) Establishment of a complete two-dimensional bioactivity map and identification of lipid-lowering active ingredients of Lindera strychnifolia The terpene enrichment obtained in step (1) was prepared into a sample solution and separated under the HSCCC chromatographic conditions in step (2). The free lipase inhibitory activity of each separated component was determined, and an HSCCC activity spectrum was established. The terpene enrichment of Lindera strychnifolia obtained in step (1) was prepared into a sample solution and eluted under the UHPLC chromatographic conditions in step (2). The eluent was collected in a 96-well plate and the free lipase inhibitory activity was determined to establish a UHPLC activity chromatogram. The HSCCC activity spectrum, UHPLC activity spectrum and the full two-dimensional HSCCC×UHPLC-MS chromatogram of step (2) were combined to draw a full two-dimensional bioactivity spectrum; the active substance was located by cross-analysis of HSCCC and UHPLC, and the lipid-lowering active ingredient of Lindera strychnifolia was identified as acetyl Lindera strychnifolia ester by comparison of molecular ion peaks and fragment ions of MS mass spectrometry.

2. The two-dimensional chromatographic screening method for the lipid-lowering active ingredients of Lindera strychnifolia as described in claim 1, characterized in that, Step (1) is as follows: Mix Lindera powder with 90% ethanol aqueous solution at a material-to-liquid ratio of 1:8, reflux for 2 h, filter, repeat extraction twice with the residue, combine the filtrates, and evaporate to dryness to obtain crude extract of Lindera; redissolve the crude extract of Lindera in 20% ethanol aqueous solution at a material-to-liquid ratio of 1:40, extract with an equal volume of petroleum ether, collect the petroleum ether layer, repeat extraction three times, combine the petroleum ether layers, and evaporate to dryness to obtain Lindera terpene enrichment.

3. The two-dimensional chromatographic screening method for the lipid-lowering active ingredients of Lindera strychnifolia as described in claim 1, characterized in that, In step (3), when establishing the HSCCC activity spectrum, the terpene enrichment of Lindera strychnifolia was dissolved in a solvent system with a volume ratio of petroleum ether / ethyl acetate / methanol / water of 5:5:6:4 to prepare a sample solution with a concentration of 20 mg / mL.

4. The two-dimensional chromatographic screening method for the lipid-lowering active ingredients of Lindera strychnifolia as described in claim 1, characterized in that, In step (3), when establishing the UHPLC activity spectrum, the terpene enrichment of Lindera strychnifolia is dissolved in methanol to prepare a sample solution with a concentration of 50 mg / mL.

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