CN103319442B - Coicenal diterpenoid compound and preparation method and application thereof in preparation of anti-inflammatory drugs - Google Patents

Abstract

The invention discloses a Coicenal diterpene compound, a preparation method thereof, and an application of the compound in the preparation of anti-inflammatory drugs. The compound provided by the present invention is shown in formula (I-1); in formula (I-1), R is the following (a) or (b) or (c): (a) H; (b) (c) In the above (a) and/or the above (b), n=2-10. The invention also protects Bipolaris coicis L437, and the preservation number is CGMCC No.7714. The invention discloses a new compound, and provides the application of the compound in inhibiting inflammation and the preparation method of the compound. The compound has inhibitory effect on various inflammations and can be developed and utilized as medicine or health product.

Description

Coicenal diterpenoid compound and its preparation method and application in the preparation of anti-inflammatory drugs

technical field

The invention relates to a Coicenal diterpene compound, a preparation method thereof, and an application of the compound in the preparation of anti-inflammatory drugs.

Background technique

Inflammation is a series of protective responses of the body to the damage caused by various inflammatory factors, and usually causes systemic reactions, such as fever, leukocytosis, and varying degrees of degeneration and necrosis of the heart, liver, kidney and other organs. Acute inflammation is often red, swollen, hot, and painful, which is a defense response of the body to stimuli, but chronic inflammation is characterized by the persistence of inflammatory factors and damage to the body, which can lead to diabetes, lung disease, cancer, cardiovascular system and Nervous system diseases and autoimmune diseases can cause serious harm to the body and even threaten the life safety of patients. Any factor that can cause tissue damage in the body can become the cause of inflammation, that is, an inflammatory factor. When inflammation occurs, some inflammatory factors directly damage vascular endothelial cells, causing increased vascular permeability, and most inflammatory factors mainly cause inflammation through the action of endogenous chemical factors. Playing a mediating role, also known as inflammatory mediator, its release and regulation is the main target of anti-inflammatory drug design.

Nitric oxide in the body is formed by the guanidine nitrogen on arginine through the catalysis of nitric oxide synthase NOS. Widely distributed in various tissues of the body, it not only functions as a second messenger and neurotransmitter, but also is an effector molecule, mediating and regulating various physiological and pathological processes including inflammation. Nitric oxide synthase can be divided into two categories, namely, structural nitric oxide synthase NOS (cNOS) and inducible nitric oxide synthase NOS (iNOS). cNOS can be continuously expressed under normal conditions, but iNOS can only be expressed in specific physiological environments, such as under the induction of certain cytokines, such as lipopolysaccharide LPS and tumor necrosis factor TNF-a. It is generally believed that NO produced by iNOS may be involved in the pathological process of various diseases. NO not only directly participates in the body's inflammatory response, but also promotes the release of other inflammatory mediators. In addition, the abnormal metabolism of NO can also lead to stroke, bronchial asthma, senile dementia, cancer and other serious diseases.

At present, anti-inflammatory drugs with relatively significant clinical efficacy mainly include three categories: non-steroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs and traditional Chinese medicine anti-inflammatory drugs, but long-term use of traditional non-steroidal anti-inflammatory drugs can cause gastric mucosal damage, Ulcers and even perforations, and steroidal anti-inflammatory drugs can cause serious disturbances in water and salt metabolism, sugar, fat, and protein metabolism. Therefore, the advantages of less side effects and multi-link effects of natural source anti-inflammatory drugs are reflected, and become a hot spot in the research and development of new drugs in the world today: such as anti-inflammatory and thrombolytic Tongmai Capsules, Liuwei Wuling Tablets, Xiaoyan Lidan Tablets, etc. . Fungi are natural product resources with a high "creation coefficient", which can produce secondary metabolites with diverse structures, novelty and wide range of activities, which are crucial to the research and development of drugs and pesticides.

Contents of the invention

The object of the present invention is to provide a kind of Coicenal diterpenoid compound and its preparation method, and the application of this compound in the preparation of anti-inflammatory drugs.

The present invention provides a compound, as shown in formula (I-1);

In formula (I-1), R is the following (a) or (b) or (c):

(a) H;

In said (a) and/or said (b), n=2-10.

When R is H, the compound represented by formula (I-1) is represented by formula (II-1).

R is And when n=2, the compound represented by formula (I-1) is represented by formula (III-1).

R is And when n=2, the compound represented by formula (I-1) is represented by formula (IV-1).

The compound represented by formula (I-1) can be specifically represented by formula (I-2).

The compound represented by formula (II-1) can be specifically represented by formula (II-2).

The compound represented by formula (III-1) can be specifically represented by formula (III-2).

The compound represented by formula (IV-1) can be specifically represented by formula (IV-2).

The present invention also protects the application of the compound in the preparation of products for preventing and/or treating inflammatory reactions. Said product may in particular be a medicament. The medicine can be tablet, powder, capsule, oral liquid, emulsion, ointment, cream, injection, suspension, tincture, granule or aerosol. The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy. The product can specifically be a health care product. The health product can be tablet, capsule, oral liquid or granule.

The present invention also protects a product for preventing and/or treating inflammatory reactions, the active ingredient of which is the compound or the pharmaceutically acceptable salt, ester or solvate of the compound. Said product may in particular be a medicament. The medicine can be tablet, powder, capsule, oral liquid, emulsion, ointment, cream, injection, suspension, tincture, granule or aerosol. The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy. The product can specifically be a health care product. The health product can be tablet, capsule, oral liquid or granule. The drug can be introduced into the body (such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue) by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated methods, or mixed with other substances or Import the body after wrapping. When necessary, one or more pharmaceutically acceptable carriers can also be added to the above drugs. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.

The present invention also protects the use of the compound in the preparation of products for preventing and/or treating allergic diseases. Said product may in particular be a medicament. The medicine can be tablet, powder, capsule, oral liquid, emulsion, ointment, cream, injection, suspension, tincture, granule or aerosol. The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy. The allergic disease may be inflammation and/or allergic inflammation caused by the release of NO as an inflammatory mediator.

The present invention also protects a product for preventing and/or treating allergic diseases, the active ingredient of which is the compound or the pharmaceutically acceptable salt, ester or solvate of the compound. Said product may in particular be a medicament. The medicine can be tablet, powder, capsule, oral liquid, emulsion, ointment, cream, injection, suspension, tincture, granule or aerosol. The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy. The allergic disease may be inflammation and/or allergic inflammation caused by the release of NO as an inflammatory mediator. The drug can be introduced into the body (such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue) by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated methods, or mixed with other substances or Import the body after wrapping. When necessary, one or more pharmaceutically acceptable carriers can also be added to the above drugs. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.

The present invention also protects Bipolaris coicis L437, which has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee (abbreviated as CGMCC, address: No. 1 Beichen West Road, Chaoyang District, Beijing) on June 5, 2013. Institute No. 3, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101), and the deposit number is CGMCC No.7714.

The present invention also protects the use of said strain L437 in the production of said compound.

The present invention also protects a method for preparing the compound, comprising the following steps: fermenting the bacterial strain L437 to obtain a fermentation product containing the compound.

The method for the compound shown in the preparation formula (II-1) comprises the following steps in sequence:

(1) Inoculate the strain L437 into the fermentation medium, and culture at 25-30°C (such as 28°C) in the dark for 30-50 days (such as 40 days);

(2) Take the fermentation product obtained in step (1), add ethyl acetate, perform ultrasonic crushing, and collect the extract, which is the extract;

(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4) Take the crude extract obtained in step (3), carry out silica gel column chromatography, and carry out gradient elution with the eluent composed of petroleum ether and acetone, and the volume ratios of petroleum ether and acetone during the elution process are respectively 100: 1, 50:1, 30:1, 20:1, 10:1, collect the post-column solution of the eluent whose volume ratio of petroleum ether and acetone is 10:1, and obtain dry substance A after rotary evaporation;

(5) Perform gel column chromatography on the dry substance A obtained in step (4), use equal volumes of mixed chloroform-methanol as the eluent, collect the post-column solution from 100mL to 140mL, and obtain 75mg dry substance A after rotary evaporation Substance C;

(6) The dry substance C obtained in step (5) was subjected to HPLC chromatography, using methanol-water (75:25 by volume) as the eluent, and the chromatographic peak with a retention time of 18.7min was collected, and after rotary evaporation, the obtained Compound represented by formula (II-1).

The method for the compound shown in the preparation formula (Ⅲ-1) comprises the following steps in sequence:

(1) Inoculate the strain L437 into the fermentation medium, and culture at 25-30°C (such as 28°C) in the dark for 30-50 days (such as 40 days);

(2) Take the fermentation product obtained in step (1), add ethyl acetate, perform ultrasonic crushing, and collect the extract, which is the extract;

(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4) Take the crude extract obtained in step (3), carry out silica gel column chromatography, and carry out gradient elution with the eluent composed of petroleum ether and acetone, and the volume ratios of petroleum ether and acetone during the elution process are respectively 100: 1. 50:1, 30:1, 20:1, 10:1, 9:1, 8:2, collect the post-column solution of the eluent with a volume ratio of petroleum ether and acetone of 8:2, and rotary evaporate After obtaining the dry matter B;

(5) Perform reverse-phase column chromatography on the dry substance B obtained in step (4), and carry out gradient elution with an eluent composed of methanol and water. The volume ratio of methanol in the eluent is 20%, 30%, 50%, 70%, collect the post-column solution of the eluent with a methanol concentration of 70% and rotary evaporate to obtain a dry substance D.

(6) Perform HPLC chromatography on the dry substance D obtained in step (5), using methanol-water (volume ratio 72:28) as the eluent, collect the chromatographic peak with a retention time of 32.9min, and obtain the formula after rotary evaporation The compound represented by (III-1).

The method for the compound shown in the preparation formula (IV-1) comprises the following steps in sequence:

(1) Inoculate the strain L437 into the fermentation medium, and culture at 25-30°C (such as 28°C) in the dark for 30-50 days (such as 40 days);

(2) Take the fermentation product obtained in step (1), add ethyl acetate, perform ultrasonic crushing, and collect the extract, which is the extract;

(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4) Take the crude extract obtained in step (3), carry out silica gel column chromatography, and carry out gradient elution with the eluent composed of petroleum ether and acetone, and the volume ratios of petroleum ether and acetone during the elution process are respectively 100: 1. 50:1, 30:1, 20:1, 10:1, 9:1, 8:2, collect the post-column solution of the eluent with a volume ratio of petroleum ether and acetone of 8:2, and rotary evaporate After obtaining the dry matter B;

(5) Perform reverse-phase column chromatography on the dry substance B obtained in step (4), and carry out gradient elution with an eluent composed of methanol and water. The volume ratio of methanol in the eluent is 20%, 30%, 50%, 70%, collect the post-column solution of the eluent with a methanol concentration of 70% and rotary evaporate to obtain a dry substance D.

(6) Perform HPLC chromatography on the dry substance D obtained in step (5), using methanol-water (volume ratio 72:28) as the eluent, collect the chromatographic peak with a retention time of 57.3min, and obtain the formula after rotary evaporation Compounds represented by (IV-2).

The preparation method of the fermentation medium can specifically be: take 80g of rice and add 120mL of water.

The method for the compound shown in the preparation formula (II-1) comprises the following steps in sequence:

(1) Inoculate the strain L437 into the fermentation medium, avoid light, 25-30°C (such as 28°C), shake culture for 30-50 days (such as 40 days);

(2) Take the fermentation system obtained in step (1), filter and collect the mycelia and fermentation supernatant respectively; add the mycelium to ethyl acetate, perform ultrasonic crushing, and collect the extract, which is the extract A; Extract the fermentation supernatant with ethyl acetate, take the organic phase, which is the extract B; combine the extract A and the extract B, and obtain the extract C;

(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4) Take the crude extract obtained in step (3), carry out silica gel column chromatography, and carry out gradient elution with the eluent composed of petroleum ether and acetone, and the volume ratios of petroleum ether and acetone during the elution process are respectively 100: 1, 50:1, 30:1, 20:1, 10:1, collect the post-column solution of the eluent whose volume ratio of petroleum ether and acetone is 10:1, and obtain dry substance A after rotary evaporation;

(5) Perform gel column chromatography on the dry substance A obtained in step (4), use equal volumes of mixed chloroform-methanol as the eluent, collect the post-column solution from 100mL to 140mL, and obtain 75mg dry substance A after rotary evaporation Substance C;

(6) The dry substance C obtained in step (5) was subjected to HPLC chromatography, using methanol-water (75:25 by volume) as the eluent, and the chromatographic peak with a retention time of 18.7min was collected, and after rotary evaporation, the obtained Compound represented by formula (II-1).

The method for the compound shown in the preparation formula (Ⅲ-1) comprises the following steps in sequence:

(1) Inoculate the strain L437 into the fermentation medium, avoid light, 25-30°C (such as 28°C), shake culture for 30-50 days (such as 40 days);

(2) Take the fermentation system obtained in step (1), filter and collect the mycelia and fermentation supernatant respectively; add the mycelium to ethyl acetate, perform ultrasonic crushing, and collect the extract, which is the extract A; Extract the fermentation supernatant with ethyl acetate, take the organic phase, which is the extract B; combine the extract A and the extract B, and obtain the extract C;

(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4) Take the crude extract obtained in step (3), carry out silica gel column chromatography, and carry out gradient elution with the eluent composed of petroleum ether and acetone, and the volume ratios of petroleum ether and acetone during the elution process are respectively 100: 1. 50:1, 30:1, 20:1, 10:1, 9:1, 8:2, collect the post-column solution of the eluent with a volume ratio of petroleum ether and acetone of 8:2, and rotary evaporate After obtaining the dry matter B;

(5) Perform reverse-phase column chromatography on the dry substance B obtained in step (4), and carry out gradient elution with an eluent composed of methanol and water. The volume ratio of methanol in the eluent is 20%, 30%, 50%, 70%, collect the post-column solution of the eluent with a methanol concentration of 70% and rotary evaporate to obtain a dry substance D.

(6) Perform HPLC chromatography on the dry substance D obtained in step (5), using methanol-water (volume ratio 72:28) as the eluent, collect the chromatographic peak with a retention time of 32.9min, and obtain the formula after rotary evaporation The compound represented by (III-1).

The method for the compound shown in the preparation formula (IV-1) comprises the following steps in sequence:

(1) Inoculate the strain L437 into the fermentation medium, avoid light, 25-30°C (such as 28°C), shake culture for 30-50 days (such as 40 days);

(2) Take the fermentation system obtained in step (1), filter and collect the mycelia and fermentation supernatant respectively; add the mycelium to ethyl acetate, perform ultrasonic crushing, and collect the extract, which is the extract A; Extract the fermentation supernatant with ethyl acetate, take the organic phase, which is the extract B; combine the extract A and the extract B, and obtain the extract C;

(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4) Take the crude extract obtained in step (3), carry out silica gel column chromatography, and carry out gradient elution with the eluent composed of petroleum ether and acetone, and the volume ratios of petroleum ether and acetone during the elution process are respectively 100: 1. 50:1, 30:1, 20:1, 10:1, 9:1, 8:2, collect the post-column solution of the eluent with a volume ratio of petroleum ether and acetone of 8:2, and rotary evaporate After obtaining the dry matter B;

(5) Perform reverse-phase column chromatography on the dry substance B obtained in step (4), and carry out gradient elution with an eluent composed of methanol and water. The volume ratio of methanol in the eluent is 20%, 30%, 50%, 70%, collect the post-column solution of the eluent with a methanol concentration of 70% and rotary evaporate to obtain a dry substance D.

(6) Perform HPLC chromatography on the dry substance D obtained in step (5), using methanol-water (volume ratio 72:28) as the eluent, collect the chromatographic peak with a retention time of 57.3min, and obtain the formula after rotary evaporation Compounds represented by (IV-2).

The preparation method of the fermentation medium (pH6.5) can be as follows: take 200g of potatoes, wash, peel and cut into pieces, add water and boil for half an hour, filter the filtrate with gauze, add 20g of glucose, and dilute to 1L with water.

The compound represented by formula (I) can be prepared by either microbial fermentation or chemical synthesis. The microorganisms used to prepare the compound represented by formula (I) are not limited to the said strain L437.

Any of the inflammatory reactions above can be contact dermatitis, skin allergy, swelling of the soles of the feet or accumulation of inflammatory cells in the bronchoalveoli of asthmatic patients.

The invention discloses a new compound, and provides the application of the compound in inhibiting inflammation and the preparation method of the compound. The compound has inhibitory effect on various inflammations and can be developed and utilized as medicine or health product.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged. RAW264.7 cells are mouse mononuclear macrophages, and the ATCC number is TIB-71.

Embodiment 1, isolation and identification of bacterial strain L437

1. Isolation of strains

On the basis of high-throughput screening of active compounds of microbial secondary metabolites, the inventors isolated a purely cultured strain from a rice leaf in Yunnan, and named it strain L437.

2. Identification of strains

1. Morphological and physiological and biochemical identification

Strain L437 can grow at 5-35°C, and the optimum temperature is around 28°C. The base of the conidiophores of strain L437 is swollen and dark brown, and the color gradually becomes lighter as it goes up. The apex is knee-shaped and unbranched. The conidia of strain L437 are brown, long fusiform, slightly curved, with 6-14 diaphragms, (63-153) mm×(14-22) mm.

2. Molecular identification

The ITS sequence of strain L437 is shown in sequence 1 of the sequence listing.

Based on the results of morphological identification, physiological and biochemical identification and molecular identification, the strain L437 belonged to Bipolaris coicis.

3. Preservation of strains

The strain L437 was deposited in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101) on June 5, 2013. , the deposit number is CGMCC No.7714. Coicis coicis (Bipolaris coicis) L437CGMCC No.7714 is referred to as strain L437.

The preparation of the compound shown in embodiment 2, formula (I)

One, the preparation of the compound shown in formula (I)

1. Pre-culture

The strain L437 was inoculated on the slant of PDA medium, and cultured in the dark at 28°C for 7 days, and the hyphae covered the entire slant.

2. Transfer the mycelium obtained in step 1 to the seed culture medium, and culture at 28° C. and 180 rpm in the dark for 4 days to obtain the seed liquid.

Seed medium: the solvent is water, the solute and its content are as follows: glucose 4.0g/L, malt extract (MaltExtract) 10.0g/L, yeast extract (Yeast Extract; purchased from Oxoid Ltd., batch number 1074139) 4.0g /L.

3. Inoculate 10 mL of seed culture solution into the fermentation medium, and culture at 28°C in the dark for 40 days.

The preparation method of the fermentation medium: put 80g of rice into a 500mL Erlenmeyer flask, add 120mL of tap water, and sterilize (121°C, 30min).

2. Extraction and separation of compounds represented by formula (I)

1. Take 1.6Kg of the fermentation product obtained in step 1, add 8000mL of ethyl acetate, and ultrasonically crush it in an ice bath (ultrasonic frequency 40Hz, time 30min), and collect the extract; repeat the same method for three times; extract three times Combined, that is the extract.

2. Perform rotary evaporation of the extract in step 1 (temperature 40°C, rotation speed 100rpm, vacuum <3mmHg) to obtain 20g of crude extract.

3. Take 20g of crude extract, and carry out silica gel column chromatography (Qingdao Ocean Chemical Co., Ltd. 200-300 mesh, column chromatography silica gel 400g, Φ7.5×100cm), and use petroleum ether and acetone as the eluent for gradient Elution, the volume ratio of petroleum ether and acetone during elution is 100:1, 50:1, 30:1, 20:1, 10:1, 9:1, 8:2, 7:3 and 6:4 , 4000mL of each eluent was eluted, and the post-column solution of the eluent with a volume ratio of petroleum ether and acetone of 10:1 was collected, and 500mg was obtained after rotary evaporation (temperature 40°C, speed 100rpm, vacuum <3mmHg) Dry substance A; collect the post-column solution of the eluent with a volume ratio of petroleum ether and acetone of 8:2, and obtain 1000mg of dry substance B after rotary evaporation (temperature 40°C, rotation speed 100rpm, vacuum degree <3mmHg);

4. Perform gel column chromatography (Sephadex LH-20, Amersham Biosciences, Φ2×90cm) on the dry substance A obtained in step 3, use equal volumes of mixed chloroform-methanol as the eluent, and collect the 100mL to 140mL column The final solution was rotary evaporated (temperature 40°C, rotation speed 100rpm, vacuum <3mmHg) to obtain 75mg of dry substance C.

5. Perform HPLC chromatography (RP-18, 250×10mm Column, 5μm, flow rate 3ml/min) on the dry substance C obtained in step 4, using methanol-water (volume ratio 75:25) as the eluent, and collect the retention time The chromatographic peak is 18.7min. After rotary evaporation (temperature 40°C, rotation speed 100rpm, vacuum <3mmHg), 16 mg of dry substance A was obtained, and the structure of dry substance A was confirmed to be the compound shown in formula (II-2).

6. Perform reverse-phase column chromatography (ODS, YMC) on the dry substance B obtained in step 3, and perform gradient elution with the eluent composed of methanol and water. The volume ratio of methanol in the eluent is 20 %, 30%, 50%, 70%, 90%, 100%, 100 ml of each methanol concentration was eluted, and the post-column solution of the eluent with a methanol concentration of 70% was collected and rotary evaporated (temperature 40°C, speed 100rpm, vacuum <3mmHg), and 235mg of dry matter D was obtained.

7. Perform HPLC chromatography (RP-18, 250×10mm Column, 5μm, flow rate 3ml/min) on the dry substance D obtained in step 6, using methanol-water (volume ratio: 72:28) as the eluent, and collect the retention time The chromatographic peak was 32.9min, and 100mg of dry substance B was obtained after rotary evaporation. The structure was confirmed to be the compound shown in formula (Ⅲ-2). The chromatographic peak with a retention time of 57.3min was collected, and 15mg of dry substance C was obtained after rotary evaporation. The structure was confirmed to be the compound shown in formula (IV-2).

3. The structure confirmation data of the compound

Confirmation data of the structure of the compound represented by formula (II-2): yellow oil; UV(n-hexane)λ _max (log ε):263nm(4.20);CD(c3.93×10 ^-4 M,n-hexane)λ _max (Δε)218(-15.4),260(+28.9) ,335(–1.04)nm;IR(ν _max ):3400,2960,2875,1656,1643,1623,1462,1397,1147,1034,931cm ^-1 ;Positive HRESIMS:m/z[M+H] ⁺ 319.2279 (calcd.for C ₂₀ H ₃₁ O ₃ , 319.2268); see Table 1 for ¹ H NMR and ¹³ C NMR data.

Confirmation data of the structure of the compound represented by formula (Ⅲ-2): yellow oil; UV(n-hexane)λ _max (log ε):263nm(4.10);CD(c1.20×10 ^-3 M,n-hexane)λ _max (Δε)219(-1.21),259(+1.88) ,344(–0.09)nm;IR(ν _max ):2963,2931,2877,1739,1657,1623,1463,1399,1200,1158,1035,983,956,932cm ^-1 ;PositiveHRESIMS:m/z[ M+H] ⁺ 419.2425 (calcd.for C ₂₄ H ₃₅ O ₆ , 419.2428); ¹ H NMR and ¹³ C NMR data are shown in Table 1.

The structural confirmation data of the compound represented by formula (IV-2): yellow oil; UV(n-hexane)λ _max (log ε):263nm(4.12);CD(c2.89×10 ^-4 M,n-hexane)λ _max (Δ ε)257(-0.75)nm;IR(ν _max ):3465,2960,2930,2876,1741,1659,1625,1462,1397,1156,1033,933cm ^-1 ;Positive HRESIMS:m/z[M+H] ⁺ 433.2587(calcd.for C ₂₅ H ₃₇ O ₆ ,433.2585); see Table 1 for ¹ H NMR and ¹³ C NMR data.

NMR data of each compound in Table 1 ( ¹ H: 500MHz; ¹³ C: 125MHz; solvent: CDCl ₃ )

Preparation of the compound shown in embodiment 3, formula (I)

One, the preparation of the compound shown in formula (I)

1. Same as step 1 of embodiment 2.

2. Same as step 1 of embodiment 2.

3. Inoculate 10 mL of seed culture solution into the fermentation medium, and incubate in the dark at 28°C and 180 rpm for 40 days with shaking.

Preparation method of fermentation medium (pH 6.5): Take 200g of potatoes, wash, peel and cut into small pieces, add water and boil for half an hour, filter the filtrate with gauze, add 20g of glucose, and dilute to 1L with water.

2. Extraction and separation of compounds represented by formula (I)

1. Take 3150ml of the fermentation system obtained in step 1, filter and collect the mycelia and fermentation supernatant respectively.

2. Take the mycelium obtained in step 1, add 1000ml of ethyl acetate, and ultrasonically crush it in an ice bath (ultrasonic frequency 40Hz, time 30min), collect the extract; add 1000ml of ethyl acetate to the mycelium, and use Perform ultrasonic crushing under the same conditions to collect the extract; add 1000ml of ethyl acetate to the mycelium, perform ultrasonic crushing under the same conditions, and collect the extract; combine the three extracts to obtain the extract A.

3. Take the fermentation supernatant obtained in step 1, extract it three times with an equal volume of ethyl acetate, and combine the organic phases extracted three times to obtain the extract solution B.

4. Combine the extract solution A and the extract solution B to obtain the extract solution C.

5. Carry out rotary evaporation of the extract solution C (temperature 40°C, rotation speed 100rpm, vacuum degree <3mmHg) to obtain 3g of crude extract.

Take the crude extract obtained in step 5, and operate according to steps 3, 4, 5, 6 and 7 of step 2 of Example 2 in sequence, the compound shown in formula (II-2), the compound shown in formula (III-2) and The yields of the compounds represented by formula (IV-2) were 2 mg, 8 mg and 2.1 mg, respectively.

Example 4. Test of the inhibitory activity of the compound represented by formula (I) on the release of NO from mouse mononuclear macrophage RAW264.7 induced by lipopolysaccharide

Inhibitory activity of compounds on lipopolysaccharide (LPS)-induced NO release from RAW264.7 cells Reference method (GiangPM, Jin HZ, Son PT, Lee JH, Hong YS, Lee JJ. activation and NO production. Journal of Natural Products, 2003, 66(9), 1217-1220.).

Since NO is extremely unstable, it is metabolized into nitrous acid-based NO ₂ ^- in the cell culture supernatant, so the concentration of NO ₂ ^- in the sample was determined by Griess method as an index to measure the NO level.

The compound to be tested is the compound represented by the formula (II-2), the compound represented by the formula (III-2) or the compound represented by the formula (IV-2) prepared in Example 2. Hydrocortisone was used as a positive control for the compounds to be tested.

Hydrocortisone: Sigma, CAS No. 50-23-7.

1. RAW264.7 cells (ATCC code: TIB-71) were cultured in 10% heat-inactivated (56°C, 30min) fetal bovine serum, 100U/ml penicillin sodium (Gibco), 100μg/ml streptomycin (Gibco) RPMI-1640 (Gibco) medium and incubated in a constant temperature incubator at 37°C and 5% CO ₂ .

2. Dilute RAW264.7 cells with RPMI-1640 culture medium to make a single cell suspension of 1×10 ⁵ cells/mL, inoculate in 96-well plate (200 μL per well), and set three parallel wells for each group. After culturing in the _CO2 incubator for 1 h, add 0.4 μL of the test compound with different concentrations dissolved in LPS (Sigma) with a final concentration of 1 μg/mL and DMSO to each well, and set the LPS group at the same time (add LPS and replace the test compound with DMSO DMSO solution, the inhibition rate of NO release is 0%) and the blank control group (the DMSO solution without LPS and DMSO instead of the test compound, the inhibition rate of NO release is 100%). Cultivate in a constant temperature incubator at 37°C and 5% CO ₂ for 24 hours, pipette 100 μL of the supernatant from each well onto the microplate, centrifuge (1000×g, 4°C, 3 min), add 100 μL of Griess reagent (Griess reagent A: 0.1%N -Aqueous solution of naphthaleneethylenediamine hydrochloride; Griess reagent B: 1% aqueous solution of sulfanilamide + 5% aqueous phosphoric acid; mix equal volumes of Griess reagent A and Griess reagent B before use) in a 96-well plate, and stand at room temperature After 10 min, the OD value was measured at 540 nm with a microplate reader.

Construct a standard curve with _NaNO2 at a concentration of 1, 5, 10, and 50 μmol/L, respectively, and calculate the concentration of ^NO2- in the cell culture supernatant and the inhibition rate of NO release according to the _NaNO2 standard curve. The formula for calculating the inhibition rate is :

The IC ₅₀ value is the concentration of the test compound or positive control corresponding to the NO inhibition rate of 50%.

Table 2 IC ₅₀ values of test compounds and hydrocortisone (μM)

serial number _IC50 (μM) Compounds represented by formula (II-2) 16.34±1.12 Compounds represented by formula (Ⅲ-2) 23.55±1.37 Compounds represented by formula (IV-2) 10.82±0.83 Hydrocortisone 46.25±2.27

The results are shown in Table 2. The compounds represented by the formula (II-2), the compound represented by the formula (III-2) and the compound represented by the formula (IV-2) all showed significant effects on the release of NO from mouse monocyte macrophages. Strong inhibitory power, that is, strong anti-inflammatory activity.

Example 5. Anti-allergic contact dermatitis induced by dinitrofluorobenzene (DNFB) activity test of the compound represented by formula (I)

Dexamethasone (DEX): Tianjin Pharmaceutical Group Xinzheng Co., Ltd. Dinitrofluorobenzene (also known as DNFB; CAS: 70-34-8): Shanghai Ruji Biotechnology Development Co., Ltd. BALB/c male mice: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

BALB/c male mice were randomly divided into model group (DNFB group), test sample group-1 (DNFB-1 group), test sample group-2 (DNFB-2 group), test sample group-3 (DNFB-3 group) and dexamethasone group (DNFB-DEX group), 10 rats in each group. From the 1st to the 7th day of the experiment, intragastric administration was administered every day; on the 8th day of the experiment, DNFB-ethanol solution (dissolving 15 mg DNFB in 1 ml ethanol) was used to subcutaneously inject sensitization on the back of the mouse, and each mouse was injected with 50 μL; On the 13th day, apply acetone-olive oil solution (that is, mix 4 parts by volume of acetone and 1 part by volume of olive oil) on the right ear of the mouse as an antigen challenge, apply 25 μL to each mouse, and the mice will develop ear contact dermatitis 24 hours later ; 24 hours after antigen challenge, ear tissue slices were removed with an 8mm diameter punch, weighed, and the inhibition rate of auricle swelling (%) was calculated. The model group was gavaged with distilled water, 0.1 ml per mouse per day. Test sample group-1 was gavaged with an aqueous solution of the compound represented by formula (II-2), at a daily dose of 50 mg/kg of mouse body weight, 0.1 ml per mouse per day. Test sample group-2 was gavaged with an aqueous solution of the compound represented by formula (III-2), at a daily dose of 50 mg/kg of mouse body weight, 0.1 ml per mouse per day. Test sample group-3 was gavaged with an aqueous solution of the compound represented by formula (IV-2), with a daily dose of 50 mg/kg mouse body weight, 0.1 ml per mouse per day. The dexamethasone group was intragastrically administered with an aqueous solution of dexamethasone, with a daily dose of 1 mg/kg mouse body weight, 0.1 ml per mouse per day.

Inhibition rate of ear swelling = [1-(difference in weight of administration group/difference in weight of model group)]*100%;

Weight difference = weight of ear tissue piece of right ear - weight of ear tissue piece of left ear.

The results are shown in Table 3. The compound represented by the formula (II-2), the compound represented by the formula (III-2) and the compound represented by the formula (IV-2) all showed good anti-DNFB-induced allergic dermatitis effects.

Table 3 compound anti-DNFB induced allergic dermatitis effect

group Weight of right ear ear tissue piece (mg) Inhibition rate(%) DNFB group 31.2±1.4 - DNFB-1 group 17.6±1.4 43.59 DNFB-2 group 24.6±2.1 21.15 DNFB-3 group 16.6±1.8 46.79 DNFB-DEX group 9.3±1.2 70.19

Example 6. Activity test of the compound represented by formula (I) against heterogeneous passive skin allergic reaction

Peritact vaccine: Chengdu Institute of Biological Products, batch number 20090403. Ovalbumin: China Pharmaceutical Group Shanghai Chemical Reagent Company, batch number F20090702. Ketotifen: sigma chemical Co. Male clean grade BALB/c mice: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Male SD rats: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

Prepare rat anti-ovalbumin serum (anti-OVA) by referring to the method of Tada et al. using ovalbumin and DPT vaccine. The specific method is: 100-200g SD rats are treated with 1% ovalbumin in normal saline solution (1g Ovalbumin was added to 100ml of normal saline) intramuscularly injected on both sides of the hind legs according to the dose of 10mg/kg rats, and at the same time intraperitoneally injected DPT vaccine 2×10 ¹⁰ U/rat, 13 days later (12-14 days was the peak of IgE time) the animals were sacrificed to collect blood, centrifuged at low speed, the serum was separated, and stored in a refrigerator.

7-week-old 18-22g male clean-grade BALB/c mice were randomly divided into 5 groups, 10 mice in each group; anti-OVA group (control group), anti-OVA-1 group, anti-OVA-2 group, anti-OVA-3 group and anti-OVA-ketotifen group.

On the 1st to 7th day of the experiment, oral administration was performed every day; on the 8th day of the experiment, 10 μl anti-OVA was injected into the skin of the mouse's right ear for sensitization (heterogeneic passive skin allergic reaction), and the same amount of anti-OVA was injected into the left ear as a physiological Saline: After 48 hours of sensitization, inject 1% ovalbumin Evans blue normal saline solution (1g Evans blue, 1g ovalbumin dissolved in 100ml normal saline) into the tail vein of mice at a dose of 0.1ml/10g body weight to attack, After 30 minutes, the animals were sacrificed by dislocation of the neck, and round ear slices were punched in the same part of the left and right ears with a puncher with a diameter of 8 mm. The anti-OVA group was orally administered distilled water, 0.1 ml per mouse per day. The anti-OVA-1 group was orally administered an aqueous solution of the compound represented by formula (II-2), with a daily dose of 50 mg/kg mouse body weight, 0.1 ml per mouse per day. The anti-OVA-2 group was orally administered an aqueous solution of the compound represented by formula (Ⅲ-2), with a daily dose of 50 mg/kg mouse body weight, 0.1 ml per mouse per day. The anti-OVA-3 group was orally administered an aqueous solution of the compound represented by formula (IV-2), with a daily dose of 50 mg/kg mouse body weight, 0.1 ml per mouse per day. The anti-OVA-ketotifen group was orally administered an aqueous solution of ketotifen, with a daily dose of 5 mg/kg mouse body weight, 0.1 ml per mouse per day.

Ear swelling inhibition rate = [1-(difference in weight of administration group/difference in weight of control group)]*100%.

Weight difference = ear piece weight of right ear - ear piece weight of left ear.

The results are shown in Table 4. The compound represented by the formula (II-2), the compound represented by the formula (III-2) and the compound represented by the formula (IV-2) all show very good anti-heterogeneic passive skin allergy effects.

The effect of table 4 compounds against heterogeneous passive skin allergic reaction

group Weight of right ear ear tissue piece (mg) Inhibition rate(%) anti-OVA group 28.8±2.5 - anti-OVA-1 group 18.8±1.1 34.7 anti-OVA-2 group 24.6±1.5 14.6 anti-OVA-3 group 13.5±2.3 53.1 anti-OVA-ketotifen group 9.2±0.9 68.1

Example 7. Activity test of the compound represented by formula (I) against carrageenan-induced paw swelling in mice

Carrageenan: sigma, 9000-07-1.

7-week-old 18-22g male clean-grade BALB/c mice were randomly divided into 5 groups, 10 mice in each group; Carrageenan group (control group), Carrageenan-1 group, Carrageenan-2 group, Carrageenan-3 group and Carrageenan group - Ketotifen group.

On the 1st day of the experiment, intragastric administration was administered for the first time. After 1 hour of administration for the first time, 0.1ml of normal saline solution of 1g/100mL carrageenan was subcutaneously injected into the left paw of each mouse to cause inflammation (subcutaneously injected into the right paw) equal volume of normal saline), administered by intragastric administration for the second time after 1 hour of inflammation, and 3 hours after the second administration, the normal paw (right) and swollen paw (right) were measured with a Digimatic caliper (resolution 0.01mm) Left), calculate the edema inhibition rate (%). The doses of the first administration and the second administration were the same. The Carrageenan group was given distilled water, 0.1 ml per mouse each time. The Carrageenan-1 group was administered with an aqueous solution of the compound represented by formula (II-2), at a dosage of 50 mg/kg of mouse body weight each time, 0.1 ml per mouse each time. The Carrageenan-2 group was given an aqueous solution of the compound represented by formula (Ⅲ-2), with a dose of 50 mg/kg mouse body weight each time, 0.1 ml per mouse each time. The Carrageenan-3 group was administered with an aqueous solution of the compound represented by formula (IV-2), at a dose of 50 mg/kg of mouse body weight each time, 0.1 ml per mouse each time. The Carrageenan-ketotifen group was given an aqueous solution of ketotifen, each dosage being 5 mg/kg mouse body weight, 0.1 ml per mouse each time.

Edema inhibition rate (%)=[1-(difference in thickness of administration group/difference in thickness of control group)]*100%.

Thickness difference = thickness of swollen sole (left) - thickness of normal sole (right).

The results are shown in Table 5. The compound represented by formula (II-2), the compound represented by formula (III-2) and the compound represented by formula (IV-2) all showed good anti-carrageenan-induced paw swelling in mice.

The compound shown in table 5 formula (I) resists the effect of carrageenan-induced mouse paw swelling

group Thickness of swollen paw (mm) Inhibition rate(%) Carrageenan group 1.56±0.16 - Carrageenan - Group 1 1.25±0.24 19.87 Carrageenan - Group 2 1.33±0.18 14.74 Carrageenan - Group 3 1.19±0.11 23.72 Carrageenan-ketotifen group 0.76±0.13 51.28

Example 8. The effect of the compound represented by formula (I) on inflammatory cells in the bronchopulmonary perfusate of sensitized mice after antigen challenge

Ovalbumin: China Pharmaceutical Group Shanghai Chemical Reagent Company, batch number F20090702. Male clean grade BALB/c mice: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

60 7-week-old 18-22g male clean-grade BALB/c mice were randomly divided into 6 groups, 10 in each group; they were: blank control group, model control group (without any treatment), experimental group-1, Experimental group-2, experimental group-3 and positive control group.

On the first day of the experiment, after ether anesthesia, each mouse was treated with 0.1% ovalbumin aluminum hydroxide gel solution (0.1g ovalbumin was added to 100ml normal saline containing 10g aluminum hydroxide) on both hind paws, two groins, A total of eight subcutaneous injections (0.4 ml per point) in the armpit, neck and back were used for sensitization; on the 10th day of the experiment, each mouse was subcutaneously injected with 0.1% ovalbumin aluminum hydroxide gel solution at the eight points (Inject 0.2 ml per point) to sensitize again; from the 21st day of the experiment, attack with 1% ovalbumin solution (0.1g ovalbumin added to 100ml normal saline) by nebulization every day, 30 minutes each time, continuous attack On the 6th day, all kinds of mice except the model control group were intragastrically administered 1 hour before each challenge; 24 hours after the last challenge, the mice were killed by dislocation, the skin of the neck was cut longitudinally, the trachea was separated and exposed, and the trachea was intubated. Tube, flush with lavage solution (Thornwell-Mcdowell solution: NaCl6.6g, KCl0.46g, _CaCl20.05g , _NaHCO32.52g , _MgCl20.09g , _Na2HPO40.1g , add water to _1000mL ), every 0.5 mL each time, 3 times in total, collect bronchoalveolar lavage fluid, take out 50 microliters after shaking well, dilute 4 times with white blood cell diluent (Hengda Parkson Biotechnology Beijing Technology Development Co., Ltd.), take a small amount and drop it on the cell counting plate , count the total number of white blood cells under a microscope; centrifuge the bronchoalveolar lavage fluid at low temperature 1000rpm for 10min, discard the supernatant, mix it with a pipette and smear, dry at room temperature, Wright's stain [basic dye methylene blue and acid The dye yellow eosin is collectively called eosin methylene blue dye, that is, Wright's dye] staining for 6-8 minutes, counterstaining with Wright's staining solution for 1 minute, rinsed the dye with tap water, and counted white blood cells under an optical microscope at 40 times , count 200 cells, count lymphomonocytes (nucleus stained purple, chromatin loose, cytoplasm stained light gray blue) and eosinophils (cytoplasm contains thick bright red granules, nuclei are mostly two leaves). The blank control group was given physiological saline, 0.1 ml per mouse each time. Experimental group-1 was administered with an aqueous solution of the compound represented by formula (II-2), at a dose of 50 mg/kg of mouse body weight each time, 0.1 ml per mouse each time. Experimental group-2 was administered with an aqueous solution of the compound represented by formula (III-2), at a dose of 50 mg/kg mouse body weight each time, 0.1 ml per mouse each time. Experimental group-3 was administered with an aqueous solution of the compound represented by formula (IV-2), at a dose of 50 mg/kg mouse body weight each time, 0.1 ml per mouse each time. The positive control group was given an aqueous solution of dexamethasone, each dosage being 1 mg/kg mouse body weight, 0.1 ml per mouse each time.

The results are shown in Table 6. Compounds represented by formula (II-2), compounds represented by formula (III-2) and compounds represented by formula (IV-2) have obvious inhibitory effect on the accumulation of inflammatory cells in the bronchoalveolar of the mouse asthma model .

The effect of table 6 compounds on bronchoalveolar inflammatory cells in mouse asthma model

group Total white blood cells (×10 ⁴ /ml) Eosinophils (%) Lymphoid mononuclear cells (%) normal control group 15.6±4.4 12.6±3.4 86.6±3.4 Model control group 123.8±22.2 70.8±9.4 43.6±8.4 Experimental group-1 63.6±18.3 53.6±11.3 47.6±9.8 Experimental group-2 77.6±10.8 52.5±6.4 45.6±7.2 Experimental group-3 52.3±12.3 41.3±4.4 55.8±5.4 positive control group 40.6±6.3 30.1±10.1 68.6±11.6

Claims (9)

1. a compound, shown in (I-1);

In formula (I-1), R is following (a) or (b) or (c):

(a)H；

(b)

(c)

In described (b) and/or described (c), n=2-10.

2. compound described in claim 1 is preparing the application prevented and/or treated in the product of inflammatory reaction.

3. prevent and/or treat a product for inflammatory reaction, its activeconstituents is compound pharmacy acceptable salt described in compound described in claim 1 or claim 1.

4. compound described in claim 1 is preparing the application prevented and/or treated in the product of allergic disorder.

5. prevent and/or treat a product for allergic disorder, its activeconstituents is compound pharmacy acceptable salt described in compound described in claim 1 or claim 1.

6. application as claimed in claim 4 or product as claimed in claim 5, is characterized in that: the inflammation that described allergic disorder causes as the release of inflammatory mediator for NO and/or allergic inflammation.

7. heart of a lotus seed Bipolaris (Bipolaris coicis) L437, its deposit number is CGMCC No.7714.

8. the application of bacterial strain described in claim 7 in compound described in production claim 1.

9. prepare a method for compound described in claim 1, comprise the steps: bacterial strain described in fermentation claim 7, obtain the tunning containing compound described in claim 1.