Use of compound PBL22 in the preparation of a drug for preventing and treating myocardial ischemia-reperfusion injury

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to application of a compound PBL22 in preparation of a drug for preventing and treating myocardial ischemia-reperfusion injury. The compound PBL22 from a plant source of Pipturus argus is found to be capable of effectively improving myocardial function after myocardial ischemia-reperfusion, reducing myocardial infarction area, reducing myocardial cell apoptosis, inhibiting release of inflammatory factors and oxidative stress response, has a clear mechanism of action, has a significant curative effect, and can be used for preventing, relieving or treating myocardial ischemia-reperfusion injury, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a new application of PBL22, a natural compound derived from Trichosanthes kirilowii leaves, and particularly to the application of compound PBL22 in the preparation of drugs for the prevention, relief or treatment of myocardial ischemia-reperfusion injury. Background Technology

[0002] In the development of ischemic heart disease, MI / R injury (myocardial ischemia / reperfusion injury) is a primary aggravation of myocardial damage after hemodynamic recovery. Its pathogenesis involves the interaction of multiple cell types and molecular pathways. Although extensive research has been conducted, its specific molecular mechanisms are not yet fully elucidated. MI / R injury typically originates from extreme metabolic disturbances in cardiomyocytes during the ischemic phase. Under the stimulation of hypoxia, malnutrition, and acidosis caused by coronary artery obstruction, cardiomyocyte mitochondrial function undergoes primary impairment, leading to ATP production depletion and ion homeostasis imbalance. This metabolic crisis makes cardiomyocytes extremely sensitive to subsequent reperfusion attacks, impairing barrier function and self-repair capabilities.

[0003] Traditional Chinese medicine and natural plant extracts have gained widespread attention in the treatment of cardiovascular diseases due to their advantages such as natural sources, fewer side effects, and synergistic effects targeting multiple targets. Piper betle L. (PBL), a medicinal and edible plant native to Hainan, contains abundant active components such as flavonoids, phenols, and alkaloids. Its extracts play a crucial role in regulating redox balance, inhibiting inflammation, and protecting organ function. In various pathological processes, the active substances in Piper betle leaf exhibit significant pharmacological activity. Its antioxidant properties stem from its direct ability to scavenge free radicals and activate endogenous antioxidant enzyme systems, which has sparked extensive research by scholars on its application in the prevention and treatment of degenerative and metabolic diseases.

[0004] To date, there have been no reports on the use of PBL22, a naturally active compound extracted from PBL, for the prevention, relief, or treatment of myocardial ischemia-reperfusion injury. This invention, based on in vivo pharmacodynamic studies of compound PBL22, reveals that it possesses significant anti-myocardial ischemia-reperfusion injury activity, effectively improving myocardial function after myocardial ischemia-reperfusion, reducing infarct size, decreasing the levels of necrosis markers in mouse peripheral serum, and inhibiting inflammatory responses. Summary of the Invention

[0005] The purpose of this invention is to provide the application of compound PBL22 in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury. This compound has significant anti-myocardial ischemia-reperfusion injury activity, can effectively improve myocardial function after myocardial ischemia-reperfusion, reduce myocardial infarction area, reduce the content of necrosis markers in mouse peripheral serum, and inhibit inflammatory response.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: The first aspect of this invention is to provide the use of compound PBL22 in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury, the chemical structure of compound PBL22 being shown in Formula I below: .

[0007] A second aspect of the present invention is to provide a method for preparing the compound PBL22, comprising the following steps: Step S1: After crushing the dried betel leaf, extract it three times with 95% ethanol solution, filter, collect the filtrate, concentrate and dry under reduced pressure to obtain ethanol extract. Step S2: Add distilled water to the ethanol extract to make a suspension, and extract it three times each with petroleum ether and ethyl acetate. Concentrate the ethyl acetate extract under reduced pressure to obtain ethyl acetate extract paste. Step S3: The ethyl acetate extract was purified by column chromatography to obtain the monomer compound PBL22.

[0008] In one optional implementation, step S3 is specifically as follows: (1) The ethyl acetate extract was subjected to MCI column chromatography with gradient elution using 30-100 vol% methanol-water solution as the eluent. The fractions Fr.a (30-60 vol% methanol-water solution), Fr.b (61-70 vol% methanol-water solution), Fr.c (71-80 vol% methanol-water solution), and Fr.d (81-90 vol% methanol-water solution) were collected. (2) The obtained fraction Fr.c was purified by ODS column elution with 40-90 vol% methanol-water solution as the eluent, and 70-90 vol% methanol-water solution was collected to obtain fraction Fr.c1; (3) The obtained fraction Fr.c1 was separated and purified by semi-preparative HPLC. 40 vol% acetonitrile-water solution was used as the eluent and the eluent was eluted at a flow rate of 2 ml / min. The eluent with a retention time of 26 to 30 min was collected and dried to obtain the monomer compound PBL22.

[0009] In one alternative implementation, a pharmaceutically acceptable carrier or excipient is also included.

[0010] The present invention has the following beneficial effects: 1. The compound PBL22 derived from Trichosanthes kirilowii leaves discovered in this invention can effectively improve myocardial function after myocardial ischemia-reperfusion, reduce myocardial infarction area, reduce myocardial cell apoptosis, inhibit the release of inflammatory factors and oxidative stress response. Its mechanism of action is clear and its efficacy is significant. It can be used to prevent, alleviate or treat myocardial ischemia-reperfusion injury and has broad application prospects.

[0011] 2. This invention is the first to discover that PBL22, a natural compound derived from Trichosanthes kirilowii leaves, has significant anti-myocardial ischemia-reperfusion injury activity, which expands the pharmaceutical applications of compound PBL22, fills the gap in the application of compound PBL22 in this field in the prior art, and provides a new drug candidate for the treatment of myocardial ischemia-reperfusion injury.

[0012] 3. The compound PBL22 of this invention is derived from natural plants. The extraction process is simple, the cost is low, and the sources are wide. Moreover, natural compounds usually have fewer side effects and better biocompatibility. Compared with chemically synthesized drugs, they have higher safety and clinical application potential. Attached Figure Description

[0013] Figure 1 The compound PBL22 of this invention 1 H-hydrogen nuclear magnetic resonance spectrum of CDCl3 (400 MHz).

[0014] Figure 2 The compound PBL22 of this invention 13 C nuclear magnetic resonance spectrum of CDCl3 (100 MHz).

[0015] Figure 3 This invention relates to the effect of compound PBL22 on the activity of cardiomyocytes in a mouse model of myocardial ischemia-reperfusion injury.

[0016] Figure 4 The effect of compound PBL22 of this invention on the release of lactate dehydrogenase (LDH) in cardiomyocytes of a mouse model of myocardial ischemia-reperfusion injury.

[0017] Figure 5 This invention relates to the effect of compound PBL22 on the level of reactive oxygen species (ROS) in cardiomyocytes of a mouse model of myocardial ischemia-reperfusion injury.

[0018] Figure 6 The effect of compound PBL22 of this invention on the RNA levels of inflammatory factors (TNF-α, IL-6, IL-1β) in cardiomyocytes of mice with myocardial ischemia-reperfusion injury model.

[0019] Figure 7 This invention relates to the evaluation of the area of ​​cardiac necrosis in a mouse model of myocardial ischemia-reperfusion injury using the compound PBL22.

[0020] Figure 8 This invention is used to evaluate the cardiac ejection fraction (EF) and cardiac short-axis shortening fraction (FS) of compound PBL22 in a mouse model of myocardial ischemia-reperfusion injury.

[0021] Figure 9 This is an evaluation of heart slices (HE staining) of a mouse model of myocardial ischemia-reperfusion injury using the compound PBL22 of this invention. Detailed Implementation

[0022] The present invention will be further illustrated below with reference to specific embodiments, but these 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. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0023] I. Extraction and Identification of Compound PBL22 (a) Extraction of compound PBL22 Step 1: Crush 2 kg of dried betel leaf leaves and extract them three times with 10 times the amount of 95% ethanol at room temperature. The first extraction takes 14 days, and the second and third extractions take 7 days each. Filter the mixture, combine the filtrates, recover the ethanol under reduced pressure, and concentrate the remaining liquid to a thick paste to obtain 94.5 g of betel leaf ethanol extract.

[0024] Step 2: Dissolve (suspend) the ethanol extract of Trichosanthes kirilowii leaves obtained in step (1) in an appropriate amount of water, and then extract it three times with 4 times the volume of petroleum ether to obtain an organic phase and an aqueous phase. Combine the organic phases to obtain a petroleum ether extract. Combine the obtained aqueous phases and extract them three times with 4 times the volume of ethyl acetate. Combine the organic phases to obtain an ethyl acetate extract. Evaporate the obtained petroleum ether extract and ethyl acetate extract under reduced pressure to obtain 15.5g of ethyl acetate extract and 35g of petroleum ether extract, respectively.

[0025] Step 3: The ethyl acetate extract was purified by column chromatography, as follows: (1) The ethyl acetate extract was subjected to MCI column chromatography with gradient elution using 30-100 vol% methanol-water solution as the eluent. The fractions Fr.a (30-60 vol% methanol-water solution), Fr.b (61-70 vol% methanol-water solution), Fr.c (71-80 vol% methanol-water solution), and Fr.d (81-90 vol% methanol-water solution) were collected. (2) The obtained fraction Fr.c was purified by ODS column elution with 40-90 vol% methanol-water solution as the eluent, and 70-90 vol% methanol-water solution was collected to obtain fraction Fr.c1; (3) The obtained fraction Fr.c1 was separated and purified by semi-preparative HPLC. 40 vol% acetonitrile-water solution was used as the eluent and the eluent was eluted at a flow rate of 2 ml / min. The eluent with a retention time of 27.8 min was collected and dried to obtain the monomer compound, which was named PBL22.

[0026] (II) Identification of compound PBL22 1 H hydrogen nuclear magnetic resonance spectrum and 13 The C-NMR spectra are as follows: Figure 1 and Figure 2 As shown, via Figure 1 and Figure 2 Structural analysis revealed that the molecular formula of compound PBL22 is C2. 19 H 20 O4, the structure is shown below.

[0027] .

[0028] II. Cellular experimental efficacy study of compound PBL22 against myocardial ischemia-reperfusion injury (a) Cell viability test for myocardial ischemia-reperfusion injury SPF-grade suckling mice aged 1-3 days were selected. After sterilization with 75% ethanol, the hearts were aseptically harvested through chest opening. The mice were immediately placed in an ice bath and repeatedly rinsed in sterile PBS to remove residual blood. The atria and major blood vessels were removed, while the ventricles were preserved and cut into pieces of approximately 1 mm³. A digestion solution of equal volumes of 0.06% trypsin and 0.1% type II collagenase was used, and the cells were digested repeatedly with short-term shaking at 37 °C. After each digestion, the supernatant was collected and the enzymatic digestion was terminated promptly with serum-containing medium. All cell suspensions were combined, filtered through a 70 μm cell sieve, centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in complete medium. Subsequently, the differential adhesion method was used for purification. The cells were pre-adhered in a 37 °C, 5% CO2 incubator for 60-90 min. The non-adhered cardiomyocyte suspension was collected and seeded into gelatin-coated culture plates. Medium containing 5-bromodeoxyuridine (BrdU) was added to inhibit the proliferation of non-cardiomyocytes. High-purity, high-activity primary suckling mouse cardiomyocytes were obtained through routine culture.

[0029] Cells cultured to a suitable condition were divided into 10 cells per well. 5Cells were seeded at high density in 96-well plates and cultured until adherence. Then, different concentrations of PBL22 (2, 5, 10 μM) were added for pre-incubation for 2 h. Next, 200 μmol / L H2O2 was added to each well for 4 h to simulate oxidative damage. After 4 h, the medium was replaced with fresh medium containing different concentrations of PBL22 for 24 h to simulate reperfusion injury (establishing a myocardial ischemia-reperfusion injury model). At the end of the experiment, 10 μL of CCK-8 assay reagent was added, the plate was gently shaken to mix, and the plates were incubated at 37 ℃ in a 5% CO2 incubator for 1 h in the dark. Wells containing only culture medium and CCK-8 without cells served as blank controls. After incubation, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The relative cell viability value can be calculated using the following formula:

[0030] The protective effect of compound PBL22 against H2O2-induced oxidative damage was evaluated by calculating relative cell viability values. Each group was divided into three replicates, and the average value was taken. Results are as follows: Figure 3 As shown, the results indicated that PBL22 significantly improved cell viability in the model group, suggesting that PBL22 has a protective effect against H2O2-induced oxidative damage, and this effect was dose-dependent.

[0031] (II) Testing the degree of cell damage in myocardial ischemia-reperfusion injury After establishing the model according to the method described in "Cell Viability Assay for Myocardial Ischemia-Reperfusion Injury" above, LDH release was detected. At the end of the experiment, cell culture supernatants from each experimental group and control group were collected, centrifuged at 1000×g for 10 min at 4 ℃ to remove cell debris, and the supernatant was mixed with the detection reagent according to the LDH detection kit instructions (C0017, Beyotime, China). The mixture was incubated at 37 ℃ in the dark for 15–20 min, and the absorbance (OD value) of each well was measured using a microplate reader at 450 nm. Calibration was performed using a blank control well, and LDH activity in the supernatant was calculated based on the standard curve. The degree of myocardial cell damage was assessed by comparing the differences in LDH activity between the experimental and control groups. Results are as follows: Figure 4 As shown (*P<0.05, **P<0.01, ***P<0.001 vsH2O2 model group).

[0032] from Figure 4 The results show that PBL22 reduces the release of LDH in cardiomyocytes, indicating that PBL22 has a significant protective effect against H2O2-induced cell necrosis in a dose-dependent manner.

[0033] (III) Detection of cellular ROS in myocardial ischemia-reperfusion injury After establishing the model according to the method described in "Cell Viability Assay for Myocardial Ischemia-Reperfusion Injury," ROS was detected using fluorescence microscopy. At the end of the experiment, the cell culture supernatant was discarded, and the cells were gently washed 2-3 times with sterile PBS. A suitable final concentration of the ROS fluorescent probe (S0033S, Beyotime, China) was added, and the cells were incubated at 37 ℃ in a 5% CO2 incubator for 20-30 min in the dark. After incubation, the cells were washed 2-3 times with PBS to remove unloaded probes. Subsequently, under a fluorescence microscope, appropriate excitation and emission wavelengths were selected (excitation wavelength approximately 488 nm, emission wavelength approximately 525 nm), and multiple fields of view were randomly selected for imaging. The results are as follows: Figure 5 As shown.

[0034] from Figure 5 As can be seen, H2O2 can induce ROS accumulation in cardiomyocytes, while PBL22 can reduce ROS levels in a dose-dependent manner.

[0035] (iv) Expression level test of inflammatory factors TNF-α, IL-1β and IL-6 in myocardial ischemia-reperfusion injury cells The expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in cardiomyocytes were detected by qPCR.

[0036] After establishing the model according to the method described in "Cell Viability Assay for Myocardial Ischemia-Reperfusion Injury" above, cardiomyocytes from each group were collected, and total RNA was extracted. Subsequently, the RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions (RK20429, ABclonal, China). qPCR was performed using the SYBR Green fluorescent dye method (RK21203, ABclonal, China), with β-actin as an internal reference gene. The relative expression levels of each inflammatory factor were calculated using the ΔΔCq method. The relative expression levels of each inflammatory factor are shown below. Figure 6 As shown (*P<0.05, **P<0.01, ***P<0.001 vs H2O2 model group). Figure 6 a represents the result of the expression level test of the inflammatory factor IL-6; Figure 6 b represents the result of the expression level test of the inflammatory factor IL-1β; Figure 6 c represents the result of the expression level test of the inflammatory factor TNF-α.

[0037] from Figure 6The results showed that the expression levels of all inflammatory factors in the control group (treated with H2O2 only) were higher than those in the blank group (treated without any treatment), indicating that myocardial ischemia-reperfusion successfully triggered the inflammatory response of the heart. In contrast, the expression levels of all inflammatory factors in the treatment group (H2O2 + compound PBL22) were lower than those in the control group (treated with H2O2 only), indicating that compound PBL22 inhibited the expression of inflammatory factors, and the anti-inflammatory effect of compound PBL22 increased with increasing dosage.

[0038] III. Animal experimental efficacy study of compound PBL22 against myocardial ischemia-reperfusion injury (I) Experimental Methods 1. Laboratory animals SPF-grade C57BL / 6 mice, male, weighing 20-25g, were purchased from Jiangsu Huano Chuangxin Pharmaceutical Technology Co., Ltd. The mice were acclimatized for one week with free access to food and water. The ambient temperature was 22-25℃, humidity 50%-60%, and a 12-hour light / dark cycle was maintained.

[0039] 2. Experimental Grouping Mice were randomly divided into 5 groups of 10 mice each: Sham surgery group: only open the chest, without ligating the coronary arteries, and administer an equal volume of normal saline by gavage after the operation; Model group (vehicle): A myocardial ischemia-reperfusion injury model was established, and an equal volume of normal saline was administered by gavage after the operation; Low-dose group of compound PBL22 (1.5 mg / kg): A myocardial ischemia-reperfusion injury model was established by intraperitoneal injection of compound PBL22 (1.5 mg / kg) for 3 consecutive days before surgery and on the day of reperfusion. Medium-dose group of compound PBL22 (5mg / kg): A myocardial ischemia-reperfusion injury model was established by intraperitoneal injection of compound PBL22 (5mg / kg) for 3 consecutive days before surgery and on the day of reperfusion. High-dose group of compound PBL22 (15 mg / kg): A myocardial ischemia-reperfusion injury model was established. Compound PBL22 (15 mg / kg) was administered intraperitoneally for 3 consecutive days before surgery and on the day of reperfusion.

[0040] 3. Establishment of a myocardial ischemia-reperfusion injury model Mice were anesthetized by intraperitoneal injection of 1.25% (20 mg / mL) solution, fixed supine on the operating table, with their necks shaved and disinfected. They were then intubated and connected to a ventilator, with respiratory parameters adjusted to a respiratory rate of 100-120 breaths / min and a tidal volume of 0.2-0.3 mL. The chest was shaved and disinfected. An incision was made in the left 4th-5th intercostal space, and the skin and muscles were dissected layer by layer to expose the pleural cavity. The pericardium was opened, exposing the left anterior descending coronary artery. The left anterior descending coronary artery was ligated with 6-0 silk suture 2-3 mm from the aortic root. After ligation, the color change of the mouse's myocardium was observed. Paleness and cyanosis of the myocardium indicated successful ischemia. After 60 minutes of sustained ischemia, the ligation was loosened to restore blood supply. The pleural cavity was closed and sutured layer by layer. Postoperatively, penicillin was administered to prevent infection, followed by reperfusion for 24 hours. In the sham surgery group, only the chest was opened and the coronary arteries were exposed; ligation was not performed.

[0041] (II) Detection Indicators and Results 1. Detection of myocardial infarction area Twenty-four hours after reperfusion, mice were anesthetized by intraperitoneal injection of 1.25% (20 mg / mL) and their hearts were perfused with 2% Evans Blue. Once the mouse veins turned blue, the heart was quickly removed, rinsed thoroughly with physiological saline, and the atria and great vessels were removed. The heart was transversely cut into five slices (each approximately 1 mm thick) and incubated in 2% triphenyltetrazolium chloride (TTC) solution at 37°C for 15 min, then stained in the dark. After staining, the slices were fixed with 4% paraformaldehyde for 10 min. Normal myocardial tissue appeared blue, normal myocardial tissue in high-risk areas appeared red, and myocardial tissue in infarcted areas appeared white. Each slice was photographed, and the total area and infarct area of ​​each myocardium were measured using Image-J software. The percentage of infarcted area to normal myocardial area in high-risk areas (IS / AAR) and the percentage of normal myocardial area in high-risk areas to total myocardial area (AAR / LV) were calculated. The results are as follows: Figure 7 As shown (*P<0.05, **P<0.01, ***P<0.001 vs H2O2 model group). Figure 7 a represents the results of stained heart sections; Figure 7 b represents the percentage of the infarcted area relative to the normal myocardial area in the danger zone (IS / AAR). Figure 7 c represents the percentage of normal myocardial area in the danger zone relative to the total myocardial area (AAR / LV).

[0042] from Figure 7 As shown in Figure a, there were more white areas in the heart slices of the model group, indicating a greater amount of myocardial tissue in the infarcted areas. After treatment with compound PBL22, the white areas decreased, and as the concentration of compound PBL22 increased, the white areas became smaller and smaller, showing a certain concentration dependence.

[0043] from Figure 7As shown in b, the infarct area in the model group accounted for 50% of the normal myocardial area in the danger zone. In the treatment group, the infarct area decreased with increasing dosage. At high dose (15 mg / kg), the proportion of the infarct area decreased to about 30%, indicating that compound PBL22 can significantly reduce the myocardial infarction area in mice with myocardial ischemia-reperfusion injury.

[0044] from Figure 7 As shown in c, the percentage of normal myocardial area in the dangerous region to the total myocardial area in the cardiac slices of the model group and the treatment group is consistent, indicating the consistency and stability of the model in all groups.

[0045] 2. Cardiac function test Twenty-four hours after reperfusion, cardiac function was assessed using ultrasound, and cardiac ejection fraction (EF) and fractional shortening (FS) were evaluated. Results were as follows: Figure 8 As shown (*P<0.05, **P<0.01, ***P<0.001 vs H2O2 model group). Figure 8 a represents echocardiography; Figure 8 b represents the cardiac ejection fraction (EF). Figure 8 c represents the fraction of cardiac short axis shortening (FS).

[0046] from Figure 8 b and Figure 8 As shown in Figure c, compared with the sham group, the EF and FS values ​​of the model group were significantly reduced (by about half), indicating that the ischemia-reperfusion operation successfully led to impaired cardiac contractile function, and the model was effectively constructed. Compared with the model group, the EF and FS values ​​of the treated mice were significantly increased, showing a certain dose-dependent effect. Combined with the results of the "myocardial infarction area" detection, this indicates that compound PBL22 has a cardioprotective effect.

[0047] 3. Detection of the degree of pathological damage to myocardial tissue Twenty-four hours after reperfusion, mouse hearts were fixed in 4% paraformaldehyde, routinely embedded in paraffin, and serially sectioned (5 μm). Sections were dewaxed to water, stained with hematoxylin for 5-10 min, differentiated with hydrochloric acid and ethanol, stained with eosin for 3-5 min, dehydrated and cleared, and then mounted. The morphological integrity of cardiomyocytes, the degree of inflammatory cell infiltration, and the disorder of fiber arrangement were observed under a light microscope. Results are as follows: Figure 9 As shown.

[0048] from Figure 9 The study showed that the damaged myocardial tissue in the model group had incomplete morphology and inflammatory cell infiltration. After PBL22 treatment, the myocardial tissue arrangement was significantly improved, and inflammatory cell infiltration was reduced. This indicates that PBL22 has a cardioprotective effect and inhibits myocardial inflammation.

[0049] 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. The application of compound PBL22 in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury, wherein the chemical structure of compound PBL22 is shown in Formula I below: 。 2. The application according to claim 1, characterized in that, The preparation method of the compound PBL22 includes the following steps: Step S1: After crushing the dried betel leaf, extract it three times with 95% ethanol solution, filter, collect the filtrate, concentrate and dry under reduced pressure to obtain ethanol extract. Step S2: Add distilled water to the ethanol extract to make a suspension, and extract it three times each with petroleum ether and ethyl acetate. Concentrate the ethyl acetate extract under reduced pressure to obtain ethyl acetate extract paste. Step S3: The ethyl acetate extract was purified by column chromatography to obtain the monomer compound PBL22.

3. The application according to claim 2, characterized in that, Step S3 is as follows: (1) The ethyl acetate extract was subjected to MCI column chromatography with gradient elution using 30-100 vol% methanol-water solution as the eluent. The fractions Fr.a (30-60 vol% methanol-water solution), Fr.b (61-70 vol% methanol-water solution), Fr.c (71-80 vol% methanol-water solution), and Fr.d (81-90 vol% methanol-water solution) were collected. (2) The obtained fraction Fr.c was purified by ODS column elution with 40-90 vol% methanol-water solution as the eluent, and 70-90 vol% methanol-water solution was collected to obtain fraction Fr.c1; (3) The obtained fraction Fr.c1 was separated and purified by semi-preparative HPLC. 40 vol% acetonitrile-water solution was used as the eluent and the eluent was eluted at a flow rate of 2 ml / min. The eluent with a retention time of 26 to 30 min was collected and dried to obtain the monomer compound PBL22.

4. The application according to claim 1, characterized in that, It also includes pharmaceutically acceptable carriers or excipients.

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