CN117142939A - 一种倍半萜化合物及其制备方法和用途及其制备的药物 - Google Patents
一种倍半萜化合物及其制备方法和用途及其制备的药物 Download PDFInfo
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Abstract
本发明提供了一种倍半萜化合物及其制备方法和用途及其制备的药物,属于药物领域。本发明环橙花烷倍半萜类化合物是式I所示的化合物、其盐、其立体异构体。本发明从钩状木霉菌中提取得到一种新的化合物,该化合物对多种农业病害真菌具有抑制效果,其中对油菜菌核、水稻纹枯具有显著的抑制活性,对黄瓜灰霉、玉米茎基腐、草莓炭疽具有良好的抑制活性,对辣椒疫霉、小麦赤霉、花生褐斑有一定抑制活性。本发明化合物可用于农业病害真菌的防治,具有良好的应用前景。式I。
Description
技术领域
本发明属于药物领域,具体涉及一种倍半萜化合物及其制备方法和用途及其制备的药物。
背景技术
钩状木霉菌(Trichoderma hamatum)为子囊菌门(Ascomycota),子囊菌纲(Sordariomycetes),肉座菌目(Hypocreales),肉座菌科(Hypocreaceae),木霉属(Trichodermasp.)真菌。
木霉属(Trichoderma spp.)真菌被广泛用于植物病害生物防治,其生物防治机制多样。抗生作用是木霉属真菌的主要生防机制之一,研究表明,钩状木霉菌产生的次生代谢产物不仅能够直接抑制病原菌的生长,还能够触发植物防御系统或促进植物营养生长从而提高植物抗病性。
但是目前对钩状木霉菌的次生代谢产物研究较少。如果能够找到钩状木霉菌的次生代谢产物中有效的抑菌成分,对于植物病害生物防治具有重要意义。
发明内容
本发明的目的是提供一种倍半萜化合物及其制备方法和用途及其制备的药物。
本发明提供了式I所示的化合物、其盐、其立体异构体:
式I。
进一步地,所述化合物为式II所示:
式II。
本发明还提供了前述化合物的制备方法,它包括如下步骤:
(1)将钩状木霉菌活化后,于培养基中培养,得钩状木霉菌菌丝和发酵液混合物;
(2)将钩状木霉菌菌丝和发酵液混合物离心,取上清发酵液用乙酸乙酯浸泡后萃取,收集乙酸乙酯层并减压浓缩,得到乙酸乙酯部位提取物;
(3)乙酸乙酯部位提取物经硅胶柱层析进行粗分,使用石油醚和丙酮混合溶液进行梯度洗脱,得到7个组分Fr.1~Fr.7;
(4)组分Fr.3经轴向压缩柱,使用甲醇和水混合溶液梯度洗脱得6个组分Fr.3-1~Fr.3-6;
(5)Fr.3-4采用凝胶柱,用无水甲醇进行等度洗脱,得到5个组分Fr.3-4-1~Fr.3-4-5;
(6)Fr.3-4-1通过半制备液相色谱进行纯化得到式II所示化合物。
进一步地,
步骤(1)中,所述将钩状木霉菌活化为将钩状木霉菌菌株接种至PDA培养基上活化1~5天;
和/或,步骤(1)中,所述培养基每1L的组分为麦芽糖20 g、味精10 g、葡萄糖10 g、酵母膏3 g、玉米浆1 g、甘露醇20 g、KH2PO40.5 g、MgSO4 .7H2O 0.3 g、CaCO320 g、5-氮杂胞苷10 mg、余量为蒸馏水;
和/或,步骤(1)中,所述培养的温度为28~30℃,培养的时间为14-21天,培养时振荡条件为120~150 r/min。
进一步地,
步骤(2)中,所述上清发酵液和乙酸乙酯体积比为1:1~5;
和/或,步骤(2)中,所述浸泡时间为24~48小时;
和/或,步骤(2)中,所述萃取次数为1~3次;
和/或,步骤(3)中,所述硅胶为200~300目硅胶;
和/或,步骤(3)中,所述梯度洗脱时,石油醚和丙酮混合溶液的体积比依次为30:1、25:1、20:1、15:1、10:1、9:1、8:1、6:1、5:1、4:1、3:1、2:1、1:1;
和/或,步骤(4)中,所述梯度洗脱时,甲醇和水混合溶液中甲醇起始体积百分比浓度为50%,甲醇浓度随时间呈梯度递增,到20min时甲醇的体积百分比浓度为100%;
和/或,步骤(4)中,所述梯度洗脱时间为20min;
和/或,步骤(4)中,所述洗脱速度为150~170mL/min。
进一步地,
步骤(6)中,所述纯化的条件为:色谱柱为Kromasil 100-5-C18柱,柱长为250 mm,内径为10 mm,填料粒径为4.5 μm;流动相为乙腈和水的混合溶液,乙腈和水的体积比为18:82;流速为3mL/min;检测波长为190nm和210 nm,柱温为30°C。
本发明还提供了前述的化合物、其盐、其立体异构体在制备预防和/或治疗农业病害真菌的药物中的用途。
进一步地,所述农业病害真菌为油菜菌核、水稻纹枯、黄瓜灰霉、玉米茎基腐、草莓炭疽、辣椒疫霉、小麦赤霉和/或花生褐斑。
本发明还提供了一种预防和/或治疗农业病害真菌的药物,它是以前述的化合物、其盐、其立体异构体为活性成分,加上药学上可接受的辅料制备而成的制剂。
进一步地,所述农业病害真菌为油菜菌核、水稻纹枯、黄瓜灰霉、玉米茎基腐、草莓炭疽、辣椒疫霉、小麦赤霉和/或花生褐斑。
与现有技术相比,本发明的有益效果:
本发明从钩状木霉菌中提取得到一种新的化合物,该化合物对多种农业病害真菌具有抑制效果,其中对油菜菌核、水稻纹枯病菌具有显著的抑制活性,对黄瓜灰霉、玉米茎基腐、草莓炭疽具有良好的抑制活性,对辣椒疫霉、小麦赤霉、花生褐斑有一定抑制活性。本发明化合物可用于农业病害真菌的防治,具有良好的应用前景。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明化合物的ESI-MS谱图。
图2为本发明化合物在氘代甲醇中的紫外谱图。
图3为本发明化合物在氘代甲醇中的红外光谱图。
图4为本发明化合物在氘代氯仿中的1H NMR谱图。
图5为本发明化合物在氘代氯仿中的13C NMR谱图。
图6为本发明化合物在氘代氯仿中的1H-1H COSY谱图。
图7为本发明化合物在氘代氯仿中的HSQC谱图。
图8为本发明化合物在氘代氯仿中的HMBC谱图。
图9为本发明化合物在氘代氯仿中的NOESY谱图。
图10为本发明化合物的圆二色谱图。
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品,通过购买市售产品获得。
1. 仪器与试剂
试剂
真菌2号液体培养基:麦芽糖20 g;味精10 g;葡萄糖10 g;酵母膏3 g;玉米浆1 g;甘露醇20 g;KH2PO40.5 g;MgSO4 .7H2O 0.3 g;CaCO320 g;5-氮杂胞苷10 mg;蒸馏水1 L;
氘代甲醇(阿达玛斯试剂有限公司);氘代氯仿(阿达玛斯贝塔(上海)化学试剂有限公司);分析级甲醇(上海星可高纯溶剂有限公司);石油醚(上海星可高纯溶剂有限公司);丙酮(成都市科隆化学品有限公司);制备级乙腈(上海星可高纯溶剂有限公司);二氯甲烷(上海星可高纯溶剂有限公司);制备级甲醇(海星可高纯溶剂有限公司);分析级乙酸乙酯(海星可高纯溶剂有限公司)。
仪器
柱层析硅胶(200-300目)(青岛海洋化工厂);Diaion HP-20(日本三菱化学公司);MCI填料(日本三菱化学公司);隔水式电热恒温培养箱(上海跃进医疗器械有限公司);超纯水器(四川优普超纯科技有限公司);QExactive UHMR复合四极杆—轨道阱质谱仪(美国赛默飞世尔公司);硅胶(200~300目,青岛海浪硅胶干燥剂有限公司);LDZX-50KBS立式压力蒸汽灭菌器(上海申安医疗器械厂);轴向动态压缩柱(中国江苏汉邦科技);NU3000型紫外检测器(中国江苏汉邦科技);真空干燥箱(DZF型,北京科伟永兴仪器有限公司);Kromasil100-5-C18(10×250mm, 4.5μm,瑞典阿克苏诺贝尔(Akzo Nobel)公司);快速制备液相仪(常州三泰科技仪器有限公司);BrukerBruker-Ascend-700-MHz核磁共振仪(德国布鲁克公司);高速离心机(湘仪离心机仪器有限公司);NP7000型半制备高效液相色谱仪(中国江苏汉邦科技);Bruker Bruker-Ascend-600-MHz核磁共振仪(德国布鲁克公司);超纯水仪(四川优普超纯科技有限公司);真空干燥箱(DZF型,北京科伟永兴仪器有限公司);Perkin-Elmer 241MC旋光仪(美国珀金埃尔默公司);chirascan圆二色谱仪(英国应用光物理有限公司);Cary 600 FT-IR红外光谱仪(美国加州安捷伦科技公司);SL302N型万分之一电子天平(上海民侨精密科学仪器有限公司);SHZ-D(III)型循环水式真空泵(巩义市予华仪器有限责任公司);DLSB-5L/20型低温冷却液循环泵(巩义市予华仪器有限责任公司)。
2. 实验菌株
菌株由生工生物工程(上海)股份有限公司鉴定(GenBank准入号:OR553890, 序列号NR134371.1)经BLAST对比,鉴定为钩状木霉菌(Trichoderma hamatum)。
实施例1、本发明化合物的制备
1. 菌株的扩培
将钩状木霉菌菌株接种至PDA培养基上活化3天,用打孔器取5个菌饼接种于已灭菌冷却的真菌2号液体培养基中(500 mL锥形瓶,每瓶200 mL),将其放置于28℃、120 r/min恒温摇床中培养7天,得到钩状木霉菌种子液。将培养好的种子液接种于已灭菌的真菌2号液体培养基中进行扩大培养,每100 mL培养基接种2.5 mL种子液,将接种后的培养基在28℃、120r/min的恒温摇床中振荡培养14天,得到菌丝和发酵液混合物。
2. 提取及分离
用高速离心机将培养好的菌丝和发酵液混合物离心,上清为发酵液,发酵液用等体积的乙酸乙酯浸泡24小时后萃取三次,收集乙酸乙酯层并减压浓缩,得到乙酸乙酯部位提取物,得到总浸膏126.0g。
浸膏经硅胶(200~300 目)柱层析进行粗分,以体积比为30:1、25:1、20:1、15:1、10:1、9:1、8:1、6:1、5:1、4:1、3:1、2:1、1:1的石油醚和丙酮混合溶液为流动相依次进行梯度洗脱,每个梯度洗脱液总体积为13.2L,根据TLC结果进行合并,共得到7个组分(Fr.1~Fr.7)。
Fr.3经轴向压缩柱洗脱得到得到6个组分(Fr.3-1~ Fr.3-6),洗脱液为甲醇和水混合溶液,洗脱流速为150mL/min,甲醇和水混合溶液中甲醇起始体积百分比浓度为50%,甲醇浓度随时间呈梯度递增,到20min时甲醇的体积百分比浓度为100%,洗脱时间为20min,轴向压缩柱连接有紫外检测器,根据紫外检测器检测到样品而呈现在电脑上的样品峰和峰的保留时间将样品分为6个组分(Fr.3-1~ Fr.3-6)。
Fr.3-4采用凝胶柱,用无水甲醇进行等度洗脱,根据TLC检识结果进行合并,得到5个组分(Fr.3-4-1~ Fr.3-4-5)。
将Fr.3-4-1通过半制备液相色谱进行纯化得到化合物,过Kromasil 100-5-C18柱(柱长为250 mm,内径为10 mm,填料粒径为4.5 μm,瑞典阿克苏诺贝尔(Akzo Nobel)公司);流动相为乙腈和水的混合溶液,乙腈和水的体积比为18:82;即样品制备全程均用体积百分比为18%的乙腈水溶液等度洗脱。流速为3mL/min;氘灯检测波长为190nm和210 nm;柱温为30°C。样品峰保留时间为26.923min。
3. 本发明化合物的表征数据
本发明化合物的结构为:,表征数据如图1~10所示。
本发明化合物为无色油状;C14H24O3;-8 (c 0.025, MeOH);IR (KBr)v max3433,2930, 1623, 1383, cm-1;1H NMR (600 MHz, CDCl3)δ6.92 – 6.85 (1H, m, H-9), 6.13(1H, d,J= 16.0 Hz, H-10), 2.45 (1H, dd,J= 14.8, 7.6 Hz, H-8a), 2.36 (1H, dd,J= 12.6, 8.2 Hz, H-8b), 2.27 (3H, s, H-12), 1.94 – 1.89(1H,m, H-5a), 1.88 –1.84 (1H, m, H-6), 1.74– 1.70 (1H, m, H-3a), 1.65 – 1.61 (1H, m, H-2), 1.58(2H,m, H-3b, H-5b), 1.27 (3H, s,H-13), 1.19 (3H, s, H-14), 1.06 (3H,J= 6.8Hz, d, H-1);13C NMR (151 MHz, CDCl3)δ198.5(C-11), 144.1(C-9), 134.2(C-10),81.4(C-3), 75.0(C-7), 54.9(C-6), 44.6(C-2), 43.8(C-8), 40.4(C-4), 27.2(C-12),26.2(C-13), 25.9(C-14), 24.6(C-5), 14.6(C-1);ESIHRMSm/z205.1588 [M-2H2O+H]+(计算值为C14H21O+[M-2H2O+H]+, 205.1587)。DP4+确定其相对构型为2S*,3R*,6R*,7R*-1,ECD计算确定其绝对构型为2S,3R,6R,7R-1
以下通过具体试验例证明本发明的有益效果。
试验例1、本发明化合物的抗菌活性测试
称取本发明化合物 3mg,溶于0.1ml的DMSO中,配成30000 ppm的母液。用移液枪吸取一定量的母液,按照浓度加入0.5%吐温-80无菌水(吐温-80的体积百分比浓度为0.5%),配成所需的浓度药液。用移液枪吸取1mL药液放入已灭菌的培养皿中,再放入9ml的PDA,摇匀,冷却。用打孔器打取圆形菌饼后用接种针挑至培养皿中央,然后将培养皿置于培养箱25℃中培养即可,48h后测量菌落直径。
试验处理3天后测量菌落直径,计算生长抑制率(%)。
(1)
式中:D表示菌落增长直径;D1表示菌落直径;D2表示菌饼直径。
(2)
式中:I表示菌丝生长抑制率;D0表示空白对照菌落增长直径;Dt表示药剂处理菌落增长直径。
表1.本发明化合物对8种常见的农业病害真菌的抑制效果
a.浓度单位为ppm。
抗菌实验表明:本发明化合物对油菜菌核、水稻纹枯具有显著的抑制活性,对黄瓜灰霉、玉米茎基腐、草莓炭疽具有良好的抑制活性,对辣椒疫霉、小麦赤霉、花生褐斑有一定抑制活性。
综上,本发明从钩状木霉菌中提取得到一种新的化合物,该化合物对多种农业病害真菌具有抑制效果,其中对油菜菌核、水稻纹枯具有显著的抑制活性,对黄瓜灰霉、玉米茎基腐、草莓炭疽具有良好的抑制活性,对辣椒疫霉、小麦赤霉、花生褐斑有一定抑制活性。本发明化合物可用于农业病害真菌的防治,具有良好的应用前景。
Claims (10)
1.式I所示的化合物、其盐、其立体异构体:
式I。
2.根据权利要求1所述的化合物、其盐、其立体异构体,其特征在于:所述化合物为式II所示:
式II。
3.权利要求2所述化合物的制备方法,其特征在于:它包括如下步骤:
(1)将钩状木霉菌活化后,于培养基中培养,得钩状木霉菌菌丝和发酵液混合物;
(2)将钩状木霉菌菌丝和发酵液混合物离心,取上清发酵液用乙酸乙酯浸泡后萃取,收集乙酸乙酯层并减压浓缩,得到乙酸乙酯部位提取物;
(3)乙酸乙酯部位提取物经硅胶柱层析进行粗分,使用石油醚和丙酮混合溶液进行梯度洗脱,得到7个组分Fr.1~Fr.7;
(4)组分Fr.3经轴向压缩柱,使用甲醇和水混合溶液梯度洗脱得6个组分Fr.3-1~Fr.3-6;
(5)Fr.3-4采用凝胶柱,用无水甲醇进行等度洗脱,得到5个组分Fr.3-4-1~Fr.3-4-5;
(6)Fr.3-4-1通过半制备液相色谱进行纯化得到式II所示化合物。
4.根据权利要求3所述的制备方法,其特征在于:
步骤(1)中,所述将钩状木霉菌活化为将钩状木霉菌菌株接种至PDA培养基上活化1~5天;
和/或,步骤(1)中,所述培养基每1L的组分为麦芽糖20 g、味精10 g、葡萄糖10 g、酵母膏3 g、玉米浆1 g、甘露醇20 g、KH2PO4 0.5 g、MgSO4 .7H2O 0.3 g、CaCO3 20 g、5-氮杂胞苷10 mg、余量为蒸馏水;
和/或,步骤(1)中,所述培养的温度为28~30℃,培养的时间为14-21天,培养时振荡条件为120~150 r/min。
5.根据权利要求3所述的制备方法,其特征在于:
步骤(2)中,所述上清发酵液和乙酸乙酯体积比为1:1~5;
和/或,步骤(2)中,所述浸泡时间为24~48小时;
和/或,步骤(2)中,所述萃取次数为1~3次;
和/或,步骤(3)中,所述硅胶为200~300目硅胶;
和/或,步骤(3)中,所述梯度洗脱时,石油醚和丙酮混合溶液的体积比依次为30:1、25:1、20:1、15:1、10:1、9:1、8:1、6:1、5:1、4:1、3:1、2:1、1:1;
和/或,步骤(4)中,所述梯度洗脱时,甲醇和水混合溶液中甲醇起始体积百分比浓度为50%,甲醇浓度随时间呈梯度递增,到20min时甲醇的体积百分比浓度为100%;
和/或,步骤(4)中,所述梯度洗脱时间为20min;
和/或,步骤(4)中,所述洗脱速度为150~170mL/min。
6.根据权利要求3所述的制备方法,其特征在于:
步骤(6)中,所述纯化的条件为:色谱柱为Kromasil 100-5-C18柱,柱长为250 mm,内径为10 mm,填料粒径为4.5 μm;流动相为乙腈和水的混合溶液,乙腈和水的体积比为18:82;流速为3 mL/min;检测波长为190nm和210 nm,柱温为30°C。
7.权利要求1或2所述的化合物、其盐、其立体异构体在制备预防和/或治疗农业病害真菌的药物中的用途。
8.根据权利要求7所述的用途,其特征在于:所述农业病害真菌为油菜菌核、水稻纹枯、黄瓜灰霉、玉米茎基腐、草莓炭疽、辣椒疫霉、小麦赤霉和/或花生褐斑。
9.一种预防和/或治疗农业病害真菌的药物,其特征在于:它是以权利要求1或2所述的化合物、其盐、其立体异构体为活性成分,加上药学上可接受的辅料制备而成的制剂。
10.根据权利要求9所述的药物,其特征在于:所述农业病害真菌为油菜菌核、水稻纹枯、黄瓜灰霉、玉米茎基腐、草莓炭疽、辣椒疫霉、小麦赤霉和/或花生褐斑。
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