CN114591269B

CN114591269B - Eremophilane type sesquiterpene and application thereof in preparation of drugs with anti-inflammatory activity

Info

Publication number: CN114591269B
Application number: CN202210281287.4A
Authority: CN
Inventors: 李正辉; 吕晓; 叶可; 石宝宝; 艾洪莲
Original assignee: South Central University for Nationalities
Current assignee: South Central Minzu University
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2022-12-13
Anticipated expiration: 2042-03-21
Also published as: CN114591269A

Abstract

The invention relates to the technical field of medicinal chemistry, and particularly discloses endophytic fungi from Hubei fritillary bulbBoeremia exiguaTo separate out the obtained oneEremophilaneThe sesquiterpene Boerenialane D and the application potential thereof in the preparation of anti-inflammatory drugs are described in more detail in the application of the sesquiterpene Boerenialane D in the preparation of drugs with significant inhibitory activity on NO inflammatory effects. According to the inventionEremophilaneThe sesquiterpene has the advantages of low toxicity and high efficiency, and can obviously inhibit NO produced by LPS stimulation in a dose-dependent manner.

Description

Eremophilane sesquiterpene and application thereof in preparation of anti-inflammatory drugs

Technical Field

The invention relates to the technical field of medicine and chemistry, in particular to Eremophilane type sesquiterpene derived from Boeremia exigua of endophytic fungus of Hubei fritillaria thunbergii and application thereof in preparing medicaments with anti-inflammatory activity.

Background

Inflammation is the body's defense response to damaging stimuli, and it regulates the body's immunity, removes pathogens to reduce cellular damage, and autonomously repairs damaged tissues by initiating a tissue repair function. When the macrophage is invaded by external pathogenic microorganisms in the body, various cytokines, chemokines, nitric oxide and inflammation-related proteins are secreted to trigger inflammation and immune response. Lipopolysaccharide (LPS) is the main component of gram-negative bacteria cell wall, is released after bacterial death and dissolution or artificial cell destruction, and the toxic component of LPS is mainly lipid-like. Under the induction of LPS, macrophages not only obviously enhance the proliferation capacity and phagocytosis capacity of cells, but also release a large amount of cytokines and inflammatory mediators, such as Nitric Oxide (NO), tumor necrosis factor (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and the like, which resist the invasion of pathogenic microorganisms, but excessive secretion can trigger inflammatory reaction, and the continuous inflammatory reaction can cause imbalance of immune homeostasis of an organism and cause tissue damage which is difficult to repair. Inflammation is one of the leading causes of human diseases such as cancer, type II diabetes, obesity, arthritis, pulmonary fibrosis, neurodegenerative diseases and cardiovascular diseases. Aiming at the problems that the clinical anti-inflammatory drugs easily cause side effects and the drug resistance is enhanced during the use of the drugs at present, the development of novel anti-inflammatory drugs with low toxicity and high efficiency is an important subject worthy of being deeply explored by the applicant.

Nitric Oxide (NO) participates in cell signal transduction processes in the human body, and expands blood vessels and protects tissues and organs. NO is produced by the enzyme NOs (nitrite oxide synthase), which is oxidized in blood and tissues to form nitrates and nitrites, which circulate physiologically in blood and tissues to form NO and other biologically active nitrogen oxides. Physiological level of NO is vital to life activities such as substance metabolism, immune regulation and signal transmission, and a proper amount of NO can treat various diseases such as hypertension, immune regulation, cardiovascular and cerebrovascular diseases and the like, but a large amount of NO generated by continuous excessive inflammation can cause body damage. Applicants have generally used drugs that target inhibition of NO production to reduce the inflammatory response, and therefore NO is commonly used as a marker for in vitro models of inflammation to screen for effective anti-inflammatory drugs.

Hubei fritillary bulb is a genuine medicinal material in Hubei province and has the effects of eliminating phlegm and stopping cough, and detoxifying and resolving masses. Mainly treats the cough caused by exogenous wind-heat and the cough caused by phlegm-heat, and is a necessary traditional Chinese medicine for common people in daily life. Endophytic fungi are a unique microbial population that can survive and multiply in living tissue of healthy plants without significant disease symptoms. The endophytic fungi and the plant host form a complex and stable dynamic micro-ecosystem in a long co-evolution process. The life activities of endophytic fungi can be involved in protecting plants from biological and non-biological attack in the growing environment. Based on the advantages of abundant resources, various varieties, short culture period, high feasibility of gene operation and the like of endophytic fungi, the terpenoid has various biological activities such as tumor resistance, antibiosis, anti-inflammation, oxidation resistance and the like, and is often used as a high-value compound to appear in the fields of health care products, cosmetics, medical products and the like. The applicant has investigated the anti-inflammatory activity of an Eremophilane type sesquiterpene isolated from the endophytic fungi of fritillaria Hupehensis.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an Eremophilane type sesquiterpene and application thereof in preparing a medicament with anti-inflammatory activity.

An Eremophilane-type sesquiterpene having anti-inflammatory activity, which has the following structural formula:

the molecular formula is as follows: c ₁₇ H ₂₂ O ₆

The application of the Eremophilane sesquiterpene in the preparation of the medicine for inhibiting the generation of NO also belongs to the protection scope of the invention.

Still another object of the present invention is to provide an application of said Eremophilane-type sesquiterpenes in the preparation of drugs for diseases related to substance metabolism, immune regulation and signaling caused by excess NO at physiological levels.

In the present invention, said anti-inflammatory activity is embodied in particular in: can obviously reduce the NO inflammatory effect generated by mouse mononuclear macrophage leukemia cells (RAW 264.7) induced by LPS. Experiments prove that the Eremophilane sesquiterpene is low in toxicity and high in efficiency, can inhibit NO (nitric oxide) stimulated by LPS (lipopolysaccharide) in a dose-dependent manner, and has potential application in preparing novel anti-inflammatory drugs.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the invention discloses an Eremophilane type sesquiterpene derived from Boeremia exigua of the endophytic fungus of Hubei fritillary, which can inhibit NO produced by LPS stimulation in a dose-dependent manner.

2. The invention provides an Eremophilane sesquiterpene with anti-inflammatory activity, which has low toxicity and high efficiency, can inhibit NO produced by LPS stimulation in a dose-dependent manner, and has potential application in preparing novel anti-inflammatory drugs.

3. The Eremophilane type sesquiterpene to be protected by the invention can be obtained by extracting and purifying from plant endophytic fungi. Has the advantages of short culture period, high operation feasibility, no chemical pollution, environmental protection and the like.

Drawings

FIG. 1 is a hydrogen spectrum (600MHz, meOD) of the compound prepared in example 1 in a specific embodiment;

FIG. 2 is a graph of a carbon spectrum (150MHz, meOD) and DEPT of the compound prepared in example 1 in this embodiment;

FIG. 3 is a HMQC plot of the compounds prepared in example 1 in a specific embodiment;

FIG. 4 is a diagram of HMBC of the compound prepared in example 1 in an embodiment;

FIG. 5 shows a diagram of a compound prepared in example 1 in a specific embodiment ¹ H- ¹ H COSY picture;

FIG. 6 is a ROESY plot of the compound prepared in example 1 in a specific embodiment;

FIG. 7 is a graph of NO inhibition of the compound prepared in example 1, in a specific embodiment, note that: # P < 0.001 compared to blank group and # P < 0.001 compared to LPS group;

FIG. 8 is a graph showing the effect of the compound prepared in example 1 on the viability of RAW264.7 cells in the presence or absence of LPS stimulation.

Detailed Description

In order to make the purpose and the summary of the invention more clear, the applicant will make a clear and complete description of the technical solution of the present invention with reference to specific embodiments.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1:

the endophytic fungi of the raw material Hubei fritillaria is separated from the root of a fresh plant of the Hubei fritillaria, the fritillaria is collected from the Bighe community in New Pond of the Enshi Earth family of the Miao nationality of the Yongshi City of Hubei province, the self number of the strain is Bei 21, the comparison of ITS sequence determination results shows that the maximum similarity of the sequence and the Boeremia exigua is up to 100 percent, and the accession number of a gene bank is as follows: MT154621.1. It was identified as Boeremia exigua, and this strain was deposited in the microbial culture Bank of the college of medicine of the university of south-middle nationality (published, see: lv Xiao, yeke, maxu Jun et al., fritillaria Hubei endophytic fungus Boeremia exigua chemical composition and its antiinflammatory activity study [ J ]. Proceedings of the university of south-middle nationality (Nature science edition), 2022, 41 (2): 174-179).

The specific separation process of the fritillaria Hupehensis endophytic fungus shellfish 21 comprises the following steps: washing Hubei Bulbus Fritillariae Cirrhosae (root of fresh plant) with tap water, cutting into small blocks, rinsing with 75% V/V ethanol for 60s, rinsing with sterile water for 3 times, soaking in 0.1% mercuric chloride (1 g mercuric chloride dissolved in 1000g distilled water) for sterilizing for 40s, rinsing with sterile water for 3 times, drying with high-pressure sterilized filter paper, cutting off the cuts at two ends of the root of Hubei Bulbus Fritillariae Cirrhosae, cutting from the middle, inoculating the small blocks to potato glucose agar (PDA) culture medium containing penicillin streptomycin mixture under sterile condition, and culturing in 25 deg.C incubator. After the fungus hypha grows out, the hypha is picked up to be purified and cultured to obtain a single bacterial strain, and the obtained single bacterial strain is transferred to a test tube filled with a PDA culture medium and is stored in a refrigerator at 4 ℃.

Adopting rice solid fermentation to carry out amplification culture on the strain shell 21: taking out the test tube from the refrigerator, placing the test tube in a sterile normal-temperature environment for 1 hour, taking out a bacterial block with the diameter of 5mm from the test tube in the sterile environment, inoculating the bacterial block to a flat PDA culture medium, and growing the bacterial block to fill the PDA after the bacterial block grows for about 8 days for later use. The rice culture medium proportion is as follows: 50 g/bottle of rice and 50 mL/bottle of distilled water are placed in a 500mL culture bottle, sterilized at high temperature of 120 ℃ for 20 minutes, cooled and inoculated on a rice culture medium by picking bacterial blocks with the diameter of 5mm from a flat PDA culture medium in a sterile environment, and the culture conditions are as follows, wherein the culture conditions are as follows, and the culture conditions are as follows: dark culture was carried out at 25 ℃ for 30d.

The process for the isolation and purification of the compound Eremophilane-type sesquiterpene of the claims and the content of the description: soaking the fermented rice solid culture medium (total 17 kg) in methanol for 5 times (each time for 8 hr). Mixing the extractive solutions after centrifuging for 5 times, concentrating under reduced pressure, evaporating to remove solvent, dissolving with small amount of water, extracting with ethyl acetate for 6 times, mixing ethyl acetate parts, and concentrating under reduced pressure to obtain crude extract 800g. Performing silica gel column Chromatography (CH) with 80-100 mesh normal phase ₂ Cl ₂ -MeOH:1, 0, 10.

Component C (component eluted at a volume ratio of dichloromethane/methanol of 10, 35.8 g) was subjected to high performance medium pressure liquid preparative chromatography (Biotage SP1, reverse phase filler material: RP-18, 20-45 μm, fuji Silysia Chemical ltd., japan) methanol water system (methanol/water =20, 80, 60, 40, 80. Detection and development by thin layer chromatography (chloroform: methanol =10 as developing solvent; thin layer chromatography silica gel plate, qingdao ocean chemical plant), and merging identical or similar components into five subfractions C1-C5. The C1 fraction (fraction eluted at a methanol/water volume ratio of 20:80, 5.8 g) was subjected to normal phase silica gel chromatography column (200-300 mesh), dichloromethane methanol system (dichloromethane/methanol =10:1-1, v/v), gradient elution yielded 4 subfractions C1-1-C1-4. C1-1 (eluted fraction at a dichloromethane/methanol volume ratio of 3:1, 678 mg) was subjected to Sephadex LH-20 gel column chromatography (Pharmacia Fine Chemical Co., ltd., sweden) with methanol, and then 108mg of the obtained product was subjected to preparative high performance liquid chromatography (Agilent 1260; chromatography column: agilent Zorbax SB-C18, specification: 9.4 mm. Times.150mm, 5 μm; acetonitrile-water, 25:75-35, 65, v/v; flow rate: 4 mL/min) for gradient elution for 30min to prepare 2mg of the compound of the present invention (retention time: 12 min).

Structural identification of the compounds: the Eremophilane sesquiterpene compound prepared in example 1 was dissolved in 0.5mL of deuterated methanol, transferred to a nuclear magnetic tube using a 200 μ L pipette, and the hydrogen, carbon and two-dimensional spectra were measured on a nuclear magnetic resonance spectrometer (600 MHz Bruker Avance III, germany) (see FIGS. 1-6). The structure of the compound is obtained by synthesizing various physicochemical data and is named as Boeremia Lane D.

Nuclear magnetic resonance data of the resulting Eremophilane-type sesquiterpenes:

other physicochemical data for Eremophilane type sesquiterpenes:

appearance: a yellow powder; specific rotation of optical axis

136.0(c 0.1,MeOH)；UV(MeOH)λ _max (log _ε ) 240 (3.47) nm; high resolution mass spectrum HR-ESI-MS m/z 345.13064[ 2 ] M + Na] ⁺ (calcd for C ₁₇ H ₂₂ O ₆ Na ⁺ ,345.13086).

According to the results of the above detection, it was confirmed that the compound obtained in this example has the structural formula:

the molecular formula is as follows: c ₁₇ H ₂₂ O ₆

Example 2: MTT detection of cell viability

The principle is as follows: the MTT method, namely the MTT colorimetric method, is a method for testing the viability of cells, which has the advantages of simple and convenient operation, high sensitivity, economy and visual result. MTT is a tetramethylazoazolium salt, collectively known as 3- (4, 5) -dimethylthiodiazolo (-z-y 1) -3, 5-di-phenylthiozolium romide, and is in the form of a yellow powder. Succinate dehydrogenase in mitochondria of living cells has the function of reducing exogenous MTT, can reduce MTT into blue (or blue-purple) crystal Formazan (Formazan) which is insoluble in water, and dead cells do not have the function. The DMSO (dimethyl sulfoxide) can dissolve the formazan deposited in the cells, and the absorbance value is measured by an enzyme labeling instrument at the wavelength of 570nm, so that the formazan content can be measured. In general, since the formazan production amount is proportional to the number of viable cells, the relative number of viable cells can be calculated from the optical density OD value (the viable cells in the blank control group are set to 100%)

In an incubator with DMEM (Hyclone, USA) medium containing 10% fetal bovine serum (FBS, PAN, germany) (5% CO) ₂ RAW264.7 macrophages (Kunming cell Bank, chinese academy of sciences) were subcultured at 37 ℃. Taking RAW264.7 macrophage with good growth state in logarithmic growth phase, preparing into cell suspension with DMEM medium containing 10% fetal calf serum, and culturing at 5 × 10 ⁴ One/well, 100 μ L per well in 96-well plates, cells were seeded only in 6 × 10 wells in the middle region to avoid edge effects, 100 μ L sterile PBS buffer (pH =7.4, 0.01M) was added per well in the peripheral wells of the cell wells, and 5% CO ₂ Culturing at 37 deg.C for 6-12h. A40 μ M solution of the compound obtained in example 1 was prepared using serum-free DMEM medium, drug group 1 (with LPS added) was incubated with 1 μ g/mL of LPS and 40 μ M of the compound obtained in example 1, drug group 2 (without LPS) was incubated with only 40 μ M of the compound obtained in example 1, blank group 1 was added with 1 μ g/mL of LPS and serum-free medium, blank group 2 was added with serum-free medium at 100 μ L per well in 3 replicate wells. After incubation in an incubator for 24 hours, the primary medium was aspirated by a pipette gun, MTT reagent (sigma) (0.5 mg/mL) was added to each well, the medium was placed in the incubator for further incubation for 4 hours, the supernatant was discarded, 150. Mu.L DMSO (Biosharp) was added to dissolve the purple crystals, and after shaking up, the absorbance at 570nm was measured by a microplate reader (German Tecan). The cell viability was calculated as: cell viability% = drug group/blank group × 100%

The experimental results are as follows: the cytotoxicity of the compound after being co-cultured with RAW264.7 macrophage is detected by an MTT method. The experimental result shows that the cell survival rate is more than 70% when the compound with the concentration of 40 mu M is added for incubation and LPS (1 mu g/mL) is added for stimulation or not added for stimulation for 24 hours, which indicates that the compound has no obvious cytotoxicity to normal and LPS-induced RAW264.7 macrophages.

Example 3: anti-inflammatory Activity assay

The principle is as follows: in the presence of H ⁺ In the environment, nitric oxide and diazonium saltsThe sulfanilamide can generate diazo reaction to generate diazo compound, the diazo compound further generates coupling reaction with naphthyl ethylene diamine to generate red compound, and the red compound has maximum absorption peak at 540-560 nm. In a proper concentration range, the concentration of the red compound is linearly related to the magnitude of the absorbance value, and the concentration of the red compound generated by the reaction has a linear relationship with the concentration of nitric oxide, so that the NO concentration can be estimated through the absorbance (OD value). Further, the inhibitory effect of the compound obtained in example 1 on the inflammatory effect of NO produced by LPS-induced RAW264.7 macrophages was investigated.

The experimental steps are as follows:

(1) Cell culture: the CO was determined in a constant temperature incubator (5% CO) using DMEM medium (Hyclone, USA) containing 10% fetal bovine serum (FBS, PAN, germany) ₂ RAW264.7 macrophages (Kunming cell Bank, chinese academy of sciences) were subcultured at 37 ℃. Taking RAW264.7 macrophage with good growth state in logarithmic growth phase, preparing into cell suspension with DMEM medium containing 10% fetal calf serum, and adjusting cell concentration to 5 × 10 ⁴ One/well, 100 μ L per well in 96-well plates, cells were seeded only in 6 × 10 wells in the middle region to avoid edge effects, 100 μ L sterile PBS buffer (pH =7.4, 0.01M) was added per well in the peripheral wells of the cell wells, and 5% CO ₂ Culturing at 37 deg.C for 6-12h.

(2) Adding medicine treatment: preparing solutions of the compound obtained in example 1 with six concentration gradients by using a DMEM medium without fetal bovine serum, wherein the solutions are respectively 40, 30, 20,10, 5 and 1 mu M, after cells are attached to the wall, the DMEM medium in the holes is sucked out and discarded, a drug group is added with LPS with the concentration of 1 mu g/mL and the solution of the compound obtained in example 1 with different concentrations for co-incubation, 100 mu L/hole is formed, each hole of a blank group is added with 100 mu L of serum-free DMEM medium, a model group is added with LPS solution with the concentration of 1 mu g/mL, a positive control group is added with PDTC50 mu M (Biyun day, S1809), the LPS and PDTC are prepared into solutions by using serum-free DMEM medium, and a 96-well plate is placed in a constant temperature incubator for further culture for 18 hours.

(3) Color development: the supernatant was transferred to a new 96-well plate 50. Mu.L per well by centrifugation at 2000r/min for 5min in a 96-well plate centrifuge. This was allowed to produce red substances using the Griess kit (Promega, USA).

(4) Color comparison: measuring the light absorption value of each hole at the 540nm wavelength of the microplate reader, calculating the NO concentration according to the OD value and the NO standard curve, and further solving the IC of each compound to be measured ₅₀ The value is obtained.

NO production rate% = (drug group-blank group)/(model group-blank group) × 100%

The experimental results are as follows:

since the toxicity of a drug affects the cell survival rate and further the production of NO, the cell survival rate needs to be considered in the study of the anti-inflammatory activity of the drug. The cell survival rate of the compound is more than 70% under the concentration of 40 mu M by detecting the cytotoxicity through MTT test, so that the deep research on the anti-inflammatory mechanism is meaningful. Anti-inflammatory results are shown in fig. 7 (LPS =1 μ g/ml), and the Eremophilane type sesquiterpene has significant inhibitory activity on NO inflammation produced by LPS-induced RAW264.7 macrophages through an in vitro activity test, and the level of NO is obviously increased after 18h of LPS stimulation compared with a blank group, which indicates that an LPS-induced inflammation model is successfully established. In the administration group, the compounds with different concentrations can remarkably inhibit NO generation in a dose-dependent manner after treatment, and the IC of the compounds ₅₀ 8.621 μ M, stronger than the positive control PDTC (IC) ₅₀ =23.1 μ M) by a factor of about 3. These results indicate that the compound has low-toxicity and high-efficiency inhibition effect on NO produced by RAW264.7 macrophage stimulated by LPS.

Claims

1. An Eremophilane type sesquiterpene, which has a structural formula:

。

2. use of an Eremophilane-type sesquiterpene of claim 1 for the preparation of a medicament for inhibiting the development of inflammation.

3. Use of an Eremophilane-type sesquiterpene of claim 1 for the preparation of a medicament for inhibiting NO production.

4. Use of an Eremophilane-type sesquiterpene according to claim 1 for the preparation of an anti-inflammatory agent.