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

Coicenal diterpenoid compound and preparation method and application thereof in preparation of anti-inflammatory drugs Download PDF

Info

Publication number
CN103319442B
CN103319442B CN201310254586.XA CN201310254586A CN103319442B CN 103319442 B CN103319442 B CN 103319442B CN 201310254586 A CN201310254586 A CN 201310254586A CN 103319442 B CN103319442 B CN 103319442B
Authority
CN
China
Prior art keywords
compound
formula
group
preparation
extract
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201310254586.XA
Other languages
Chinese (zh)
Other versions
CN103319442A (en
Inventor
刘宏伟
王全新
宝丽
韩俊杰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Microbiology of CAS
Original Assignee
Institute of Microbiology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Microbiology of CAS filed Critical Institute of Microbiology of CAS
Priority to CN201310254586.XA priority Critical patent/CN103319442B/en
Publication of CN103319442A publication Critical patent/CN103319442A/en
Application granted granted Critical
Publication of CN103319442B publication Critical patent/CN103319442B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明公开了一种Coicenal类二萜化合物及其制备方法,以及该化合物在制备抗炎药物中的应用。本发明提供的化合物,如式(Ⅰ-1)所示;式(Ⅰ-1)中,R为如下(a)或(b)或(c):(a)H;(b)(c)所述(a)和/或所述(b)中,n=2-10。本发明还保护薏平脐蠕孢(Bipolaris coicis)L437,保藏编号为CGMCC No.7714。本发明公开了一种新化合物,并提供了该化合物在抑制炎症中的应用以及该化合物的制备方法,该化合物对多种炎症具有抑制作用,可作为药物或保健品开发利用。 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类二萜化合物及其制备方法和在制备抗炎药物中的应用Coicenal diterpenoid compound and its preparation method and application in the preparation of anti-inflammatory drugs

技术领域technical field

本发明涉及一种Coicenal类二萜化合物及其制备方法,以及该化合物在制备抗炎药物中的应用。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.

背景技术Background technique

炎症是机体对各种致炎因子引起的损伤所发生的一系列保护性应答,通常会引起全身反应,常见发热、白细胞增多及心、肝、肾等器官不同程度的变性和坏死等。急性炎症常见红、肿、热、痛,是机体对刺激的一种防御反应,但慢性炎症则表现为致炎因子持续存在并且损伤机体,可导致糖尿病、肺部疾病、癌症、心血管系统和神经系统的疾病及自身免疫性疾病等,对机体产生严重危害,甚至威胁患者生命安全。任何能引起机体组织损伤的因素都可成为炎症的原因,即致炎因子。在炎症发生时,有些致炎因子直接损伤血管内皮细胞,引起血管通透性升高,大多数致炎因子主要是通过内源性化学因子的作用而导致炎症,这些化学因子在炎症发展过程中起着介导作用,又称为炎症介质,其释放及调控是抗炎药物设计的主要靶点。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.

机体内一氧化氮是由精氨酸上的胍基氮经过一氧化氮合成酶NOS的催化而形成的。广泛分布于机体各个组织中,既有第二信使和神经递质的功能,又是效应分子,介导和调节包括炎症在内的多种生理和病理过程。一氧化氮合成酶可分为两类,即结构型一氧化氮合成酶NOS(cNOS)和诱导型一氧化氮合成酶NOS(iNOS)。cNOS在正常情况下可持续表达,但是iNOS只有在特定的生理环境下才能表达,如在某些细胞因子,如脂多糖LPS、肿瘤坏死因子TNF-a等诱导下表达。一般认为由iNOS所产生的NO可能参与多种疾病发生的病理过程。NO不但直接参与了机体的炎症反应,而且还可以促进其他炎症介质的释放,此外,NO的代谢异常还能导致中风、支气管哮喘、老年痴呆、癌症等严重疾病。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.

目前临床上疗效比较显著的抗炎药物主要有非甾体抗炎药、甾体抗炎药和中药抗炎药三大类,但长期使用传统的非甾体抗炎药会造成胃粘膜损伤、溃疡甚至穿孔,而甾体抗炎药会造成水盐代谢和糖、脂肪、蛋白质代谢的严重紊乱。因此,天然来源抗炎药物的副作用少、多环节作用的优势体现出来,成为当今世界新药研发的热点:如临床上常用的抗炎溶栓通脉胶囊、六味五灵片、消炎利胆片等。真菌属于“创造系数”很高的天然产物资源,可以产生结构多样、新颖,活性广泛的次级代谢产物,对药物和农药的研发都至关重要。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.

发明内容Contents of the invention

本发明的目的是提供一种Coicenal类二萜化合物及其制备方法,以及该化合物在制备抗炎药物中的应用。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.

本发明提供了一种化合物,如式(Ⅰ-1)所示;The present invention provides a compound, as shown in formula (I-1);

式(Ⅰ-1)中,R为如下(a)或(b)或(c):In formula (I-1), R is the following (a) or (b) or (c):

(a)H;(a) H;

所述(a)和/或所述(b)中,n=2-10。In said (a) and/or said (b), n=2-10.

R为H时,式(Ⅰ-1)所示化合物如式(Ⅱ-1)所示。When R is H, the compound represented by formula (I-1) is represented by formula (II-1).

R为且n=2时,式(Ⅰ-1)所示化合物如式(Ⅲ-1)所示。R is And when n=2, the compound represented by formula (I-1) is represented by formula (III-1).

R为且n=2时,式(Ⅰ-1)所示化合物如式(Ⅳ-1)所示。R is And when n=2, the compound represented by formula (I-1) is represented by formula (IV-1).

式(Ⅰ-1)所示化合物具体可如式(Ⅰ-2)所示。The compound represented by formula (I-1) can be specifically represented by formula (I-2).

式(Ⅱ-1)所示化合物具体可如式(Ⅱ-2)所示。The compound represented by formula (II-1) can be specifically represented by formula (II-2).

式(Ⅲ-1)所示化合物具体可如式(Ⅲ-2)所示。The compound represented by formula (III-1) can be specifically represented by formula (III-2).

式(Ⅳ-1)所示化合物具体可如式(Ⅳ-2)所示。The compound represented by formula (IV-1) can be specifically represented by formula (IV-2).

本发明还保护所述化合物在制备预防和/或治疗炎症反应的产品中的应用。所述产品具体可为药物。所述药物可为片剂、粉剂、胶囊、口服液、乳剂、膏剂、霜剂、注射剂、混悬剂、酊剂、颗粒剂或气雾剂。上述各种剂型的药物均可以按照药学领域的常规方法制备。所述产品具体可为保健品。所述保健品可为片剂、胶囊、口服液或颗粒剂。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.

本发明还保护一种预防和/或治疗炎症反应的产品,其活性成分为所述化合物或所述化合物药学上可接受的盐、酯或溶剂合物。所述产品具体可为药物。所述药物可为片剂、粉剂、胶囊、口服液、乳剂、膏剂、霜剂、注射剂、混悬剂、酊剂、颗粒剂或气雾剂。上述各种剂型的药物均可以按照药学领域的常规方法制备。所述产品具体可为保健品。所述保健品可为片剂、胶囊、口服液或颗粒剂。所述药物可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体(如肌肉、皮内、皮下、静脉、粘膜组织),或是被其他物质混合或包裹后导入机体。需要的时候,在上述药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。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.

本发明还保护所述化合物在制备预防和/或治疗变态反应性疾病的产品中的应用。所述产品具体可为药物。所述药物可为片剂、粉剂、胶囊、口服液、乳剂、膏剂、霜剂、注射剂、混悬剂、酊剂、颗粒剂或气雾剂。上述各种剂型的药物均可以按照药学领域的常规方法制备。所述变态反应性疾病可为NO作为炎症介质的释放所导致的炎症和/或过敏性炎症。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.

本发明还保护一种预防和/或治疗变态反应性疾病的产品,其活性成分为所述化合物或所述化合物药学上可接受的盐、酯或溶剂合物。所述产品具体可为药物。所述药物可为片剂、粉剂、胶囊、口服液、乳剂、膏剂、霜剂、注射剂、混悬剂、酊剂、颗粒剂或气雾剂。上述各种剂型的药物均可以按照药学领域的常规方法制备。所述变态反应性疾病可为NO作为炎症介质的释放所导致的炎症和/或过敏性炎症。所述药物可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体(如肌肉、皮内、皮下、静脉、粘膜组织),或是被其他物质混合或包裹后导入机体。需要的时候,在上述药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。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.

本发明还保护薏平脐蠕孢(Bipolaris coicis)L437,已于2013年6月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101),保藏编号为CGMCC No.7714。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.

本发明还保护所述菌株L437在生产所述化合物中的应用。The present invention also protects the use of said strain L437 in the production of said compound.

本发明还保护一种制备所述化合物的方法,包括如下步骤:发酵所述菌株L437,得到含有所述化合物的发酵产物。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.

制备式(Ⅱ-1)所示化合物的方法依次包括如下步骤:The method for the compound shown in the preparation formula (II-1) comprises the following steps in sequence:

(1)将所述菌株L437接种至发酵培养基,25-30℃(如28℃)避光静置培养30-50天(如40天);(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)取步骤(1)得到的发酵产物,加入乙酸乙酯,进行超声破碎,收集提取液,即为浸提液;(2) Take the fermentation product obtained in step (1), add ethyl acetate, perform ultrasonic crushing, and collect the extract, which is the extract;

(3)将步骤(2)得到的浸提液进行旋转蒸发,得到粗浸膏;(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4)取步骤(3)得到的粗浸膏,进行经硅胶柱层析,以石油醚和丙酮组成的洗脱液进行梯度洗脱,洗脱过程中石油醚和丙酮的体积比分别为100:1、50:1、30:1、20:1、10:1,收集石油醚和丙酮的体积比为10:1的洗脱液的过柱后溶液,旋转蒸发后得到干物质A;(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)将步骤(4)得到的干物质A进行凝胶柱层析,以等体积混合的氯仿-甲醇为洗脱液,收集第100mL至140mL的过柱后溶液,旋转蒸发后得到75mg干物质C;(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)将步骤(5)得到的干物质C进行HPLC层析,以甲醇-水(体积比为75:25)为洗脱液,收集保留时间为18.7min的色谱峰,旋转蒸发后,得到式(Ⅱ-1)所示化合物。(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).

制备式(Ⅲ-1)所示化合物的方法依次包括如下步骤:The method for the compound shown in the preparation formula (Ⅲ-1) comprises the following steps in sequence:

(1)将所述菌株L437接种至发酵培养基,25-30℃(如28℃)避光静置培养30-50天(如40天);(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)取步骤(1)得到的发酵产物,加入乙酸乙酯,进行超声破碎,收集提取液,即为浸提液;(2) Take the fermentation product obtained in step (1), add ethyl acetate, perform ultrasonic crushing, and collect the extract, which is the extract;

(3)将步骤(2)得到的浸提液进行旋转蒸发,得到粗浸膏;(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4)取步骤(3)得到的粗浸膏,进行经硅胶柱层析,以石油醚和丙酮组成的洗脱液进行梯度洗脱,洗脱过程中石油醚和丙酮的体积比分别为100:1、50:1、30:1、20:1、10:1、9:1、8:2,收集石油醚和丙酮的体积比为8:2的洗脱液的过柱后溶液,旋转蒸发后得到干物质B;(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)将步骤(4)得到的干物质B进行反相柱层析,以甲醇和水组成的洗脱液进行梯度洗脱,甲醇在洗脱液中所占的体积比依次为20%、30%、50%、70%,收集甲醇浓度为70%的洗脱液的过柱后溶液并旋转蒸发,得到干物质D。(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)将步骤(5)得到的干物质D进行HPLC层析,以甲醇-水(体积比为72:28)为洗脱液,收集保留时间为32.9min的色谱峰、旋转蒸发后得到式(Ⅲ-1)所示化合物。(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).

制备式(Ⅳ-1)所示化合物的方法依次包括如下步骤:The method for the compound shown in the preparation formula (IV-1) comprises the following steps in sequence:

(1)将所述菌株L437接种至发酵培养基,25-30℃(如28℃)避光静置培养30-50天(如40天);(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)取步骤(1)得到的发酵产物,加入乙酸乙酯,进行超声破碎,收集提取液,即为浸提液;(2) Take the fermentation product obtained in step (1), add ethyl acetate, perform ultrasonic crushing, and collect the extract, which is the extract;

(3)将步骤(2)得到的浸提液进行旋转蒸发,得到粗浸膏;(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4)取步骤(3)得到的粗浸膏,进行经硅胶柱层析,以石油醚和丙酮组成的洗脱液进行梯度洗脱,洗脱过程中石油醚和丙酮的体积比分别为100:1、50:1、30:1、20:1、10:1、9:1、8:2,收集石油醚和丙酮的体积比为8:2的洗脱液的过柱后溶液,旋转蒸发后得到干物质B;(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)将步骤(4)得到的干物质B进行反相柱层析,以甲醇和水组成的洗脱液进行梯度洗脱,甲醇在洗脱液中所占的体积比依次为20%、30%、50%、70%,收集甲醇浓度为70%的洗脱液的过柱后溶液并旋转蒸发,得到干物质D。(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)将步骤(5)得到的干物质D进行HPLC层析,以甲醇-水(体积比为72:28)为洗脱液,收集保留时间为57.3min的色谱峰、旋转蒸发后得到式(Ⅳ-2)所示化合物。(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).

所述发酵培养基的制备方法具体可为:取80g大米,加入120mL水。The preparation method of the fermentation medium can specifically be: take 80g of rice and add 120mL of water.

制备式(Ⅱ-1)所示化合物的方法依次包括如下步骤:The method for the compound shown in the preparation formula (II-1) comprises the following steps in sequence:

(1)将所述菌株L437接种至发酵培养基,避光、25-30℃(如28℃)、振荡培养30-50天(如40天);(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)取步骤(1)得到的发酵体系,过滤分别收集菌丝体和发酵上清;将所述菌丝体加入乙酸乙酯,进行超声破碎,收集提取液,即为浸提液甲;将所述发酵上清用乙酸乙酯萃取,取有机相,即为浸提液乙;将所述浸提液甲和所述浸提液乙合并,即为浸提液丙;(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)将步骤(2)得到的浸提液进行旋转蒸发,得到粗浸膏;(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4)取步骤(3)得到的粗浸膏,进行经硅胶柱层析,以石油醚和丙酮组成的洗脱液进行梯度洗脱,洗脱过程中石油醚和丙酮的体积比分别为100:1、50:1、30:1、20:1、10:1,收集石油醚和丙酮的体积比为10:1的洗脱液的过柱后溶液,旋转蒸发后得到干物质A;(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)将步骤(4)得到的干物质A进行凝胶柱层析,以等体积混合的氯仿-甲醇为洗脱液,收集第100mL至140mL的过柱后溶液,旋转蒸发后得到75mg干物质C;(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)将步骤(5)得到的干物质C进行HPLC层析,以甲醇-水(体积比为75:25)为洗脱液,收集保留时间为18.7min的色谱峰,旋转蒸发后,得到式(Ⅱ-1)所示化合物。(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).

制备式(Ⅲ-1)所示化合物的方法依次包括如下步骤:The method for the compound shown in the preparation formula (Ⅲ-1) comprises the following steps in sequence:

(1)将所述菌株L437接种至发酵培养基,避光、25-30℃(如28℃)、振荡培养30-50天(如40天);(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)取步骤(1)得到的发酵体系,过滤分别收集菌丝体和发酵上清;将所述菌丝体加入乙酸乙酯,进行超声破碎,收集提取液,即为浸提液甲;将所述发酵上清用乙酸乙酯萃取,取有机相,即为浸提液乙;将所述浸提液甲和所述浸提液乙合并,即为浸提液丙;(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)将步骤(2)得到的浸提液进行旋转蒸发,得到粗浸膏;(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4)取步骤(3)得到的粗浸膏,进行经硅胶柱层析,以石油醚和丙酮组成的洗脱液进行梯度洗脱,洗脱过程中石油醚和丙酮的体积比分别为100:1、50:1、30:1、20:1、10:1、9:1、8:2,收集石油醚和丙酮的体积比为8:2的洗脱液的过柱后溶液,旋转蒸发后得到干物质B;(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)将步骤(4)得到的干物质B进行反相柱层析,以甲醇和水组成的洗脱液进行梯度洗脱,甲醇在洗脱液中所占的体积比依次为20%、30%、50%、70%,收集甲醇浓度为70%的洗脱液的过柱后溶液并旋转蒸发,得到干物质D。(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)将步骤(5)得到的干物质D进行HPLC层析,以甲醇-水(体积比为72:28)为洗脱液,收集保留时间为32.9min的色谱峰、旋转蒸发后得到式(Ⅲ-1)所示化合物。(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).

制备式(Ⅳ-1)所示化合物的方法依次包括如下步骤:The method for the compound shown in the preparation formula (IV-1) comprises the following steps in sequence:

(1)将所述菌株L437接种至发酵培养基,避光、25-30℃(如28℃)、振荡培养30-50天(如40天);(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)取步骤(1)得到的发酵体系,过滤分别收集菌丝体和发酵上清;将所述菌丝体加入乙酸乙酯,进行超声破碎,收集提取液,即为浸提液甲;将所述发酵上清用乙酸乙酯萃取,取有机相,即为浸提液乙;将所述浸提液甲和所述浸提液乙合并,即为浸提液丙;(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)将步骤(2)得到的浸提液进行旋转蒸发,得到粗浸膏;(3) Perform rotary evaporation on the extract obtained in step (2) to obtain crude extract;

(4)取步骤(3)得到的粗浸膏,进行经硅胶柱层析,以石油醚和丙酮组成的洗脱液进行梯度洗脱,洗脱过程中石油醚和丙酮的体积比分别为100:1、50:1、30:1、20:1、10:1、9:1、8:2,收集石油醚和丙酮的体积比为8:2的洗脱液的过柱后溶液,旋转蒸发后得到干物质B;(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)将步骤(4)得到的干物质B进行反相柱层析,以甲醇和水组成的洗脱液进行梯度洗脱,甲醇在洗脱液中所占的体积比依次为20%、30%、50%、70%,收集甲醇浓度为70%的洗脱液的过柱后溶液并旋转蒸发,得到干物质D。(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)将步骤(5)得到的干物质D进行HPLC层析,以甲醇-水(体积比为72:28)为洗脱液,收集保留时间为57.3min的色谱峰、旋转蒸发后得到式(Ⅳ-2)所示化合物。(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).

发酵培养基(pH6.5)的制备方法具体可为:取200g马铃薯,洗净去皮切成块,加水煮沸半个小时,用纱布过滤收集滤液,加入20g葡萄糖,用水定容至1L。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.

式(Ⅰ)所示化合物既可以采用微生物发酵法制备得到,也可以通过化学合成法制备得到。用于制备式(Ⅰ)所示化合物的微生物不限于所述菌株L437。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.

以上任一所述炎症反应可为接触性皮炎、皮肤过敏、足跖肿胀或哮喘患者支气管肺泡的炎症细胞聚集。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.

本发明公开了一种新化合物,并提供了该化合物在抑制炎症中的应用以及该化合物的制备方法,该化合物对多种炎症具有抑制作用,可作为药物或保健品开发利用。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.

具体实施方式Detailed ways

以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。RAW264.7细胞即小鼠单核巨噬细胞,ATCC编号为TIB-71。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.

实施例1、菌株L437的分离和鉴定Embodiment 1, isolation and identification of bacterial strain L437

一、菌株的分离1. Isolation of strains

发明人在微生物次生代谢产物活性化合物的高通量筛选基础上,从云南一株水稻叶片上分离得到一株纯培养的菌株,将其命名为菌株L437。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.

二、菌株的鉴定2. Identification of strains

1、形态和生理生化鉴定1. Morphological and physiological and biochemical identification

菌株L437在5-35℃均可生长,以28℃左右最适。菌株L437的分生孢子梗基部膨大暗褐色,往上渐细颜色渐淡,顶端曲膝状,不分枝,最长可达600mm,宽4-8mm。菌株L437的分生孢子为褐色,长梭形,微弯曲,6-14个膈,(63-153)mm×(14-22)mm。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、分子鉴定2. Molecular identification

菌株L437的ITS序列如序列表的序列1所示。The ITS sequence of strain L437 is shown in sequence 1 of the sequence listing.

综合形态鉴定、生理生化鉴定和分子鉴定的结果,菌株L437属于薏平脐蠕孢(Bipolaris coicis)。Based on the results of morphological identification, physiological and biochemical identification and molecular identification, the strain L437 belonged to Bipolaris coicis.

三、菌株的保藏3. Preservation of strains

菌株L437已于2013年6月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101),保藏编号为CGMCC No.7714。薏平脐蠕孢(Bipolaris coicis)L437CGMCC No.7714简称菌株L437。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.

实施例2、式(Ⅰ)所示化合物的制备The preparation of the compound shown in embodiment 2, formula (I)

一、式(Ⅰ)所示化合物的制备One, the preparation of the compound shown in formula (I)

1、预培养1. Pre-culture

将菌株L437接种到PDA培养基斜面上,28℃避光培养7天,菌丝长满整个斜面。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、将步骤1得到的菌丝体转接到种子培养基中,28℃、180rpm避光培养4天,得到种子液。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.

种子培养基:溶剂为水,溶质及其含量如下:葡萄糖4.0g/L、麦芽提取物(MaltExtract)10.0g/L、酵母提取物(Yeast Extract;购自Oxoid Ltd.,批号为1074139)4.0g/L。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、将10mL种子培养液接种至发酵培养基中,28℃避光静置培养40天。3. Inoculate 10 mL of seed culture solution into the fermentation medium, and culture at 28°C in the dark for 40 days.

发酵培养基的制备方法:取80g大米放入500mL锥形瓶,加入120mL自来水,灭菌(121℃,30min)。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).

二、式(Ⅰ)所示化合物的提取分离2. Extraction and separation of compounds represented by formula (I)

1、取1.6Kg步骤一得到的发酵产物,加入8000mL乙酸乙酯,在冰浴中超声破碎(超声频率40Hz,时间为30min),收集提取液;相同的方法重复提取三次;将三次的提取液合并,即为浸提液。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、将步骤1中浸提液进行旋转蒸发(温度40℃,转速100rpm,真空度<3mmHg),得到20g粗浸膏。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、取20g粗浸膏,进行经硅胶柱层析(青岛海洋化工有限公司200-300目,柱层析硅胶400g,Φ7.5×100cm),以石油醚和丙酮组成的洗脱液进行梯度洗脱,洗脱过程中石油醚和丙酮的体积比分别为100:1、50:1、30:1、20:1、10:1、9:1、8:2、7:3和6:4,每种洗脱液洗脱4000mL,收集石油醚和丙酮的体积比为10:1的洗脱液的过柱后溶液,旋转蒸发(温度40℃,转速100rpm,真空度<3mmHg)后得到500mg干物质A;收集石油醚和丙酮的体积比为8:2的洗脱液的过柱后溶液,旋转蒸发(温度40℃,转速100rpm,真空度<3mmHg)后得到1000mg干物质B;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、将步骤3得到的干物质A进行凝胶柱层析(Sephadex LH-20,AmershamBiosciences,Φ2×90cm),以等体积混合的氯仿-甲醇为洗脱液,收集第100mL至140mL的过柱后溶液,旋转蒸发(温度40℃,转速100rpm,真空度<3mmHg)后得到75mg干物质C。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、将步骤4得到的干物质C进行HPLC层析(RP-18,250×10mm Column,5μm,流速3ml/min),以甲醇-水(体积比为75:25)为洗脱液,收集保留时间为18.7min的色谱峰,旋转蒸发(温度40℃,转速100rpm,真空度<3mmHg)后,得到16mg干物质甲,干物质甲经结构确证为式(Ⅱ-2)所示化合物。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、将步骤3得到的干物质B进行反相柱层析(ODS,YMC),以甲醇和水组成的洗脱液进行梯度洗脱,甲醇在洗脱液中所占的体积比依次为20%,30%,50%,70%,90%,100%,每种甲醇浓度洗脱100毫升,收集甲醇浓度为70%的洗脱液的过柱后溶液并旋转蒸发(温度40℃,转速100rpm,真空度<3mmHg),得到235mg干物质D。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、将步骤6得到的干物质D进行HPLC层析(RP-18,250×10mm Column,5μm,流速3ml/min),以甲醇-水(体积比为72:28)为洗脱液,收集保留时间为32.9min的色谱峰、旋转蒸发后得到100mg干物质乙、经结构确证为式(Ⅲ-2)所示化合物,收集保留时间为57.3min的色谱峰、旋转蒸发后得到15mg干物质丙、经结构确证为式(Ⅳ-2)所示化合物。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).

三、化合物的结构确证数据3. The structure confirmation data of the compound

式(Ⅱ-2)所示化合物的结构确证数据:黄色油状;UV(n-hexane)λmax(log ε):263nm(4.20);CD(c3.93×10-4M,n-hexane)λmax(Δ ε)218(-15.4),260(+28.9),335(–1.04)nm;IR(νmax):3400,2960,2875,1656,1643,1623,1462,1397,1147,1034,931cm-1;Positive HRESIMS:m/z[M+H]+319.2279(calcd.for C20H31O3,319.2268);1H NMR和13C NMR数据见表1。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.

式(Ⅲ-2)所示化合物的结构确证数据:黄色油状;UV(n-hexane)λmax(log ε):263nm(4.10);CD(c1.20×10-3M,n-hexane)λmax(Δ ε)219(-1.21),259(+1.88),344(–0.09)nm;IR(νmax):2963,2931,2877,1739,1657,1623,1463,1399,1200,1158,1035,983,956,932cm-1;PositiveHRESIMS:m/z[M+H]+419.2425(calcd.for C24H35O6,419.2428);1H NMR和13C NMR数据见表1。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.

式(Ⅳ-2)所示化合物的结构确证数据:黄色油状;UV(n-hexane)λmax(log ε):263nm(4.12);CD(c2.89×10-4M,n-hexane)λmax(Δ ε)257(-0.75)nm;IR(νmax):3465,2960,2930,2876,1741,1659,1625,1462,1397,1156,1033,933cm-1;Positive HRESIMS:m/z[M+H]+433.2587(calcd.for C25H37O6,433.2585);1H NMR和13C NMR数据见表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.

表1各个化合物的NMR数据(1H:500MHz;13C:125MHz;溶剂:CDCl3NMR data of each compound in Table 1 ( 1 H: 500MHz; 13 C: 125MHz; solvent: CDCl 3 )

实施例3、式(Ⅰ)所示化合物的制备Preparation of the compound shown in embodiment 3, formula (I)

一、式(Ⅰ)所示化合物的制备One, the preparation of the compound shown in formula (I)

1、同实施例2的步骤一的1。1. Same as step 1 of embodiment 2.

2、同实施例2的步骤一的2。2. Same as step 1 of embodiment 2.

3、将10mL种子培养液接种至发酵培养基中,避光、28℃、180rpm振荡培养40天。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.

发酵培养基(pH6.5)的制备方法:取200g马铃薯,洗净去皮切成小块,加水煮沸半个小时,用纱布过滤收集滤液,加入20g葡萄糖,用水定容至1L。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.

二、式(Ⅰ)所示化合物的提取分离2. Extraction and separation of compounds represented by formula (I)

1、取3150ml步骤一得到的发酵体系,过滤并分别收集菌丝体和发酵上清。1. Take 3150ml of the fermentation system obtained in step 1, filter and collect the mycelia and fermentation supernatant respectively.

2、取步骤1得到的菌丝体,加入1000ml乙酸乙酯,在冰浴中超声破碎(超声频率40Hz,时间为30min),收集提取液;在菌丝体中再加入1000ml乙酸乙酯,采用相同的条件进行超声破碎,收集提取液;在菌丝体中再加入1000ml乙酸乙酯,采用相同的条件进行超声破碎,收集提取液;将三次的提取液合并,即为浸提液甲。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、取步骤1得到的发酵上清,用等体积乙酸乙酯萃取三次,合并三次萃取的有机相,即为浸提液乙。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、将浸提液甲和浸提液乙合并,即为浸提液丙。4. Combine the extract solution A and the extract solution B to obtain the extract solution C.

5、将浸提液丙进行旋转蒸发(温度40℃,转速100rpm,真空度<3mmHg),得到3g粗浸膏。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.

取步骤5得到的粗浸膏,依次按实施例2的步骤二的3、4、5、6和7进行操作,式(Ⅱ-2)所示化合物、式(Ⅲ-2)所示化合物和式(Ⅳ-2)所示化合物的产量分别为2mg、8mg和2.1mg。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.

实施例4、式(Ⅰ)所示化合物对脂多糖诱导的小鼠单核巨噬细胞RAW264.7释放NO的抑制活性测试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

化合物对脂多糖(LPS)诱导RAW264.7细胞释放NO的抑制活性参考文献方法(GiangPM,Jin HZ,Son PT,Lee JH,Hong YS,Lee JJ.ent-Kauranediterpenoids from Crotontonkinensis inhibit LPS-induced NF-κB activation and NO production.Journalof Natural Products,2003,66(9),1217-1220.)。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.).

由于NO极不稳定,在细胞培养上清液中代谢成为亚硝酸基NO2 -,故采用Griess法测定样品中NO2 -的浓度作为衡量NO水平的指标。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.

待测化合物即为实施例2制备的式(Ⅱ-2)所示化合物、式(Ⅲ-2)所示化合物或式(Ⅳ-2)所示化合物。将氢化可的松作为待测化合物的阳性对照。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.

氢化可的松:Sigma,CAS号为50-23-7。Hydrocortisone: Sigma, CAS No. 50-23-7.

1、RAW264.7细胞(ATCC编号为TIB-71)培养于含10%热灭活(56℃、30min)胎牛血清、100U/ml青霉素钠(Gibco)、100μg/ml链霉素(Gibco)的RPMI-1640(Gibco)培养液中,37℃、5%CO2的恒温培养箱中孵育。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、用RPMI-1640培养液将RAW264.7细胞稀释制成1×105个/mL的单细胞悬液,接种于96孔板中(每孔200μL),每组设三个平行孔。CO2培养箱中培养1h后每孔加入终浓度为1μg/mL的LPS(Sigma)和DMSO溶解的不同浓度的待测化合物0.4μL,同时设定LPS组(加入LPS并且用DMSO代替待测化合物的DMSO溶液,对NO释放的抑制率为0%)和空白对照组(不加LPS并且用DMSO代替待测化合物的DMSO溶液,对NO释放的抑制率为100%)。37℃、5%CO2的恒温培养箱中培养24h,每孔吸取上清100μL到酶标板上,离心(1000×g、4℃、3min),加入100μL Griess reagent(Griess reagentA:0.1%N-萘乙二胺盐酸盐水溶液;Griess reagent B:1%对氨基苯磺酰胺水溶液+5%磷酸水溶液;使用前将Griess reagent A和Griess reagent B等体积混合)于96孔板混合,室温静止10min后,用酶标仪在540nm处测定OD值。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.

用浓度分别为1、5、10、50μmol/L的NaNO2绘制标准曲线,根据NaNO2标准曲线计算细胞培养上清液中NO2-的浓度以及对NO释放的抑制率,抑制率计算公式为: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 :

IC50值即为NO抑制率为50%时对应的待测化合物或阳性对照的浓度。The IC 50 value is the concentration of the test compound or positive control corresponding to the NO inhibition rate of 50%.

表2待测化合物和氢化可的松的IC50值(μM)Table 2 IC 50 values of test compounds and hydrocortisone (μM)

编号serial number IC50(μM) IC50 (μM) 式(Ⅱ-2)所示化合物Compounds represented by formula (II-2) 16.34±1.1216.34±1.12 式(Ⅲ-2)所示化合物Compounds represented by formula (Ⅲ-2) 23.55±1.3723.55±1.37 式(Ⅳ-2)所示化合物Compounds represented by formula (IV-2) 10.82±0.8310.82±0.83 氢化可的松Hydrocortisone 46.25±2.2746.25±2.27

结果如表2所示,式(Ⅱ-2)所示化合物、式(Ⅲ-2)所示化合物和式(Ⅳ-2)所示化合物均对小鼠单核巨噬细胞的NO释放显示出较强的抑制力,即具有较强的抗炎活性。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.

实施例5、式(Ⅰ)所示化合物抗二硝基氟苯(DNFB)诱发的变应性接触性皮炎的活性测试Example 5. Anti-allergic contact dermatitis induced by dinitrofluorobenzene (DNFB) activity test of the compound represented by formula (I)

地塞米松(DEX):天津药业集团新郑股份有限公司。二硝基氟苯(又称DNFB;CAS:70-34-8):上海如吉生物科技发展有限公司。BALB/c雄性小鼠:北京维通利华实验动物技术有限公司。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.

将BALB/c雄性小鼠随机分为模型组(DNFB组)、测试样品组-1(DNFB-1组)、测试样品组-2(DNFB-2组)、测试样品组-3(DNFB-3组)和地塞米松组(DNFB-DEX组),每组10只。实验第1至第7天,每天灌胃给药;实验第8天,用DNFB-乙醇溶液(即将15mg DNFB溶于1ml乙醇)在小鼠背部皮下注射致敏,每只小鼠注射50μL;实验第13天,在小鼠右耳上涂丙酮-橄榄油溶液(即将4体积份丙酮与1体积份橄榄油混合)作为抗原攻击,每只小鼠涂抹25μL,24h后小鼠产生耳接触性皮炎;抗原攻击24小时后,用直径8mm打孔器取下耳组织片,称重,计算耳廓肿胀抑制率(%)。模型组用蒸馏水灌胃,每天每只小鼠0.1毫升。测试样品组-1用式(Ⅱ-2)所示化合物的水溶液灌胃,每天的剂量为50mg/kg小鼠体重,每天每只小鼠0.1毫升。测试样品组-2用式(Ⅲ-2)所示化合物的水溶液灌胃,每天的剂量为50mg/kg小鼠体重,每天每只小鼠0.1毫升。测试样品组-3用式(Ⅳ-2)所示化合物的水溶液灌胃,每天的剂量为50mg/kg小鼠体重,每天每只小鼠0.1毫升。地塞米松组用地塞米松的水溶液灌胃,每天的剂量为1mg/kg小鼠体重,每天每只小鼠0.1毫升。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.

耳肿胀抑制率=【1-(给药组重量差/模型组重量差)】*100%;Inhibition rate of ear swelling = [1-(difference in weight of administration group/difference in weight of model group)]*100%;

重量差=右耳耳组织片重量-左耳耳组织片重量。Weight difference = weight of ear tissue piece of right ear - weight of ear tissue piece of left ear.

结果见表3。式(Ⅱ-2)所示化合物、式(Ⅲ-2)所示化合物和式(Ⅳ-2)所示化合物均显示了很好的抗DNFB诱发变应性皮炎的作用。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.

表3化合物抗DNFB诱发变应性皮炎作用Table 3 compound anti-DNFB induced allergic dermatitis effect

组别group 右耳耳组织片重量(mg)Weight of right ear ear tissue piece (mg) 抑制率(%)Inhibition rate(%) DNFB组DNFB group 31.2±1.431.2±1.4 -- DNFB-1组DNFB-1 group 17.6±1.417.6±1.4 43.5943.59 DNFB-2组DNFB-2 group 24.6±2.124.6±2.1 21.1521.15 DNFB-3组DNFB-3 group 16.6±1.816.6±1.8 46.7946.79 DNFB-DEX组DNFB-DEX group 9.3±1.29.3±1.2 70.1970.19

实施例6、式(Ⅰ)所示化合物抗异种被动皮肤过敏反应的活性测试Example 6. Activity test of the compound represented by formula (I) against heterogeneous passive skin allergic reaction

百日破疫苗:成都生物制品研究所,批号20090403。卵白蛋白:中国医药集团上海化学试剂公司,批号F20090702。酮替芬:sigma chemical Co.。雄性清洁级BALB/c小鼠:北京维通利华实验动物技术有限公司。雄性SD大鼠:北京维通利华实验动物技术有限公司。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.

参照Tada等人方法利用卵白蛋白及百白破疫苗制备大鼠抗卵白蛋白血清(anti-OVA),具体方法为:100-200g的SD大鼠,用1%卵白蛋白的生理盐水溶液(即1g卵白蛋白加入到100ml生理盐水中)按照10mg/kg大鼠的剂量肌肉注射于后腿两侧,同时腹腔注射百白破疫苗2×1010U/只,13天后(12-14天为IgE高峰时间)处死动物采血,低速离心,分离血清,置于冰箱中保存。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.

将7周龄18~22g雄性清洁级BALB/c小鼠随机分成5组,每组10只;分别为anti-OVA组(对照组)、anti-OVA-1组、anti-OVA-2组、anti-OVA-3组和anti-OVA-酮替芬组。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.

实验第1至第7天,每天口服给药;实验第8天,将10μl anti-OVA注射到小鼠右耳皮内进行致敏(异种被动皮肤过敏反应),左耳皮内注射等量生理盐水;致敏48h后,按0.1ml/10g体重的剂量向小鼠尾静脉内注射1%卵白蛋白伊文斯兰生理盐水溶液(1g伊文斯兰、1g卵白蛋白溶于100ml生理盐水)进行攻击,30min后脱颈处死,用8mm直径打孔器分别在左右两耳同一部位打下圆耳片,测定左、右耳组织块重量差,计算耳肿胀抑制率(%)。anti-OVA组口服蒸馏水,每天每只小鼠0.1毫升。anti-OVA-1组口服式(Ⅱ-2)所示化合物的水溶液,每天的剂量为50mg/kg小鼠体重,每天每只小鼠0.1毫升。anti-OVA-2组口服式(Ⅲ-2)所示化合物的水溶液,每天的剂量为50mg/kg小鼠体重,每天每只小鼠0.1毫升。anti-OVA-3组口服式(Ⅳ-2)所示化合物的水溶液,每天的剂量为50mg/kg小鼠体重,每天每只小鼠0.1毫升。anti-OVA-酮替芬组口服酮替芬的水溶液,每天的剂量为5mg/kg小鼠体重,每天每只小鼠0.1毫升。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.

耳肿胀抑制率=【1-(给药组重量差/对照组重量差)】*100%。Ear swelling inhibition rate = [1-(difference in weight of administration group/difference in weight of control group)]*100%.

重量差=右耳耳片重量-左耳耳片重量。Weight difference = ear piece weight of right ear - ear piece weight of left ear.

结果见表4。式(Ⅱ-2)所示化合物、式(Ⅲ-2)所示化合物和式(Ⅳ-2)所示化合物均显示了很好的抗异种被动皮肤过敏反应的作用。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.

表4化合物抗异种被动皮肤过敏反应的作用The effect of table 4 compounds against heterogeneous passive skin allergic reaction

组别group 右耳耳组织片重量(mg)Weight of right ear ear tissue piece (mg) 抑制率(%)Inhibition rate(%) anti-OVA组anti-OVA group 28.8±2.528.8±2.5 -- anti-OVA-1组anti-OVA-1 group 18.8±1.118.8±1.1 34.734.7 anti-OVA-2组anti-OVA-2 group 24.6±1.524.6±1.5 14.614.6 anti-OVA-3组anti-OVA-3 group 13.5±2.313.5±2.3 53.153.1 anti-OVA-酮替芬组anti-OVA-ketotifen group 9.2±0.99.2±0.9 68.168.1

实施例7、式(Ⅰ)所示化合物抗角叉菜胶诱发小鼠足跖肿胀的活性测试Example 7. Activity test of the compound represented by formula (I) against carrageenan-induced paw swelling in mice

角叉菜胶:sigma,9000-07-1。Carrageenan: sigma, 9000-07-1.

将7周龄18~22g雄性清洁级BALB/c小鼠随机分成5组,每组10只;分别为Carrageenan组(对照组)、Carrageenan-1组、Carrageenan-2组、Carrageenan-3组和Carrageenan-酮替芬组。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.

实验第1天,第一次灌胃给药,第一次给药1小时后,每只小鼠左足跖皮下注射1g/100mL角叉菜胶的生理盐水溶液0.1ml以致炎(右足跖皮下注射等体积生理盐水),致炎1小时后第二次灌胃给药,第二次给药3小时后,用Digimatic卡尺(分辨率为0.01mm)测量正常足跖(右)和肿胀足跖(左)的厚度,计算浮肿抑制率(%)。第一次给药和第二次给药的剂量相同。Carrageenan组给予蒸馏水,每次每只小鼠0.1毫升。Carrageenan-1组给予式(Ⅱ-2)所示化合物的水溶液,每次的剂量为50mg/kg小鼠体重,每次每只小鼠0.1毫升。Carrageenan-2组给予式(Ⅲ-2)所示化合物的水溶液,每次的剂量为50mg/kg小鼠体重,每次每只小鼠0.1毫升。Carrageenan-3组给予式(Ⅳ-2)所示化合物的水溶液,每次的剂量为50mg/kg小鼠体重,每次每只小鼠0.1毫升。Carrageenan-酮替芬组给予酮替芬的水溶液,每次的剂量为5mg/kg小鼠体重,每次每只小鼠0.1毫升。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.

浮肿抑制率(%)=【1-(给药组厚度差/对照组厚度差)】*100%。Edema inhibition rate (%)=[1-(difference in thickness of administration group/difference in thickness of control group)]*100%.

厚度差=肿胀足跖(左)的厚度-正常足跖(右)的厚度。Thickness difference = thickness of swollen sole (left) - thickness of normal sole (right).

结果见表5。式(Ⅱ-2)所示化合物、式(Ⅲ-2)所示化合物和式(Ⅳ-2)所示化合物均显示了很好的抗角叉菜胶诱发小鼠足跖肿胀的作用。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.

表5式(Ⅰ)所示化合物抗角叉菜胶诱发小鼠足跖肿胀的作用The compound shown in table 5 formula (I) resists the effect of carrageenan-induced mouse paw swelling

组别group 肿胀足跖的厚度(mm)Thickness of swollen paw (mm) 抑制率(%)Inhibition rate(%) Carrageenan组Carrageenan group 1.56±0.161.56±0.16 -- Carrageenan-1组Carrageenan - Group 1 1.25±0.241.25±0.24 19.8719.87 Carrageenan-2组Carrageenan - Group 2 1.33±0.181.33±0.18 14.7414.74 Carrageenan-3组Carrageenan - Group 3 1.19±0.111.19±0.11 23.7223.72 Carrageenan-酮替芬组Carrageenan-ketotifen group 0.76±0.130.76±0.13 51.2851.28

实施例8、式(Ⅰ)所示化合物对致敏小鼠抗原攻击后支气管肺灌流液中炎症细胞的影响Example 8. The effect of the compound represented by formula (I) on inflammatory cells in the bronchopulmonary perfusate of sensitized mice after antigen challenge

卵白蛋白:中国医药集团上海化学试剂公司,批号F20090702。雄性清洁级BALB/c小鼠:北京维通利华实验动物技术有限公司。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.

7周龄18-22g雄性清洁级BALB/c小鼠60只,随机分为6组,每组10只;分别为:空白对照组、模型对照组(不进行任何处理)、实验组-1、实验组-2、实验组-3和阳性对照组。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.

实验第1天,每只小鼠乙醚麻醉后用0.1%卵白蛋白氢氧化铝凝胶溶液(0.1g卵白蛋白加入到含有10g氢氧化铝的100ml生理盐水中)于两后足掌、两腹股沟、两腋下、颈部以及背部共八点皮下注射(每点注射0.4毫升)进行致敏;实验第10天,每只小鼠在所述八点皮下注射0.1%卵白蛋白氢氧化铝凝胶溶液(每点注射0.2毫升)再次致敏;实验第21天开始,每天用1%的卵白蛋白溶液(0.1g卵白蛋白加入到100ml生理盐水中)雾化吸入进行攻击,每次30分钟,连续攻击6天,每次攻击前1小时除模型对照组以外的各种小鼠通过灌胃给药;末次攻击24小时后脱臼处死小鼠,纵行剪开颈部皮肤,分离暴露气管,行气管插管,用灌洗液(Thornwell-Mcdowell溶液:NaCl6.6g、KCl0.46g、CaCl20.05g、NaHCO32.52g、MgCl20.09g、Na2HPO40.1g,加水至1000mL)灌洗,每次0.5mL,共3次,收集支气管肺泡灌洗液,摇匀后取出50微升,用白细胞稀释液(恒大百盛生物北京科技发展有限公司)稀释4倍,取少量滴于细胞计数板上,在显微镜下计数白细胞总数;将支气管肺泡灌洗液低温1000rpm离心10min,弃去上清液,用移液器打匀后涂片,室温干燥,瑞氏染液[碱性染料美蓝和酸性染料黄色伊红合称伊红美蓝染料,即瑞氏染料]染色6-8min,用瑞氏染液复染1分钟,用自来水将染料冲洗干净,在光学显微镜40倍镜下进行白细胞分类计数,计数200个细胞,计算淋巴单核细胞(核染成紫色,染色质疏松,胞浆染成浅灰蓝色)以及嗜酸性粒细胞(胞浆内含有粗大的鲜红色颗粒,核多数为两叶)的百分比。空白对照组给予生理盐水,每次每只小鼠0.1毫升。实验组-1给予式(Ⅱ-2)所示化合物的水溶液,每次的剂量为50mg/kg小鼠体重,每次每只小鼠0.1毫升。实验组-2给予式(Ⅲ-2)所示化合物的水溶液,每次的剂量为50mg/kg小鼠体重,每次每只小鼠0.1毫升。实验组-3给予式(Ⅳ-2)所示化合物的水溶液,每次的剂量为50mg/kg小鼠体重,每次每只小鼠0.1毫升。阳性对照组给予地塞米松的水溶液,每次的剂量为1mg/kg小鼠体重,每次每只小鼠0.1毫升。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.

结果见表6,式(Ⅱ-2)所示化合物、式(Ⅲ-2)所示化合物和式(Ⅳ-2)所示化合物对小鼠哮喘模型支气管肺泡的炎症细胞聚集有明显的抑制作用。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 .

表6化合物对鼠哮喘模型支气管肺泡炎症细胞的作用The effect of table 6 compounds on bronchoalveolar inflammatory cells in mouse asthma model

组别group 白细胞总数(×104/ml)Total white blood cells (×10 4 /ml) 嗜酸性粒细胞(%)Eosinophils (%) 淋巴单核细胞(%)Lymphoid mononuclear cells (%) 正常对照组normal control group 15.6±4.415.6±4.4 12.6±3.412.6±3.4 86.6±3.486.6±3.4 模型对照组Model control group 123.8±22.2123.8±22.2 70.8±9.470.8±9.4 43.6±8.443.6±8.4 实验组-1Experimental group-1 63.6±18.363.6±18.3 53.6±11.353.6±11.3 47.6±9.847.6±9.8 实验组-2Experimental group-2 77.6±10.877.6±10.8 52.5±6.452.5±6.4 45.6±7.245.6±7.2 实验组-3Experimental group-3 52.3±12.352.3±12.3 41.3±4.441.3±4.4 55.8±5.455.8±5.4 阳性对照组positive control group 40.6±6.340.6±6.3 30.1±10.130.1±10.1 68.6±11.668.6±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.
CN201310254586.XA 2013-06-25 2013-06-25 Coicenal diterpenoid compound and preparation method and application thereof in preparation of anti-inflammatory drugs Expired - Fee Related CN103319442B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310254586.XA CN103319442B (en) 2013-06-25 2013-06-25 Coicenal diterpenoid compound and preparation method and application thereof in preparation of anti-inflammatory drugs

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310254586.XA CN103319442B (en) 2013-06-25 2013-06-25 Coicenal diterpenoid compound and preparation method and application thereof in preparation of anti-inflammatory drugs

Publications (2)

Publication Number Publication Date
CN103319442A CN103319442A (en) 2013-09-25
CN103319442B true CN103319442B (en) 2015-04-08

Family

ID=49188534

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310254586.XA Expired - Fee Related CN103319442B (en) 2013-06-25 2013-06-25 Coicenal diterpenoid compound and preparation method and application thereof in preparation of anti-inflammatory drugs

Country Status (1)

Country Link
CN (1) CN103319442B (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1884559A (en) * 2006-06-06 2006-12-27 中国水稻研究所 Goosegrass bipolaris crude toxin extraction process and formulation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1884559A (en) * 2006-06-06 2006-12-27 中国水稻研究所 Goosegrass bipolaris crude toxin extraction process and formulation

Also Published As

Publication number Publication date
CN103319442A (en) 2013-09-25

Similar Documents

Publication Publication Date Title
CN104825462B (en) The antiphlogistic use of plain boiled pork Ganodenna Lucidum P.E
CN102516344B (en) Compound with antitumor activity and preparation method and application thereof
WO2017092230A1 (en) Biflavone compound and uses thereof for treating cancers and preparing drugs
CN102731276B (en) Diterpene compound possessing antitumor activity, preparation method thereof and application thereof
CN101367802B (en) Beta-kabarin alkaloids in quassia wood, preparation method and application thereof
CN111377994A (en) Seven withanolides compounds from cape gooseberry and preparation method and application thereof
CN101803531B (en) China cryptoporus sinensis and solid cultural method and application thereof
CN103319442B (en) Coicenal diterpenoid compound and preparation method and application thereof in preparation of anti-inflammatory drugs
CN107011302A (en) Phenol acid amides I and its extracting method and application
CN117658996A (en) Cinnamoyl flavan alkaloids and preparation methods and applications thereof
CN103275138B (en) Sixteen carbon diacetylated no double bond lactone type sophorolipid and its application
CN103275139B (en) 16 carbon diacetyl one double bond lactone type sophorolipid and application thereof
CN106632378A (en) Compound capable of inhibiting mast cell degranulation and preparation method and application thereof
CN106333969A (en) Agaricus gennadii HDAC1 enzyme inhibition effective part and preparation method and application
CN108276363B (en) Secondary metabolites of Aspergillus brucellis and their application in the preparation of antifungal drugs
CN114591286A (en) Novel macrolide compound acautalides A-C and preparation method and application thereof
CN106236842B (en) Effective part of tinospora sinensis for inhibiting HDAC1 enzyme, preparation method and application
CN107177513B (en) Penicillium commune T3-1 and its fermented cpds, method for extraction and purification and the application in antiallergy
CN103275140B (en) The two key lactone type sophorolipids of 18 one of carbon diacetyl and application thereof
CN103450142B (en) A kind of chroman compound and its extraction method and application
CN105085221B (en) Compound with antifungal and anti-tumor activity and preparation method and application
CN101805699B (en) Preparation method and application of gliocladicin C
CN112047887B (en) A kind of wide gluten amide and preparation method and application thereof
CN106176982A (en) Black top Herba corydalis racemosae effective site and its preparation method and application
CN107362238A (en) A kind of Schima superba extract and its application

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20150408

Termination date: 20200625