Use of 4,5-di-o-caffeoylquinic acid methyl ester in preparation of anti-hepatitis B virus drugs

Abstract

The application belongs to the technical field of natural medicinal chemistry and antiviral drugs, and provides application of 4,5-CQME in preparation of an anti-hepatitis B virus drug. The compound is separated from honeysuckle flower buds, the compound can inhibit replication and transcription of HBV DNA, and down-regulate HBsAg, HBeAg and HBxAg antigen expression in a dose-dependent manner, and does not cause obvious cytotoxicity in HepG2.2.15 cells. Mechanism research shows that 4,5-CQME plays a role by inhibiting activation of an NF-κB signal pathway, and down-regulating expression of HBx, HNF1α, HNF4α and PGC1α and the like. In-vivo research shows that 4,5-CQME can inhibit replication of HBV. Therefore, 4,5-CQME shows anti-HBV activity in vitro and in vivo, and provides a new choice for development of an anti-hepatitis B virus candidate drug.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry and antiviral drug technology, and relates to the application of methyl 4,5-di-O-caffeoylquinic acid ester, specifically to the application of methyl 4,5-di-O-caffeoylquinic acid ester in the preparation of anti-hepatitis B virus drugs. Background Technology

[0002] Chronic hepatitis B virus (HBV) infection is a prominent global public health problem. According to the World Health Organization, approximately 254 million people worldwide are chronically infected with HBV. Chronic infection can gradually progress to liver failure, cirrhosis, and hepatocellular carcinoma, and about one million people die from HBV-related liver diseases each year. HBV replication not only depends on the virus's own proteins but also requires the participation of HBx proteins and host transcription factors such as HNF1α, HNF4α, and PGC1α. Activation of the NF-κB signaling pathway also accelerates viral replication and liver disease progression. Targeting these host molecules is an important strategy for developing anti-HBV drugs.

[0003] Currently, first-line anti-HBV treatments primarily utilize nucleoside (acid) polymerase inhibitors, with representative drugs including entecavir and lamivudine. These drugs effectively reduce viral load by inhibiting viral polymerase; however, they only target viral replication and cannot completely eliminate the virus, requiring long-term or even lifelong medication. Long-term use can easily induce drug-resistant mutants in HBV, leading to viral rebound and disease exacerbation. Therefore, developing anti-HBV candidate drugs with mechanisms of action distinct from nucleoside analogs and derived from natural products can address the shortcomings of existing treatment options.

[0004] Honeysuckle (Lonicera japonica Thunb.), also known as golden honeysuckle, is traditionally used in clinical practice for antiviral treatments, with caffeoylquinic acid derivatives being its main active substances. Current research confirms that some caffeoylquinic acid components possess anti-HBV activity, but compounds with different substitution structures exhibit vastly different activities; for example, methyl 3,4,5-tri-O-caffeoylquinate, belonging to the same family, can actually promote HBV replication. To date, the anti-HBV activity and mechanism of action of methyl 4,5-di-O-caffeoylquinate from honeysuckle remain unclear, hindering the development and utilization of this natural compound in the field of anti-hepatitis B drugs. Summary of the Invention

[0005] The purpose of this invention is to provide the application of methyl 4,5-di-O-caffeoylquinic acid in the preparation of anti-hepatitis B virus drugs. The anti-HBV potential of 4,5-CQME derived from honeysuckle was evaluated by in vitro experiments, aiming to provide a preliminary experimental basis for further elucidating the antiviral mechanism of quinic acid and its derivatives and exploring potential anti-hepatitis B virus drugs.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention provides the application of methyl 4,5-di-O-caffeoylquinic acid ester in the preparation of anti-hepatitis B virus drugs.

[0007] Preferably, the methyl 4,5-di-O-caffeoylquinic acid ester exerts its anti-hepatitis B virus activity by inhibiting the activation of the NF-κB signaling pathway and downregulating the expression of HBx, HNF1α, HNF4α, and PGC1α proteins.

[0008] Preferably, the methyl 4,5-di-O-caffeoylquinic acid is derived from honeysuckle flower buds.

[0009] Preferably, the active ingredient of the drug is methyl 4,5-di-O-caffeoylquinic acid.

[0010] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0011] Preferably, the pharmaceutically acceptable excipient is selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and controlled-release materials.

[0012] Preferably, the drug includes various acceptable dosage forms.

[0013] Preferably, the dosage form of the drug is any one of capsules, granules, tablets, powders, ointments, powders, pills, or liquids.

[0014] The beneficial effects of this invention are: 1. This invention discloses the medicinal use of methyl 4,5-di-O-caffeoylquinic acid ester against hepatitis B virus. This compound is derived from the flower buds of honeysuckle (Lonicera japonica), a traditional Chinese medicine. The raw material is readily available, and the extraction and purification process is mature, yielding a monomer with a purity of over 95%. In vitro cell experiments show that it has no significant toxicity to HepG2.2.15 hepatocytes at effective dosage concentrations. In vivo animal experiments show no significant damage to major organs such as the heart, liver, spleen, lungs, and kidneys, indicating good safety profile.

[0015] 2. The methyl 4,5-di-O-caffeoylquinate provided by this invention can dose-dependently inhibit HBV DNA replication and RNA transcription, and downregulate the expression of HBsAg, HBeAg, and HBxAg viral antigens. In an in vivo duck hepatitis B virus model, it can inhibit viral replication and reduce virus-induced liver tissue damage, exhibiting both antiviral and hepatoprotective effects. Its mechanism of action differs from nucleoside analogs; it does not target viral polymerase but rather exerts its effect by inhibiting NF-κB signaling pathway activation and downregulating host transcription factors such as HBx, HNF1α, HNF4α, and PGC1α. This mechanism is independent of the PI3K / AKT / mTOR / p70S6K pathway, providing a target for anti-HBV drugs. Attached Figure Description

[0016] Figure 1 This invention presents the chemical and biological characterization of 4,5-CQME (A: HPLC analysis chromatogram of 4,5-CQME; B: Chemical structure of 4,5-CQME; C: Effect of 4,5-CQME on HepG2.2.15 cell viability; D: Cytotoxicity of 4,5-CQME on HepG2.2.15 cells; Data are expressed as mean ± standard deviation; Compared with the control group, ** p <0.01, *** p <0.001); Figure 2 This invention describes the effects of 4,5-CQME on in vitro HBV replication (AC: effects of 4,5-CQME on secreted HBsAg (A), HBeAg (B), and HBV DNA (C) in the supernatant of HepG2.2.15 cells; DG: effects of 4,5-CQME on HBsAg expression (D), HBeAg expression (E), HBV DNA replication (F), and pgRNA transcription (G) in HepG2.2.15 cells; H and I: effects of 4,5-CQME on the protein expression levels of HBsAg, HBcAg, and HBx in HepG2.2.15 cells; data are expressed as mean ± standard deviation; compared with the control group, * p <0.05,** p <0.01, *** p <0.001); Figure 3 This invention describes the mechanism by which 4,5-CQME inhibits HBV replication (AD: Effects of 4,5-CQME on the activation of the PI3K / AKT / mTOR signaling pathway (A), MAPK signaling pathway (B), TAK1 / NF-κB signaling pathway (CD), and the expression levels of HBV-related transcription factors (CD) in HepG2.2.15 cells; data are expressed as mean ± standard deviation; compared with the model group, * p <0.05,**p <0.01); Figure 4 This invention relates to the effects of 4,5-CQME on HBV replication in vivo (A and B: effects of 4,5-CQME treatment on liver body index (HSI) (A) and DHBV viral titer (B); C: H&E staining showing the therapeutic effect of 4,5-CQME on liver damage caused by HBV replication; D: H&E staining showing that 4,5-CQME has no significant toxicity to other normal tissues; data are expressed as mean ± standard error; compared with the control group, ### p <0.001; compared to the model group, ** p <0.01, *** p <0.001); Figure 5 This is a schematic diagram illustrating how 4,5-CQME inhibits HBV replication by downregulating HBV-related transcription factors in this invention. Detailed Implementation

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0018] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] The materials used in the following embodiments are as follows: Antibodies: Anti-hepatitis B surface antigen (HBsAg) (sc-53299), anti-hepatitis B core antigen (HBcAg) (sc-23947), and anti-hepatitis B virus X protein (HBx) (sc-57760) antibodies were purchased from Santa Cruz Biotechnology (Dallas, USA). Anti-human PI3K (42570), AKT (4691), phosphorylated Ser473-AKT (4060), mTOR (2972), phosphorylated Ser2481-mTOR (2974), p70 S6K (2708), phosphorylated Thr389-p70 S6K (9205), ERK1 / 2 (4695), phosphorylated Thr202 / Tyr204-ERK1 / 2 (4370), TAK1 (5206), phosphorylated Thr184 / 187-TAK1 (4531), p65 (8242), phosphorylated Ser536-NF-κB The p65 (3033), JNK (9252), phosphorylated Thr183 / Tyr185-JNK (9255), p38 (8690), phosphorylated Thr180 / Tyr182-p38 MAPK (4511), HNF1α (89670), HNF4α (3113), and PGC1α (2178) antibodies were purchased from Cell Signaling Technology (Danvers University, Massachusetts, USA). The anti-human GAPDH (60004-1-Ig) antibody was purchased from Proteintech (Wuhan, China). The horseradish peroxidase (HRP)-labeled secondary antibodies, anti-mouse IgG (7076S) and anti-rabbit IgG (7074S), were purchased from Cell Signaling Technology.

[0021] Reagents: Cell Counting Kit-8 (Apexbio, K1018) was purchased from APE×BIO (Shanghai, China). Cytotoxicity assay kit (G1780) was purchased from Promega (Madison, USA). Enzyme-linked immunosorbent assay (ELISA) kits, including ARCHITECT Hepatitis B e antigen detection kit (6C32) and ARCHITECT Hepatitis B surface antigen detection kit (6C36), were purchased from Abbott Laboratories (Chicago, USA). Quantitative fluorescence diagnostic kits for Hepatitis B virus DNA and Hepatitis B virus RNA were purchased from Sansure Biotech (Changsha, China). EASYspin rapid RNA extraction kit for tissues / cells (RN07) was purchased from Adelaide (Beijing, China). HiFi-MMLV cDNA synthesis kit (CW0744M) was purchased from CWBiotech (Beijing, China). Forget-Me-Not™ EvaGreen® real-time quantitative PCR (qPCR) premix (31042-1) was purchased from Biotium (Hayward, USA).

[0022] Example 1: Isolation of methyl 4,5-di-O-caffeoylquinate (4,5-CQME) 1. Separation and extraction 1.1 Raw Materials and Extraction Take 6.5 kg of dried honeysuckle (Lonicera japonica Thunb.) flower buds, add 50 L of 75% ethanol aqueous solution, and soak and extract 3 times at room temperature; combine all extracts, evaporate and concentrate under reduced pressure to remove ethanol, and obtain 1.5 kg of ethanol extract.

[0023] 1.5 kg of the ethanol extract was suspended and dispersed in water, and then fractionally extracted with cyclohexane, ethyl acetate, and n-butanol. The ethyl acetate extract phase was collected to obtain 83 g of ethyl acetate extract.

[0024] 1.2 Column Chromatography Coarse Separation 83 g of the ethyl acetate extract was loaded onto a silica gel column for chromatography (column size 5 cm × 45 cm, 100-200 mesh). The elution system was a dichloromethane-methanol gradient elution (volume ratio 100:1 to 1:0). The eluent was collected in fractions to obtain 20 fractions Fr.1 to Fr.20. 37.0 g of the Fr.16 fraction was used for further purification.

[0025] 1.3 ODS Inverted Column Subdivision Fr.16 (37.0 g) was loaded onto an ODS reversed-phase column (5.5 cm × 28 cm) and eluted with a methanol-water gradient (volume ratio 10:90 to 100:0) to obtain 9 subfractions Fr.16-1 to Fr.16-9. The target monomer was prepared by selecting the Fr.16-5 fraction.

[0026] 1.4 Preparative HPLC Purification Chromatographic column: Cosmosil 5C18-MS-II (5 μm, 20 mm × 250 mm); mobile phase: methanol-water = 40:60 (v / v); flow rate: 8 mL / min; detection wavelength: 330 nm; Fr.16-5 sample solution was injected, and the chromatographic peak fraction with retention time tR = 59.2 min was collected. The eluents were combined and dried under reduced pressure to obtain 500 mg of pale yellow powder 4,5-CQME. Approximately 2 g of 4,5-CQME was obtained from the total purified Fr.16 fraction. In addition, 3-CQA, 3-CQME, and 4,5-CQA were also isolated from Fr.16. The chemical structures of each compound were determined by... 1 H NMR and 13 Confirmed by ¹³C NMR, and determined by HPLC analysis, the purity was higher than 95%.

[0027] 2. Structural identification and purity testing use 1 H NMR and 13 The chemical structure was confirmed by C18 NMR; the molecular formula of 4,5-CQME is C18. 26 H 26 O 12 The molecular weight was 530.48. Purity was determined by analytical HPLC. Using methanol-water (40:60, v / v) as the eluent, the fraction was purified by preparative HPLC (Cosmosil 5C18-MS-II, 5 μm, 20×250 mm, flow rate 8 mL / min, detection wavelength 330 nm) to obtain 4,5-CQME (retention time tR: 9.8 min), with a purity greater than 95%. Figure 1 China A- Figure 1 (Middle B). Approximately 2 g of 4,5-CQME was purified from 6.5 kg of honeysuckle, and the 4,5-CQME content in the honeysuckle raw material was approximately 0.31 g / kg of dried flower buds.

[0028] Example 2: Screening experiment on in vitro cytotoxicity and safe dosing concentration of 4,5-CQME 1. Experimental materials and cells Cell line: HepG2.2.15 (cell bank); Complete culture medium: DMEM basal medium + 10% fetal bovine serum + 100 U / mL penicillin + 100 μg / mL streptomycin; Culture conditions: 37 ℃, 5% CO2 saturated humidity incubator.

[0029] Detection kits: CCK-8 cell viability kit, LDH lactate dehydrogenase cytotoxicity kit.

[0030] 2. Cell viability assay The cells were diluted with culture medium to a concentration of 5 × 10⁻⁶. 4 Single-cell suspensions of 100 μL / mL were seeded into each well of a 96-well plate. After culturing in a CO2 incubator for 24 h, DMEM medium containing different concentrations (0, 20, 50, 100, 200, 400 μg / mL) of 4,5-CQME was added, and blank wells were also included. After culturing for another 72 h, the medium was removed, and 100 μL of medium containing 10% CCK-8 working solution was added to each well. The culture was terminated by incubation at 37 ℃ in the dark for 2 h. After shaking, the OD value was measured at 490 nm, the results were recorded, and cell viability was calculated. The experiment was repeated three times, and the average value was taken.

[0031] Cell viability (%) = [(OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%.

[0032] 3. Cytotoxicity test Cell culture and drug administration were performed as described above. After 72 h of culture, an equal volume of untreated cells was retained in the control wells, followed by the addition of 10 μL of lysis buffer. The cells were incubated at 37 °C for 10 min, and this lysis buffer was transferred to a 96-well plate as a control for maximum lactate dehydrogenase (LDH) release. Culture medium was taken from the blank wells as a control for spontaneous LDH release. 50 μL of culture medium was transferred from all test wells to a new 96-well plate. 50 μL of substrate mixture was added to each well, and the plates were incubated at 37 °C in the dark for 10 min. Then, 50 μL of stop solution was added, and the absorbance at 450 nm was recorded. The experiment was repeated three times, and the average value was taken.

[0033] Cytotoxicity (%) = [(OD value of experimental group - OD value of spontaneous release group) / (OD value of maximum release group - OD value of spontaneous release group)] × 100%.

[0034] 4. Results Analysis The results showed that when the concentration was below 100 μg / mL, 4,5-CQME had no significant effect on the viability of HepG2.2.15 cells; however, when the concentration was above 100 μg / mL, it began to significantly inhibit the viability of HepG2.2.15 cells. Figure 1(C). The results of the cytotoxicity assay were consistent with those of the CCK-8 assay: at concentrations below 100 μg / mL, 4,5-CQME did not produce significant cytotoxicity in HepG2.2.15 cells; however, at concentrations above 100 μg / mL, significant cytotoxicity began to occur. Figure 1 (D). Therefore, in subsequent experiments, the concentration of 4,5-CQME did not exceed 100 μg / mL.

[0035] Example 3: Evaluation and Mechanism Analysis of the In Vitro Inhibitory Activity of 4,5-CQME on HBV Replication 1. Grouping and Dosing Cells diluted to 2×10 4 Cells / mL were seeded into 24-well culture plates at 1 mL per well. After adhesion, the cells were injected with 4,5-CQME at different concentrations: 0 (blank control), 20, 50, and 100 μg / mL. The cells were cultured for 72 h. The cell culture supernatant and cell pellet were collected separately.

[0036] ELISA detection of HBsAg and HBeAg secretion levels The ARCHITECT series HBsAg (hepatitis B surface antigen (6C36)) and HBeAg (hepatitis B e antigen (6C32)) quantitative ELISA kits were used to detect the content of the two antigens in the supernatant and cell lysate, and the S / CO value was recorded to evaluate the inhibitory effect on viral protein secretion.

[0037] qPCR quantification of HBV DNA and RNA levels Sansure Biotech's HBV DNA and HBV RNA Real-Time PCR Kit extracts total nucleic acids from cells, reverse transcribes (RNA sample), and then quantifies them using real-time fluorescence PCR to detect HBV genomic DNA and RNA levels.

[0038] 4. Western blot method Cells were lysed using RIPA lysis buffer after treatment with 4,5-CQME. After separation by SDS-PAGE electrophoresis, cells were transferred to a PVDF membrane. The membrane was blocked in blocking buffer (5% BSA) at room temperature for 1 h, followed by overnight incubation with primary antibody at 4 °C. After washing three times with TBST solution, the membrane was incubated with secondary antibody at room temperature for 1 h, then developed using ECL chemiluminescence solution and exposed using a chemiluminescence imaging system.

[0039] 5. Results Analysis Experimental results showed that 4,5-CQME significantly inhibited the levels of HBsAg, HBeAg, and HBV DNA secreted by HepG2.2.15 cells in a dose-dependent manner. Figure 2 China A- Figure 2(C). Similar results were also observed in HepG2.2.15 cells regarding HBsAg, HBeAg, and HBV DNA levels. Figure 2 D- Figure 2 (F). Furthermore, these observations were validated in HepG2.2.15 cells using Western blotting. HBV pregenomic RNA (pgRNA) is a direct transcription product of the HBV viral genome and reflects viral activity. The results showed that 4,5-CQME significantly inhibited HBV pgRNA levels (F). Figure 2 These results were also confirmed by Western blotting in HepG2.2.15 cells (G). Figure 2 H- Figure 2 (I). In summary, 4,5-CQME can inhibit HBV replication in vitro.

[0040] To verify the effect of 4,5-CQME treatment on the AKT / mTOR / p70S6K signaling pathway, this invention also examined the expression of PI3K and the phosphorylation levels of AKT, mTOR, and p70S6K in HepG2.2.15 cells. The results showed that 4,5-CQME treatment had no significant effect on PI3K expression or AKT / mTOR / p70S6K phosphorylation levels. Figure 3 (A). Furthermore, 4,5-CQME had no effect on the expression of key proteins in the MAPK pathway, which also regulates HBV replication. Figure 3 (B)

[0041] To verify the effect of 4,5-CQME treatment on NF-κB signaling pathway activation, this invention examined the expression level of phosphorylated NF-κB p65 subunits. The results showed that 4,5-CQME treatment significantly inhibited NF-κB phosphorylation (…). Figure 3 C- Figure 3 (D). HNF1α and HNF4α belong to the hepatocyte nuclear factor (HNF) family and play an important role in the regulation of HBV replication. Inhibition of PGC1α has been shown to suppress HBV replication. The above results indicate that 4,5-CQME treatment can downregulate the expression of HNF1α, HNF4α, and PGC1α.

[0042] In summary, 4,5-CQME may exert its anti-HBV activity by inhibiting the NF-κB pathway and the expression of HNF1α, HNF4α and PGC1α.

[0043] Example 4 In vivo experiment 1. Modeling Thirty-two 1-day-old male Guangzhou ducks (purchased from Guangzhou Jiangcun Duck Farm) were used; 24 ducklings were injected intravenously with 0.2 mL of DHBV-positive serum to establish the DHBV model, and were infected for 2 weeks; the remaining 8 ducks were injected with physiological saline as a blank control group.

[0044] Two weeks after infection, the model ducklings were randomly divided into 3 groups of 8 ducklings each: Model group: administered physiological saline by gavage; Positive control group: Entecavir 10 mg / kg by gavage; 4,5-CQME experimental group: 40 mg / kg by gavage; Administer once daily for 14 consecutive days, with free access to food and water.

[0045] 2. Sample collection and testing After 14 days of continuous administration, the ducklings were weighed. Each group of ducklings was euthanized by electric shock followed by decapitation or cervical dislocation. Organs were harvested and immediately flash-frozen in liquid nitrogen. Blood samples were collected via the carotid artery, serum was separated, and stored at -70 °C for later analysis. Duck livers were weighed. Simultaneously, the heart, liver, spleen, lungs, kidneys, and duodenum were collected for HE staining.

[0046] 3. H&E staining Tissues and organs were fixed with paraformaldehyde, embedded in paraffin, and cut into 5 μm thick sections. The sections were placed on glass slides and dewaxed sequentially with xylene and ethanol, followed by staining with Mayer hematoxylin and 0.2% eosin or Sirius red. The stained sections were imaged using an Olympus VS200 image acquisition system.

[0047] 4. Results Analysis The liver body index (HSI), which is the ratio of liver weight to body weight, is commonly used to reflect the liver's regenerative capacity and the degree of liver damage. Experimental results show ( Figure 4 (A) Short-term DHBV infection and drug treatment did not significantly alter the liver index in ducks. Furthermore, 4,5-CQME treatment reduced the DHBV content in the blood of Muscovy ducks. Figure 4 (See Figure B), indicating that 4,5-CQME also possesses certain anti-HBV replication activity in vivo. This invention further investigated whether 4,5-CQME causes damage to tissues and organs using HE staining; the results showed that 4,5-CQME has a certain protective effect against HBV-induced liver damage. Figure 4 (C). At a dose of 40 mg / kg body weight, 4,5-CQME did not cause significant damage to major tissues and organs. Figure 4 (D). The above results indicate that 4,5-CQME can inhibit DHBV replication in vivo without causing significant tissue damage.

[0048] In summary, 4,5-CQME is a natural caffeoylquinic acid compound purified from honeysuckle buds. 4,5-CQME significantly inhibits HBV replication both in vitro and in vivo, with minimal impact on hepatocytes. The mechanism suggests that 4,5-CQME treatment reduces NF-κB phosphorylation levels and the expression of HNF1α, HNF4α, and PGC1α. This invention confirms that the caffeoylquinic acid compound 4,5-CQME inhibits HBV replication by suppressing NF-κB phosphorylation and the expression of HNF1α, HNF4α, and PGC1α. Figure 5 Therefore, 4,5-CQME may have development value as a potential anti-HBV treatment.

[0049] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

Application of methyl 1,4,5-di-O-caffeoylquinic acid in the preparation of anti-hepatitis B virus drugs.

2. The application according to claim 1, characterized in that, The methyl 4,5-di-O-caffeoylquinic acid ester exerts its anti-hepatitis B virus activity by inhibiting the activation of the NF-κB signaling pathway and downregulating the expression of HBx, HNF1α, HNF4α, and PGC1α proteins.

3. The application according to claim 1, characterized in that, The methyl 4,5-di-O-caffeoylquinic acid ester is derived from honeysuckle flower buds.

4. The application according to claim 1, characterized in that, The active ingredient of the drug is methyl 4,5-di-O-caffeoylquinic acid.

5. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

7. The application according to claim 1, characterized in that, The drug includes various acceptable dosage forms.

8. The application according to claim 7, characterized in that, The dosage form of the drug is any one of capsules, granules, tablets, powders, ointments, powders, pills, or liquids.

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