A iridoid for resisting myocardial cell injury extracted from brown bell flower tree, preparation method and application thereof

Abstract

A secoiridoid that can resist myocardial cell damage and is extracted from brown bellflower tree, with the molecular formula C 17 H 20 O7, is extracted, isolated and identified as avelladoid I (Avd I) from the water extract of the dried endothelium of brown bellflower tree. It can improve the viability of myocardial cells, reduce the LDH level in damaged myocardial cells, and reduce the abnormally elevated level of inflammatory factors in damaged cells, proving that Avd I can improve myocardial cell damage and relieve myocardial cell inflammatory damage, with significant social and economic benefits.

Description

Technical Field

[0001] The present invention relates to medicine, in particular to a secoiridoid for resisting myocardial cell injury extracted from brown bellflower tree, a preparation method and an application thereof. Background Art

[0002] According to the latest statistical data of the World Health Organization, cardiovascular diseases rank first among the main causes of global human death. The latest "Report on Cardiovascular Health and Diseases in China 2022" issued by the National Center for Cardiovascular Diseases pointed out that the prevalence of cardiovascular diseases in China is in a continuous upward stage, and the estimated number of current patients is 330 million. The heart is one of the most important organs in the human body. It promotes blood flow throughout the body, supplies oxygen, nutrients, hormones, etc. to tissues and organs, maintains the normal metabolism and function of cells, realizes the body's humoral regulation, and ensures the relative constancy of the body's internal environment. Cardiomyocytes are non-renewable cells. When the number of cardiomyocytes decreases to a certain extent, the collagen scaffold breaks, resulting in thinning of the ventricular wall and accompanied by local movement incoordination, leading to reduced cardiac function and eventually heart failure. Doxorubicin is an anti-cancer drug commonly used in the treatment of solid tumors such as breast cancer and liver cancer or hematological malignancies. Its advantages are a broad anti-tumor spectrum, high clinical efficacy, and effectiveness against hypoxic cells. However, it has toxic damage to the heart, and its mechanism may involve oxidative stress, mitochondrial damage, abnormal energy metabolism, calcium overload, apoptosis, autophagy, etc. The mechanism of myocardial injury is relatively complex. Current research mainly focuses on inflammation, oxidative stress, apoptosis, etc. These pathological processes affect and promote each other, and ultimately form a vicious cycle, resulting in apoptosis and necrosis of cells, leading to results such as cardiac tissue damage and dysfunction. In the case of damaged cardiomyocytes, it will lead to an increase in inflammatory factors, an increase in the level of lipid peroxidation, and the destruction of the stability and integrity of the cell membrane, ultimately resulting in cardiomyocyte apoptosis. Apoptosis is an effective strategy that cells are forced to adopt in order to better adapt to the surrounding living environment. It is a natural, non-inflammatory, energy-requiring, programmed form of cell death. It has been reported that cardiomyocyte apoptosis is involved in the occurrence and development of various heart diseases such as ischemia-reperfusion injury, heart failure, and myocardial infarction. Therefore, drugs with anti-cardiomyocyte apoptosis effects may be used to treat various heart diseases and have great development potential. Reactive oxygen species (ROS), a by-product generated during mitochondrial aerobic metabolism in the body, under physiological conditions, low concentrations of ROS in mitochondria control cardiomyocyte differentiation and excitation-contraction coupling, which is beneficial to cardiovascular function. However, excessive generation of ROS will in turn trigger the production of mitochondrial ROS, cause oxidative damage to proteins, lipids, and DNA, induce an increase in the level of MDA, a decrease in the levels of SOD and GSH-Px, trigger a decrease in mitochondrial transmembrane potential, release of cytochrome C, and activate a series of Caspase enzymes, inducing apoptosis of cells, and ultimately leading to impaired cardiac excitation-contraction coupling, arrhythmia, myocardial hypertrophy, apoptosis, necrosis, and fibrosis of cells. The endogenous mitochondrial apoptosis pathway of cells is triggered by intracellular stress such as oxidative stress, calcium overload, and DNA damage.Previous studies have shown that ROS induced by certain chemotherapeutic drugs can increase the Bax / Bcl-2 ratio, thereby participating in apoptosis via the mitochondrial pathway, while overexpression of the anti-apoptotic gene Bcl-2 can reduce the production of oxygen free radicals. In a normal organism, the production and clearance of ROS are in dynamic equilibrium; when the production of ROS increases or the clearance ability decreases, oxidative stress occurs. During oxidative stress, the levels of ROS and MDA reflect the level of free radical damage to cells; on the other hand, organisms have developed effective anti-ROS systems during evolution, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and antioxidant substances (such as vitamin C, vitamin E), etc. The final state of the cell depends on the balance between ROS and antioxidant substances.

[0003] Tabebuia avellanedae is a plant of the genus Tabebuia in the family Bignoniaceae. It has been used to treat inflammation, cancer, depression and other diseases in its native South and Central America for more than 1,000 years. Tabebuia avellanedae is considered to be the most effective species in the genus Tabebuia, and its inner bark is usually used medicinally. Tabebuia avellanedae was introduced into China from South America more than a decade ago. Currently, it is widely used as a street tree, courtyard tree and landscape tree in Guangdong, Hainan and other provinces. The common species of Tabebuia introduced and applied in South China and the tropical regions of Southwest China mainly include: Tabebuia chrysantha, Tabebuia impetiginosa and Tabebuia rosea, etc. At present, the research on Tabebuia plants in China mainly focuses on their biological characteristics, seed germination, seedling raising and cultivation techniques, horticultural and greening applications, etc. Research results on Tabebuia plants abroad show that plants of this genus are rich in compounds such as quinones, cyclopentenes, iridoids, lignins, flavonoids, phenylpropanoids, saponins and coumarins. Tabebuia plants generally have activities such as anti-inflammatory, anti-cancer, antioxidant, anti-Parkinson's disease, anti-ulcer, promoting skin healing, anti-microbial, anti-depressant, lipid-lowering, anti-fatty liver, inhibiting melanin production, anti-hyperuricemia, regulating immune response, anti-leukemia, analgesia, etc. However, through literature review, it is found that there has been no relevant research report on the protective effect of iridoid derivatives in Tabebuia avellanedae on cardiomyocytes so far. Summary of the Invention

[0004] In view of the above situation, to overcome the deficiencies of the prior art, the object of the present invention is to provide an iridoid for protecting cardiomyocytes from damage extracted from Tabebuia avellanedae, its preparation method and application, which can effectively solve the problem of therapeutic drug use for diseases related to cardiomyocyte damage caused by cardiomyocyte inflammation, oxidative stress damage of cardiomyocytes, and abnormal apoptosis of cardiomyocytes.

[0005] To achieve the above object, the technical solution adopted by the present invention is a iridoid for anti-myocardial cell injury extracted from brown tabebuia, with the molecular formula C 17 H 20 O 7 , and the molecular structural formula is:

[0006]

[0007] The preparation method of the iridoid for anti-myocardial cell injury extracted from brown tabebuia includes the following steps:

[0008] S1. Add 3 - 5 times the weight - volume of boiling water to the dried endothelium of brown tabebuia, extract three times, combine the extracts and perform vacuum concentration and freeze - drying to obtain a crude extract;

[0009] S2. Dissolve the crude extract in 2 - 4 times the weight - volume of distilled water, ultrasonically treat it in an ice bath for 10 - 30 min to obtain a homogeneous suspension, and perform liquid - liquid extraction with n - hexane, EtOAc, and n - butanol in sequence. Among them, the volume of each solvent is 3 - 4 times that of the suspension, extract 3 times respectively, combine each extraction liquid respectively, and perform vacuum concentration and freeze - drying to obtain n - hexane fraction, EtOAc fraction, n - butanol fraction, and water fraction extracts;

[0010] S3. Take the EtOAc fraction extract and perform silica gel column chromatography. The amount of silica gel used is 30 - 50 times the mass of the extract, and elute it successively with gradient solvent systems CHCl 3 , CHCl 3 / MeOH with a volume ratio of 50:1, CHCl 3 / MeOH with a volume ratio of 20:1, CHCl 3 / MeOH with a volume ratio of 10:1. Perform vacuum concentration to obtain 4 fractions. Among them, the fraction eluted with CHCl 3 / MeOH 20:1 is subjected to ODS column chromatography, and elute it successively with gradient solvent systems MeOH / H 2 O with a volume ratio of 1:3, MeOH / H 2 O with a volume ratio of 1:2, MeOH / H 2 O with a volume ratio of 1:1, MeOH / H 2 O with a volume ratio of 2:1, and MeOH. Perform vacuum concentration to obtain 5 fractions. Among them, the fraction eluted with MeOH / H 2 O with a volume ratio of 1:2 is purified by Sephadex LH - 20 column chromatography, and then purified by high - performance liquid chromatography using a COSMOSIL 5C18 - AR - II liquid chromatography column and a mobile phase of 45% MeOH to obtain the iridoid avelladoid I (Avd I).

[0011] The dosage of the eluent used in step S3 is 3-5 column volumes, and the dosage of ODS is 20-40 times the mass of the eluted part.

[0012] In step S3, the column height of the Sephadex LH-20 column is more than one meter, and the column diameter is not limited.

[0013] Application of the iridoid extracted from Tabebuia avellanedae Griseb. for anti-myocardial cell injury prepared by the method in the preparation of drugs for anti-myocardial cell injury.

[0014] Avelladoid I (Avd I) was extracted, separated and identified from the dried endothelium aqueous extract of Tabebuia avellanedae Griseb. in the present invention. It can improve the viability of myocardial cells, reduce the LDH level in damaged myocardial cells, and reduce the abnormally elevated inflammatory factor level in damaged cells, proving that Avd I can improve myocardial cell injury and relieve myocardial cell inflammatory injury, with significant social and economic benefits. Description of the Drawings

[0015] Figure 1 It is the structural formula of compound Avd I of the present invention and the related structural formulas of 1H-1H COSY, HMBC and NOE.

[0016] Figure 2 It is the effect diagram of compound Avd I of the present invention on the cell viability (A), LDH (B) and inflammatory factor (C) levels of DOX-induced H9c2 myocardial cells.

[0017] Figure 3 It is the effect diagram of compound Avd I of the present invention on the reactive oxygen species level, SOD, MDA and GSH-Px levels of DOX-induced H9c2 myocardial cells.

[0018] Figure 4 It is the effect diagram of compound Avd I of the present invention on the apoptosis (A) and mitochondrial membrane potential (B) levels of DOX-induced H9c2 myocardial cells.

[0019] Figure 5 It is the effect diagram of compound Avd I of the present invention on the expression levels of key proteins in the mitochondrial apoptosis pathway of DOX-induced H9c2 myocardial cells. Detailed Embodiments

[0020] The following detailed description of the specific embodiments of the present invention is provided in conjunction with the drawings and examples.

[0021] Example 1

[0022] A preparation method of an iridoid for anti-myocardial cell injury extracted from Tabebuia avellanedae Griseb., comprising the following steps:

[0023] S1. Add the dried inner bark of the brown bellflower tree to 3 times its weight in volume of boiling water, extract three times, combine the extracts and carry out vacuum concentration and freeze-drying to obtain a crude extract;

[0024] S2. Dissolve the crude extract in 2 times its weight in volume of distilled water, ultrasonically treat it in an ice bath for 10 min to obtain a homogeneous suspension, and carry out liquid-liquid extraction successively with n-hexane, EtOAc and n-butanol. Among them, the volume of each solvent is 3 times that of the suspension, extract 3 times respectively, combine the respective extraction liquids, and carry out vacuum concentration and freeze-drying to obtain the n-hexane fraction, EtOAc fraction, n-butanol fraction and water fraction extracts;

[0025] S3. Take the EtOAc fraction extract and perform silica gel column chromatography. The amount of silica gel used is 30 times the mass of the extract. Successively use the gradient solvent systems CHCl 3 , CHCl 3 / MeOH with a volume ratio of 50:1, CHCl 3 / MeOH with a volume ratio of 20:1, CHCl 3 / MeOH with a volume ratio of 10:1 for elution. The amount of eluent used is 3 column volumes each. Carry out vacuum concentration to obtain 4 fractions. Among them, the fraction eluted with CHCl 3 / MeOH 20:1 is subjected to ODS column chromatography. The amount of ODS used is 20 times the mass of the eluted fraction. Successively use the gradient solvent systems MeOH / H 2 O with a volume ratio of 1:3, MeOH / H 2 O with a volume ratio of 1:2, MeOH / H 2 O with a volume ratio of 1:1, MeOH / H 2 O with a volume ratio of 2:1, MeOH for elution. The amount of eluent used is 5 column volumes each. Carry out vacuum concentration to obtain 5 fractions. Among them, the fraction eluted with MeOH / H 2 O with a volume ratio of 1:2 is purified by Sephadex LH-20 column chromatography, and then high performance liquid chromatography is used. With a COSMOSIL 5C18-AR-II liquid chromatography column and a mobile phase of 45% MeOH for purification, iridoids are obtained.

[0026] Example 2

[0027] A preparation method of iridoids for anti-myocardial cell injury extracted from the brown bellflower tree, comprising the following steps:

[0028] S1. Add the dried inner bark of the brown bellflower tree to 4 times its weight in volume of boiling water, extract three times, combine the extracts and carry out vacuum concentration and freeze-drying to obtain a crude extract;

[0029] S2. Dissolve the crude extract in distilled water at a weight - to - volume ratio of 3 times, and ultrasonically treat it in an ice bath for 20 min to obtain a uniformly textured suspension. Then, perform liquid - liquid extraction successively with n - hexane, EtOAc, and n - butanol. Among them, the volume of each solvent is 3.5 times that of the suspension, and each extraction is carried out 3 times. Then, combine the respective extraction solutions, perform vacuum concentration and freeze - drying to obtain the extracts of the n - hexane fraction, EtOAc fraction, n - butanol fraction, and water fraction;

[0030] S3. Take the EtOAc - fraction extract and subject it to silica gel column chromatography. The amount of silica gel used is 40 times the mass of the extract. Elute successively with the gradient solvent systems CHCl 3 , CHCl 3 / MeOH with a volume ratio of 50:1, CHCl 3 / MeOH with a volume ratio of 20:1, and CHCl 3 / MeOH with a volume ratio of 10:1. The amount of the eluent used is 4 column volumes for each. Perform vacuum concentration to obtain 4 fractions. Among them, the fraction eluted with CHCl 3 / MeOH 20:1 is subjected to ODS column chromatography. The amount of ODS used is 30 times the mass of the eluted fraction. Elute successively with the gradient solvent systems MeOH / H 2 O with a volume ratio of 1:3, MeOH / H 2 O with a volume ratio of 1:2, MeOH / H 2 O with a volume ratio of 1:1, MeOH / H 2 O with a volume ratio of 2:1, and MeOH. The amount of the eluent used is 5 column volumes for each. Perform vacuum concentration to obtain 5 fractions. Among them, the fraction eluted with MeOH / H 2 O with a volume ratio of 1:2 is purified by Sephadex LH - 20 column chromatography, and then purified by high - performance liquid chromatography using a COSMOSIL 5C18 - AR - II liquid chromatography column with a mobile phase of 45% MeOH to obtain iridoids.

[0031] Example 3

[0032] A method for preparing iridoids with anti - myocardial cell injury extracted from the brown bell - flower tree, comprising the following steps:

[0033] S1. Add boiling water at a weight - to - volume ratio of 5 times to the dried endothelium of the brown bell - flower tree, extract three times, combine the extraction solutions and perform vacuum concentration and freeze - drying to obtain a crude extract;

[0034] S2. Dissolve the crude extract in distilled water at a weight-to-volume ratio of 4 times, and ultrasonically treat it in an ice bath for 30 min to obtain a uniformly textured suspension. Then perform liquid-liquid extraction successively with n-hexane, EtOAc, and n-butanol. Herein, the volume of each solvent is 4 times that of the suspension, and each extraction is carried out 3 times. Combine the respective extraction liquids, and perform vacuum concentration and freeze-drying to obtain extracts of the n-hexane fraction, EtOAc fraction, n-butanol fraction, and water fraction;

[0035] S3. Subject the EtOAc fraction extract to silica gel column chromatography. The amount of silica gel used is 50 times the mass of the extract. Elute successively with gradient solvent systems of CHCl 3 , CHCl 3 / MeOH with a volume ratio of 50:1, CHCl 3 / MeOH with a volume ratio of 20:1, and CHCl 3 / MeOH with a volume ratio of 10:1. The amount of eluent used is 5 column volumes for each. Perform vacuum concentration to obtain 4 fractions. Among them, the fraction eluted with CHCl 3 / MeOH 20:1 is subjected to ODS column chromatography. The amount of ODS used is 40 times the mass of the eluted fraction. Elute successively with gradient solvent systems of MeOH / H 2 O with a volume ratio of 1:3, MeOH / H 2 O with a volume ratio of 1:2, MeOH / H 2 O with a volume ratio of 1:1, MeOH / H 2 O with a volume ratio of 2:1, and MeOH. The amount of eluent used is 5 column volumes for each. Perform vacuum concentration to obtain 5 fractions. Among them, the fraction eluted with MeOH / H 2 O with a volume ratio of 1:2 is purified by Sephadex LH-20 column chromatography, and then purified by high performance liquid chromatography using a COSMOSIL 5C18-AR-II liquid chromatography column with a mobile phase of 45% MeOH to obtain iridoids.

[0036] A new iridoid avelladoid I (Avd I) was extracted, separated, and identified from the water extract of the endothelium of Tabebuia avellanedae. It solves the problem of therapeutic drug use for myocardial cell damage caused by myocardial cell inflammation, oxidative stress injury of myocardial cells, and apoptosis of myocardial cells, and has been proven through experiments. The relevant experimental data are as follows:

[0037] 1. Materials and Methods

[0038] 1.1 Instruments, Materials, and Reagents

[0039] NMR spectrometer JEOL ECA-600 (JEOL, Japan); mass spectrometer JEOL JMS-700 (JEOL, Japan); mass spectrometer JEOL JMS SX-102 (JEOL, Japan); IR spectrophotometer IR-460 (Shimadzu, Japan); UV-visible spectrometer UV-1600 (Shimadzu, Japan); high-sensitivity polarimeter SEPA-3000 (Horiba, Japan); rotary evaporator (EYELA SB-1100); cooling water circulation device (EYELA CA-1115, Japan); freeze dryer (EYELA PDU-2110, Japan); water aspirator (EYELA A-1000S, Japan); forced air drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd., China); ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd., China); ELIEⅡ cell incubator (Revco, USA); NIKON ECLIPSE TS100 inverted microscope (Nikon, Japan); Arium 611VF ultrapure water system (Sartorius, Germany); micropipette (Eppendorf, Germany); AB204-N one-ten-thousandth precision analytical balance (Mettler Toledo, Sweden); Centrifuge-5810 centrifuge (Eppendorf, Germany); two-way constant temperature magnetic stirrer (Shanghai Zhenrong Scientific Instrument Co., Ltd.); iMark microplate reader (Bio-Rad, USA); ELIEⅡ cell incubator (Revco, USA); BD FACSAriaⅢ flow cytometer (BD, USA); Aria computer workstation (BD, USA); Odyssey CLx dual-color infrared laser imaging system (LI-COR, USA).

[0040] Silica gel 63 - 210 μm (Kantou Kagaku, Japan); ODS 63 - 212 μm (Wako Pure Chemical, Japan); Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden); silica gel 60F 254TLC plates (Merck Co., Germany); RP-18F254S TLC plates (Merck Co., Germany); C18-AR-II 5 μm chromatographic column (Nacalai Tesque., Japan). The ferric chloride developer (FeCl3-k3[Fe(CN)6]=1:1) was prepared in the laboratory; 1% anisaldehyde-sulfuric acid developer was prepared in the laboratory; DMEM medium (Gibco); fetal bovine serum FBS (Hangzhou Sijiqing Bioengineering Research Institute); trypsin (biosharp); doxorubicin hydrochloride for injection (Shenzhen Wanle Pharmaceutical Co., Ltd.); resveratrol (Shanghai Yuanye); MTT (biosharp), DMSO (Solarbio), LDH kit (Nanjing Jiancheng); IL-6, IL-1β, TNF-α kits (Jiangsu Enzyme Immunoassay); reactive oxygen species detection ROS kit (Solarbio); apoptosis detection kit (BD), JC-1 mitochondrial membrane potential kit (Solarbio); Triton (Solarbio); DAPI (Solarbio); Caspase 9 (abcam); Caspase 3 (abcam); Cleaved Caspase 3 (Cell Signaling); Bax (abcam); Bcl-2 (abcam); P53 (abcam); β-actin (ABclonal); goat anti-rabbit secondary antibody (Li-COR); goat anti-mouse secondary antibody (Li-COR); the rest of the reagents were domestic or imported commercially available products of chromatographic pure or chemical pure grade reagents.

[0041] 1.2 Extraction, isolation, and purification of avelladoid I (Avd I)

[0042] The brown Tabebuia avellanedae Lorentz ex Griseb bark was a generous gift from Professor Ota Tomohisa of Kanazawa University, Japan. It was identified by Professor Ota Tomohisa as the bark of Tabebuia avellanedae Lorentz ex Griseb, a plant of the genus Tabebuia in the Bignoniaceae family. The plant sample was stored in the laboratory of Henan University of Traditional Chinese Medicine, with the registration number T-340.

[0043] The dried inner bark of 10 kg of brown bellflower tree was extracted with boiling water three times (30 L each time), and the extracts were combined and concentrated under reduced pressure and freeze-dried to obtain a crude extract; 350 g of the crude extract was dissolved in 1 L of distilled water and sonicated in an ice bath for 20 min to obtain a homogeneous suspension, and liquid-liquid extraction was carried out successively with n-hexane, EtOAc and n-butanol (3.5 L each, extracted three times respectively) to obtain 2.3 g of the n-hexane fraction, 48.6 g of the EtOAc fraction, 103.7 g of the n-butanol fraction and 190.0 g of the water fraction; 47.0 g of the EtOAc fraction was subjected to silica gel column chromatography and eluted successively with a gradient solvent system (CHCl 3 3, CHCl 3 3 / MeOH 50:1, 20:1, 10:1) to obtain four fractions by concentration under reduced pressure. Among them, 4.3 g of the fraction eluted with CHCl 3 3 / MeOH 20:1 was subjected to ODS column chromatography and eluted successively with a gradient solvent system (MeOH / H 2 2O 1:3, 1:2, 1:1, 2:1, MeOH) to obtain five fractions by concentration under reduced pressure. Among them, the fraction eluted with MeOH / H 2 2O 1:2 was purified by Sephadex LH-20 column chromatography and then by silica gel column chromatography, and finally purified by high performance liquid chromatography using a COSMOSIL 5C18-AR-II liquid chromatography column and a mobile phase of 45% MeOH to obtain 1.8 mg of iridoid avelladoid I (Avd I).

[0044] 1.3. Protective effect experiment of Avd I on H9c2 cardiomyocytes induced by DOX

[0045] 1.3.1 Cell culture and grouping for drug administration

[0046] The H9c2 rat cardiomyocyte cell line was purchased from Beijing Beina. High-glucose DMEM high-glucose culture medium containing 10% FBS was used, and the culture medium was changed every 2 - 3 days. Cells in the logarithmic growth phase were used for the experiment. H9c2 cells were inoculated into 96-well plates at 4×10 4 cells·mL -1 -1, 200 μL per well.

[0047] After 24 h of inoculation, the cells were randomly divided into four groups: blank control group (NC), DOX model group (1 μg·mL -1 -1 DOX), resveratrol group (1 μg·mL -1 -1 DOX + 10 μmol·L -1 -1 RSV), compound group (1 μg·mL -1 -1 DOX + 1, 5, 10, 20, 40 μmol·L -1The (compound) was incubated for 24 h, and the supernatant was collected to measure LDH. The cells were used for MTT assay and Incell-Western experiment.

[0048] H9c2 cells were seeded in 24-well plates at a density of 2×10 5 , 1 mL per well. After 24 h of seeding, the drugs were administered to each group as described above. Among them, the compound group was treated with a medium containing 1 μg·mL -1 DOX and 1 μmol·L -1 compound for 24 h. The cells or supernatant were collected for measuring the levels of IL-6, IL-1β, TNF-α, SOD, MDA, and GSH-Px.

[0049] Alternatively, H9c2 cells were seeded in 6-well plates at a density of 8×10 4 cell·mL -1 , 3 mL per well. After 24 h of seeding, the drugs were administered to each group as described in the previous paragraph. After 24 h, the cells were collected for flow cytometry analysis of the target indicators.

[0050] 1.3.2 Detection of cell viability by MTT assay

[0051] After the treatment with drugs in each group, 200 μL of fresh culture medium containing 10% MTT was added to each well. After 4 h, the absorbance at 490 nm was measured using a microplate reader, and the cell viability was calculated. Cell survival rate (%) = (ODexperimental group / ODcontrol group) × 100%.

[0052] 1.3.3 Detection of the levels of cell inflammatory factors IL-6, IL-1β, and TNF-α by ELISA

[0053] The detection was performed according to the ELISA kit instructions, and the OD value was measured at 450 nm.

[0054] 1.3.4 Detection of the levels of LDH, SOD, MDA, and GSH-Px in cell supernatant

[0055] The operation was performed according to the instructions of the LDH, SOD, MDA, and GSH-Px kits from Nanjing Jiancheng Bioengineering Institute.

[0056] 1.3.5 Detection of cell ROS level by DCFH-DA probe method

[0057] After the treatment with drugs in each group, the cells were collected, washed once with PBS, and adjusted to a density of 1×10 -1 using the working solution of DCHF-DA probe at a final concentration of 10 μmol·L 6 cell·mL -1, incubate at 37°C in the dark for 20 min, gently flick and mix the cell suspension every 5 min. Wash the cells twice with the culture medium, and adjust the cell concentration to 1×10 5 cell·mL -1 , detect using a flow cytometer, and analyze the results using an Aria computer workstation.

[0058] 1.3.6 Detection of cell apoptosis level by AnnexinV-FITC / PI double staining method

[0059] After the grouping and drug administration treatment is completed, collect the culture supernatant, wash the cells once with PBS, combine and collect the supernatant and the washing solution, add 200 μL of 0.25% trypsin to each well for digestion, terminate the digestion with the collected solution, and centrifuge at 1000 r·min -1 for 5 min to collect the cells. Wash once with PBS, and adjust the cell density to 1×10 6 cell·mL -1 , pipette 100 μL of the cell suspension into a 5 mL flow cytometry tube, add AnnexinV-FITC / PI, and incubate in the dark at room temperature for 15 min. Add 400 μL of 1×Binding Buffer to each tube, detect using a flow cytometer within 1 h, and analyze the results using an Aria computer workstation.

[0060] 1.3.7 Detection of cell mitochondrial membrane potential level by JC-1 probe method

[0061] After the grouping and drug administration treatment is completed, collect the cells. Wash once with PBS, dilute the JC-1 probe with 1×Assay Buffer to a final concentration of 10 μg·mL -1 , add 500 μL of the JC-1 probe working solution to each tube of the sample, and incubate at 37°C in the dark for 15 min. Wash the samples twice with 1×Assay Buffer, and adjust the cell concentration to 1×10 5 cell·mL -1 , detect using a flow cytometer, and analyze the results using an Aria computer workstation.

[0062] 1.3.8 Detection of apoptosis-related proteins in cells by Incell-Western method

[0063] After the grouped drug administration treatment was completed, the supernatant was discarded, and 150 μL of 10% formaldehyde was added to each well and fixed at room temperature for 20 min. 150 μL of 0.1% Triton-x-100 was added to each well and washed at room temperature 5 times, 5 min each time. 150 μL of blocking solution was added to each well and blocked at room temperature for 1.5 h. After blocking, 50 μL of primary antibody dilution (1:200) was added to each well, and the primary antibodies of Caspase 3, Cleaved Caspase3, Bax, Bcl-2, and β-actin were incubated overnight at 4°C. Then, PBST washing was performed 5 times, 5 min each time. 50 μL of secondary antibody dilution (1:500) was added to each well and incubated on a shaker at room temperature for 1 h. Then, PBST was washed 4 times and PBS was washed 1 time, 5 min each time. The washing solution was completely aspirated, and dual-channel detection and analysis at 700 nm and 800 nm were performed using a dual-color infrared laser imaging system (Odyssey CLx, USA), and the results were analyzed using Image Studio working software.

[0064] 1.4 Statistical analysis

[0065] All experimental data were expressed as mean ± standard deviation and the difference analysis was processed using SPSS 26.0 statistical software. One-way ANOVA was used, and P < 0.05 represented significant difference, and P < 0.01 represented extremely significant difference.

[0066] 2. Results

[0067] 2.1 Structural identification of Avd I

[0068] Compound Avd I was a yellow oily liquid and soluble in chloroform; specific rotation -33.62 (c 0.10, MeOH); UV (MeOH) λ max (logε) 258 (0.49), 194 (0.26) nm; IR υ max (KBr) 3360, 2943, 2840, 1705, 1607, 1512, 1259, 1170, 1119, 1026, 986, 772 cm –1 ; HR-FAB-MS m / z 335.1139 [M-H] - (C 17 H 19 O 7 calculated value 335.1131); 1 H and 13 C NMR data are shown in Table 1. Its chemical formula is as follows:

[0069]

[0070] Table 1 of Compound Avd I 13 C NMR and 1 H NMR data

[0071]

[0072] The liquid compound Avd I is a yellow oil, soluble in chloroform; HRFABMS m / z 335.1139 [M-H] - (calcd. 335.1131, C 17 H 19 O 7 ), and its molecular formula was determined to be C 17 H 20 O 7 . The IR spectrum indicated the presence of hydroxyl (3360 cm – 1 ) and carbonyl (1705 cm –1 ) groups. 1 The H NMR spectrum showed two AA'BB' system aromatic hydrogen signals [δ H 7.96 (2H, d, J = 8.6) and δ H 6.91 (2H, d, J = 8.6)], one methoxy signal δ H 3.85 (s), and HMQC showed that there was also an oxygenated methylene hydrogen signal in the compound [δ H 3.96 (d, 9.6) and 3.87 (d, 9.6)], two methylene hydrogen signals [δ H 1.63 (m) and 1.99 (m)] and [δ H 2.04 (m) and 2.76 (m)], three oxygenated methine hydrogen signals [δ H 5.60 (d, 5.5), 5.50 (m) and 5.27 (m)], two methine hydrogen signals [δ H 2.54 (dd, 5.8, 13.1) and 2.64 (dd, 5.5, 10.3)]. 13 C NMR (Table 1) combined with the HSQC spectrum observed 17 carbon signals, including 6 aromatic carbon signals (δ C 163.6, 131.7, 131.7, 122.1, 113.7, 113.7), and the remaining 11 carbon signals, including 1 methoxy carbon signal (δ C 55.5), 1 carbonyl carbon signal (δ C 165.9), 9 aliphatic carbon signals (δ C 98.7, 90.2, 86.1, 78.0, 77.1, 52.5, 43.6, 38.7 and 28.0), combined with Figure 1HMBC signals and 1 H- 1 H COSY signals. By comparing with the literature, it was found that they were basically consistent with the data of Catalpin Triacetate. It was speculated that compound Avd I contained a cyclopentane-fused pyran ring type iridoid group. The difference was that the C-3, C-8 and C-4’ positions of Catalpin Triacetate were connected with acetyl groups, while there was no acetyl group signal in Avd I, and it was speculated that the C-3 and C-8 positions were substituted by hydroxyl groups. Moreover, the HMBC spectrum showed that δ H 3.85 (H-4’-OCH3) had a long-range correlation with δ C 163.6 (C-4’), indicating that there was a methoxy substitution at the C-4’ position of the benzene ring. In addition, the HMBC spectrum showed that δ H 7.96 (H-2’ and H-6’) had a long-range correlation with δ C 165.9 (C-7’), indicating that there was a carbonyl substitution at the C-1’ position of the benzene ring. Combining with δ H 5.27 (H-6) and δ C 165.9 (C-7’) having a long-range correlation, it was indicated that the iridoid group was connected to the benzoyl group. The relative configuration of compound Avd I was determined by the NOE spectrum. The NOE spectrum showed that there was a NOE correlation between H-5 and H-9, and there were NOE correlations among H-5, H-6 and H-10α. In addition, there were NOE correlations among H-1 and H-3, and there was a NOE correlation between H-1 and H-10β, determining its relative stereoconfiguration as Figure 1 shown. Its 1 H-NMR and 13 C-NMR data are shown in Table 1. Its HMBC, 1 H- 1 H COSY, NOE correlation signals are shown in Figure 1 . Therefore, it was determined that this compound was 2a,6-dihydroxyoctahydro-2H-4,5-dioxacyclopenta[cd]inden-1-yl

[0073] 4-methoxycyclohexa-1,3-diene-1-carboxylate, named avelladoid I.

[0074] 2.2 Effects of Avd I on cell viability, LDH level and inflammatory factor level of H9c2 cardiomyocytes induced by DOX

[0075] The results are shown in Figure 2As shown, compared with the NC group, the cell viability in the DOX group was significantly decreased (P<0.01), and the levels of LDH and inflammatory factors were significantly increased (P<0.01 or P<0.05). Compared with the model group, at 1 - 20 μmol·L -1 Avd I significantly increased the cell viability (P<0.01) and simultaneously significantly decreased the LDH level in the cell supernatant (P<0.01 or P<0.05). The effective concentration of the compound was 1 - 20 μmol·L -1 , and to save the usage amount of the compound, 1 μmol·L -1 Avd I was used for the subsequent research. Compared with the model group, 1 μmol·L -1 Avd I could significantly decrease the levels of IL-6 (P<0.05) and IL-1β (P<0.01) to levels close to those of the normal group cells.

[0076] Compared with the normal group, * P≤0.05, ** P≤0.01; compared with the model group. # P≤0.05, ## P≤0.01( n = 3)

[0077] 2.3 Effects of Avd I on the ROS level, SOD, MDA, and GSH-Px levels in DOX-induced H9c2 cardiomyocytes

[0078] The results are shown in Figure 3 As shown, compared with the NC group, the ROS level in the DOX group was extremely significantly increased (P<0.01), and the SOD level (P<0.01) and GSH-Px level (P<0.05) were significantly decreased; compared with the DOX group, 1 μmol·L -1 Avd I could effectively decrease the intracellular ROS level induced by DOX (P<0.05) and significantly increase the SOD level in the cells (P<0.05).

[0079] Compared with the normal group, * P≤0.05, **P≤0.01; compared with the model group. # P≤0.05, ## P≤0.01 (x±s, n = 3)

[0080] 2.4 Effects of Avd I on the apoptosis level and mitochondrial membrane potential level in DOX-induced H9c2 cardiomyocytes

[0081] The results are shown in Figure 4As shown, compared with the NC group, the apoptosis rate of cells in the DOX group increased extremely significantly (P<0.01), and the level of mitochondrial membrane potential of cells decreased extremely significantly (P<0.01); compared with the DOX group, 1 μmol·L -1 Avd I could extremely significantly reduce the apoptosis level of H9c2 cardiomyocytes (P<0.01), and there was a tendency to increase the mitochondrial membrane potential level in H9c2 cardiomyocytes, but there was no significant difference.

[0082] Compared with the normal group, * P≤0.05, **P≤0.01; compared with the model group. # P≤0.05, ## P≤0.01 (x±s, n = 3)

[0083] 2. Effect of 2.5Avd I on the mitochondrial apoptosis pathway of H9c2 cardiomyocytes induced by DOX

[0084] The results are shown in Figure 5 As shown, compared with the NC group, the ratios of the key proteins Bax / Bcl-2 and Cleaved Caspase 3 / Caspase 3 in the apoptosis pathway in H9c2 cardiomyocytes increased extremely significantly in the DOX group (P<0.01). Compared with the DOX group, 1 μmol·L -1 Avd I could reduce the ratios of Bax / Bcl-2 and Cleaved Caspase 3 / Caspase 3 to near normal levels (P<0.01).

[0085] Compared with the normal group, **P≤0.01; compared with the model group. ## P≤0.01 (x±s, n = 3)

[0086] 3. Conclusion

[0087] Avelladoid I (Avd I) was extracted, isolated and identified from the dried endothelial aqueous extract of Tabebuia avellanedae. In the adriamycin-induced cardiomyocyte injury model, Avelladoid I could improve the viability of cardiomyocytes after modeling and reduce the LDH level in damaged cardiomyocytes, demonstrating that Avelladoid I could improve cardiomyocyte injury. At the same time, after adriamycin modeling, the levels of inflammatory factors IL-6, IL-1β, and TNF-α in cells increased significantly, and the compound Avelladoid I could reduce the abnormally elevated levels of inflammatory factors in damaged cells and relieve myocardial cell inflammatory injury.

[0088] To confirm whether the compounds isolated from *Tabebuia avellanedae* can improve cardiomyocyte apoptosis, the present invention uses the AnnexinV-FITC / PI double staining method to detect the effect of each compound on the apoptosis level of H9c2 cardiomyocytes. The results show that Avelladoid I can inhibit doxorubicin-induced excessive apoptosis of cardiomyocytes. Cell apoptosis is mainly initiated and executed through the extrinsic pathway (death receptor pathway) and the intrinsic pathway (mitochondrial pathway). The intrinsic apoptotic pathway of cells is triggered by various stress conditions, chemical reagents or drugs. When the intrinsic apoptosis is initiated, the expression levels of members of the B-cell lymphoma-2 gene (Bcl-2) family change, resulting in an increase in the ratio of the expression levels of the pro-apoptotic protein human Bcl-2-associated X protein (Bax) to the anti-apoptotic protein Bcl-2, activating the pro-apoptotic trigger proteins (BAX / BAK) on the mitochondrial membrane, causing a decrease in the mitochondrial membrane potential, resulting in the release of cytochrome c in the mitochondria. Cytochrome c and dATP in the cytoplasm further cause the oligomerization of the apoptosis protease activating factor-1 (Apaf-1) protein, promoting the binding of caspase-9 to form an apoptosome, activating the Caspase-3 cascade pathway, and ultimately leading to cell apoptosis. To further confirm the mechanism of the compounds in *Tabebuia avellanedae* against cardiomyocyte apoptosis, the present invention uses the JC-1 probe method to detect the mitochondrial membrane potential of cells on a flow cytometer, and uses the In-cell Western method to detect the protein expression levels of mitochondrial apoptosis pathway-related proteins such as Cleaved-caspase-3, Caspase-3, Bax, and Bcl-2, to study the effect of the compounds in *Tabebuia avellanedae* on the apoptosis-related pathways. The present invention detects the apoptosis level in cells and the key proteins in the mitochondrial apoptosis pathway signaling pathway. The results show that after treatment with Avelladoid I, the apoptosis level of cells decreases significantly, the mitochondrial membrane potential level is increased to varying degrees, and the levels of key proteins in the mitochondrial apoptosis pathway also decrease, and the mitochondrial apoptosis pathway of cells is inhibited. It can be seen that Avelladoid I can reduce the apoptosis level of cells and relieve apoptosis damage through the mitochondrial apoptosis pathway. The present invention uses the DCFH-DA probe method to detect the intracellular reactive oxygen species level on a flow cytometer, and further detects the contents of MDA, total SOD, and GSH-PX in cells to reflect the state of intracellular oxidative stress.

[0089] In summary, avelladoid I (Avd I) was extracted, isolated, and identified from the water extract of the dried inner bark of Tabebuia avellanedae. Avelladoid I (Avd I) can improve the viability of cardiomyocytes, reduce the LDH level in damaged cardiomyocytes, and decrease the abnormally elevated levels of inflammatory factors in damaged cells, demonstrating that Avd I can improve cardiomyocyte injury and alleviate inflammatory injury of cardiomyocytes. Avelladoid I (Avd I) can reduce the level of apoptosis, increase the level of mitochondrial membrane potential, and regulate the levels of key proteins in the mitochondrial apoptosis pathway, suggesting that Avd I can inhibit the mitochondrial apoptosis pathway of cardiomyocytes. Avelladoid I (Avd I) can regulate the oxidative stress level of cardiomyocytes, solving the problem of therapeutic drug use for cardiomyocyte injury caused by cardiomyocyte inflammation, oxidative stress injury of cardiomyocytes, and cardiomyocyte apoptosis. It is a major innovation in anti-cardiomyocyte injury drugs, with significant social and economic benefits.

[0090] It should be noted that the above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any person skilled in the art, without departing from the scope of the technical solution of the present invention, can make changes or modifications to equivalent embodiments with equivalent changes by using the disclosed technical content, and all fall within the protection scope of the present invention.

Claims

1. An icroid terpenoid extract from brown bellflower tree for preventing myocardial cell damage, characterized in that: The molecular formula is C 17 H 20 O7, molecular structure formula is: .

2. The method for preparing the iridoid ether terpenoids for preventing myocardial cell damage extracted from brown bellflower tree according to claim 1, characterized in that: The following steps are involved: S1. Add 3-5 times the weight volume of boiling water to the dried endobark of brown bellflower tree, extract three times, combine the extracts and perform vacuum concentration and freeze drying to obtain a crude extract; S2, dissolving the crude extract in 2-4 times the weight volume of distilled water, ultrasonically treating in an ice bath for 10-30 min to obtain a suspension with a uniform texture, and sequentially performing liquid-liquid extraction with n-hexane, EtOAc and n-butanol, wherein the volume of each solvent is 3-4 times that of the suspension, respectively, extracting 3 times, and combining the extracts, respectively, and performing reduced pressure concentration and freeze-drying to obtain extracts of n-hexane fraction, EtOAc fraction, n-butanol fraction and water fraction; S3. The extract of the EtOAc fraction was subjected to silica gel column chromatography, wherein the amount of silica gel was 30-50 times the mass of the extract, and eluted with a gradient solvent system of CHCl3, CHCl3 / MeOH with a volume ratio of 50:1, CHCl3 / MeOH with a volume ratio of 20:1, and CHCl3 / MeOH with a volume ratio of 10:1 in sequence, and concentrated under reduced pressure to obtain 4 fractions, wherein the CHCl3 / MeOH 20:1 elution fraction was subjected to ODS column chromatography, and eluted with a gradient solvent system of MeOH / H2O with a volume ratio of 1:3, MeOH / H2O with a volume ratio of 1:2, MeOH / H2O with a volume ratio of 1:1, MeOH / H2O with a volume ratio of 2:1, and MeOH in sequence, and concentrated under reduced pressure to obtain 5 fractions, wherein the MeOH / H2O elution fraction with a volume ratio of 1:2 was purified by Sephadex LH-20 column chromatography, and then HPLC was used to obtain COSMOSIL 5C18-AR-II liquid chromatography column and 45% MeOH mobile phase were used for purification to obtain cyclopentadiene ether terpenes.

3. The method for preparing the iridoid ether terpenoids for resisting myocardial cell damage extracted from brown bellflower tree according to claim 2, characterized in that: The following steps are involved: S1. Add 3 times the weight volume of boiling water to the dried endobark of brown bellflower tree, extract three times, combine the extracts and perform vacuum concentration and freeze drying to obtain a crude extract; S2, dissolving the crude extract in 2 times the weight volume of distilled water, ultrasonically treating in an ice bath for 10 min to obtain a uniform suspension, sequentially performing liquid-liquid extraction with n-hexane, EtOAc and n-butanol, wherein the volume of each solvent is 3 times that of the suspension, extracting three times respectively, combining each extract, concentrating under reduced pressure and freeze-drying to obtain extracts of n-hexane fraction, EtOAc fraction, n-butanol fraction and water fraction; S3. The extract of the EtOAc fraction was subjected to silica gel column chromatography, wherein the amount of silica gel used was 30 times the mass of the extract, and eluted with a gradient solvent system of CHCl3, CHCl3 / MeOH with a volume ratio of 50:1, CHCl3 / MeOH with a volume ratio of 20:1, and CHCl3 / MeOH with a volume ratio of 10:1 in sequence, and concentrated under reduced pressure to obtain 4 fractions, of which the CHCl3 / MeOH 20:1 elution fraction was subjected to ODS column chromatography, and eluted with a gradient solvent system of MeOH / H2O with a volume ratio of 1:3, MeOH / H2O with a volume ratio of 1:2, MeOH / H2O with a volume ratio of 1:1, MeOH / H2O with a volume ratio of 2:1, and MeOH in sequence, and concentrated under reduced pressure to obtain 5 fractions, of which the MeOH / H2O elution fraction was subjected to Sephadex column chromatography. The product was purified by LH-20 column chromatography, and then purified by high performance liquid chromatography using a COSMOSIL5C18-AR-II liquid chromatography column and 45% MeOH mobile phase to obtain cyclopentadiene.

4. The method for preparing the iridoid ether terpenoids for resisting myocardial cell damage extracted from brown bellflower tree according to claim 2, characterized in that: The following steps are involved: S1. Add 4 times the weight volume of boiling water to the dried endobark of brown bellflower tree, extract three times, combine the extracts and perform vacuum concentration and freeze drying to obtain a crude extract; S2, dissolving the crude extract in 3 times the weight volume of distilled water, ultrasonically treating in an ice bath for 20 min to obtain a uniform suspension, sequentially performing liquid-liquid extraction with n-hexane, EtOAc and n-butanol, wherein the volume of each solvent is 3.5 times that of the suspension, extracting three times respectively, combining each extract, concentrating under reduced pressure and freeze-drying to obtain extracts of n-hexane fraction, EtOAc fraction, n-butanol fraction and water fraction; S3. The extract of the EtOAc fraction was subjected to silica gel column chromatography, wherein the amount of silica gel used was 40 times the mass of the extract, and eluted with a gradient solvent system of CHCl3, CHCl3 / MeOH with a volume ratio of 50:1, CHCl3 / MeOH with a volume ratio of 20:1, and CHCl3 / MeOH with a volume ratio of 10:1 in sequence, and concentrated under reduced pressure to obtain 4 fractions, of which the CHCl3 / MeOH 20:1 elution fraction was subjected to ODS column chromatography, and eluted with a gradient solvent system of MeOH / H2O with a volume ratio of 1:3, MeOH / H2O with a volume ratio of 1:2, MeOH / H2O with a volume ratio of 1:1, MeOH / H2O with a volume ratio of 2:1, and MeOH in sequence, and concentrated under reduced pressure to obtain 5 fractions, of which the MeOH / H2O elution fraction was subjected to Sephadex column chromatography. The product was purified by LH-20 column chromatography, and then purified by high performance liquid chromatography using a COSMOSIL5C18-AR-II liquid chromatography column and 45% MeOH mobile phase to obtain cyclopentadiene.

5. The method for preparing the iridoid ether terpenoids for resisting myocardial cell damage extracted from brown bellflower tree according to claim 2, characterized in that: The following steps are involved: S1. Add 5 times the weight volume of boiling water to the dried endobark of brown bellflower tree, extract three times, combine the extracts and perform vacuum concentration and freeze drying to obtain a crude extract; S2, dissolving the crude extract in 4 times the weight volume of distilled water, ultrasonically treating in an ice bath for 30 min to obtain a uniform suspension, sequentially performing liquid-liquid extraction with n-hexane, EtOAc and n-butanol, wherein the volume of each solvent is 4 times that of the suspension, extracting three times respectively, combining each extract, concentrating under reduced pressure and freeze-drying to obtain extracts of n-hexane fraction, EtOAc fraction, n-butanol fraction and water fraction; S3. The extract of the EtOAc fraction was subjected to silica gel column chromatography, wherein the amount of silica gel used was 50 times the mass of the extract, and eluted with a gradient solvent system of CHCl3, CHCl3 / MeOH with a volume ratio of 50:1, CHCl3 / MeOH with a volume ratio of 20:1, and CHCl3 / MeOH with a volume ratio of 10:1 in sequence, and concentrated under reduced pressure to obtain 4 fractions, of which the CHCl3 / MeOH 20:1 elution fraction was subjected to ODS column chromatography, and eluted with a gradient solvent system of MeOH / H2O with a volume ratio of 1:3, MeOH / H2O with a volume ratio of 1:2, MeOH / H2O with a volume ratio of 1:1, MeOH / H2O with a volume ratio of 2:1, and MeOH in sequence, and concentrated under reduced pressure to obtain 5 fractions, of which the MeOH / H2O elution fraction was subjected to Sephadex column chromatography. The product was purified by LH-20 column chromatography, and then purified by high performance liquid chromatography using a COSMOSIL5C18-AR-II liquid chromatography column and 45% MeOH mobile phase to obtain cyclopentadiene.

6. The method for preparing the iridoid ether terpenoids for resisting myocardial cell damage extracted from brown bellflower tree according to any one of claims 2 to 5, characterized in that: The amount of eluent used in step S3 is 3-5 column volumes, and the amount of ODS used is 20-40 times the mass of the elution site.

7. The method for preparing the iridoid ether terpenoids for preventing myocardial cell damage extracted from the brown bell tree according to any one of claims 2 to 5, characterized in that: The column height of the Sephadex LH-20 column in step S3 is more than one meter.

8. Use of the iridoid terpenoids for preventing myocardial cell injury extracted from the brown bellflower tree prepared by the method according to any one of claims 2 to 5 in the preparation of drugs for preventing myocardial cell injury.

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