CN115991690B - 一种倍半萜酰胺衍生物及其制备方法和应用 - Google Patents
一种倍半萜酰胺衍生物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种倍半萜酰胺衍生物及其制备方法和应用,该制备方法将提取完冰片的龙脑樟叶干燥后粉碎,取粉碎后的龙脑樟叶,用有机溶剂浸泡搅拌提取,旋转蒸发,减压浓缩后得到龙脑樟叶有机溶剂粗提物,将粗提物用温水打散,加入等量萃取剂萃取,减压浓缩得到萃取部位浸膏,用活性炭对萃取部位浸膏进行脱色处理,之后冷冻干燥,得到脱色样品,通过硅胶柱层析,用二氯甲烷和甲醇按体积比100:0、100:2、100:4、10:1、5:1、3:1梯度洗脱得到5个组分,取其中的第3个组分过硅胶柱,用石油醚和乙酸乙酯按体积比9:1、8:2梯度洗脱得到4个亚组份,第2个亚组份再经洗脱即为倍半萜酰胺衍生物。
Description
技术领域
本发明属于植物提取技术领域,涉及一种倍半萜酰胺衍生物及其制备方法和应用。
背景技术
龙脑樟是目前获取天然冰片的最佳植物选择,但对于提取冰片之后的龙脑樟的应用研究很少,利用率极低,造成了一定程度的资源浪费和环境污染,亟待深入研究。对提取冰片之后的龙脑樟叶进行研究,有望从中挖掘出功能分子,实现龙脑樟资源的多功能产业化,最大化利用植物资源。
CN110123853A公开了一种龙脑樟叶片粗提物的提取法,包括如下步骤:S1、在龙脑樟树上采摘新鲜的叶片,并对采摘的新鲜叶片进行清洗;S2、将清洗过后的龙脑樟叶片均摊在大圆簸箕中,并放置在阴凉处,自然晾干龙脑樟叶片表面上的水分,并且在晾干的过程中,需要翻动龙脑樟叶片2-5次;S3、将自然晾干后的龙脑樟叶片放置在粉碎设备中粉碎3-5分钟,然后取出备用;S4、将粉碎后的龙脑樟叶片放置在容器中,并向容器中加入石油醚溶剂,要确保石油醚溶剂能够完全的淹没龙脑樟叶片,并将容器放置在水浴锅中进行水浴加热,让容器中的温度持续保持在40-80度,浸泡24-48小时,然后将龙脑樟叶片捞出,石油醚溶剂仍保留在容器中;S5、再另取一个新的容器,向新容器中倒入新的石油醚溶剂,然后将步骤S4中捞出的龙脑樟叶片放入到新容器中,继续按照步骤S4中的方法进行浸泡,并且重复操作3-6次;S6、最后一次浸泡结束后,将剩余的龙脑樟叶片杂质捞出,并且完全控干龙脑樟叶片杂质上的石油醚溶剂,将每次浸泡使用的石油醚溶剂合并在一起即可得到龙脑樟叶片的粗提物。
CN110123853A是直接提取龙脑樟叶片的粗提物,而没有对提取产物作深入研究,也不是使用提取冰片之后的龙脑樟叶作为原料。
发明内容
为了开发提取冰片之后的龙脑樟叶的价值,本发明的目的在于提供一种从龙脑樟叶中制备倍半萜酰胺衍生物的方法,提取得到了一种倍半萜酰胺衍生物。
本发明采用了下述技术方案:
一种倍半萜酰胺衍生物,其化学结构式如下:
。
本发明还提供了一种从龙脑樟叶中制备倍半萜酰胺衍生物的方法,将提取完冰片的龙脑樟叶干燥后粉碎,取粉碎后的龙脑樟叶,用有机溶剂浸泡搅拌提取,减压浓缩后得到龙脑樟叶有机溶剂粗提物,将粗提物用温水打散,加入等量萃取剂萃取,之后进行旋转蒸发、减压浓缩得到萃取部位浸膏,用活性炭对萃取部位浸膏进行脱色处理,之后冷冻干燥,得到脱色样品,通过硅胶柱层析,用二氯甲烷和甲醇按体积比100:0、100:2、100:4、10:1、5:1、3:1梯度洗脱得到5个组分,取其中的第3个组分过硅胶柱,用石油醚和乙酸乙酯按体积比9:1、8:2梯度洗脱得到4个亚组份,第2个亚组份再经洗脱得到单体化合物,即为倍半萜酰胺衍生物。
进一步优选,所述有机溶剂为乙醇。
进一步优选,所述萃取剂为二氯甲烷。
本发明还提供了倍半萜酰胺衍生物在抗菌药物中应用。
特别是本发明提供了倍半萜酰胺衍生物在抗铜绿假单孢菌、耐甲氧西林金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌、白色念珠菌中的应用。
本发明的有益效果:提取得到了一种具有抗菌作用的倍半萜酰胺衍生物。特别是倍半萜酰胺衍生物对四个致病细菌铜绿假单孢菌、耐甲氧西林金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌和一个致病真菌白色念珠菌表现广谱抗菌活性,活性与阳性对照环丙沙星和两性霉素相当,其中对大肠杆菌表现比阳性对照环丙沙星更强的抑制作用。
附图说明
图1是 Bl-B主要COSY和HMBC相关图(粗实线代表COSY;细线箭头代表HMBC)
图2是实施例1所提取的倍半萜酰胺衍生物的氢谱。
图3是实施例1所提取的倍半萜酰胺衍生物Bl-B的碳谱。
图4是实施例1所提取的倍半萜酰胺衍生物的Dept谱。
图5 是实施例1所提取的倍半萜酰胺衍生物的HSQC谱。
图6是实施例1所提取的倍半萜酰胺衍生物的COSY谱。
图7是实施例1所提取的倍半萜酰胺衍生物的HMBC谱。
图8是实施例1所提取的倍半萜酰胺衍生物的HR-MS谱。
图9是实施例1所提取的倍半萜酰胺衍生物的NOESY谱。
图2-图7和图9中出现的f1、f2代表化学位移。
实施方式
下面结合实施例进一步详细阐明本发明。
实施例
样品的提取与分离:龙脑樟叶,由江西樟乡天然冰片有限责任公司提供,将提取完冰片的龙脑樟叶彻底干燥后粉碎,取粉碎后的龙脑樟叶 4 kg,95%的乙醇浸泡搅拌提取3次,用旋转蒸发仪减压浓缩后得到龙脑樟叶乙醇粗提物(1335g),将粗提物用温水打散,加入等量二氯甲烷萃取,之后进行旋转蒸发、减压浓缩得到二氯甲烷萃取部位浸膏。用活性炭对二氯甲烷部位浸膏进行脱色处理,之后冷冻干燥,得到脱色后的二氯甲烷部位样品(497g),通过硅胶柱层析,用二氯甲烷和甲醇按体积比100:0、100:2、100:4、10:1、5:1、3:1梯度洗脱得到5个组分(L-1~L-5),取其中的组分3(L-3)过硅胶柱,用石油醚和乙酸乙酯按体积比9:1、8:2梯度洗脱得到4个亚组份(L-3-1~L-3-4),L-3-2再经C18柱, 60%甲醇等度洗脱得到单体化合物,经一维、二维核磁和质谱鉴定为新化合物Borneolactone B(20.1g)。
参照图2-图8,Borneolactone B(Bl-B)的结构鉴定:化合物 Bl-B,在紫外灯254nm和高效液相波长254nm下吸收很淡,波长210nm下吸收较为明显。TLC斑点遇碘化铋钾显色剂显橘黄色,可能含氮。氢谱(甲醇)显示峰信号位移分布在δH 0.90~4.93ppm,表明至少二十三个氢的存在,其中包括两个甲基信号(2.00, s, 3H)和(0.90, s, 3H),六个亚甲基[其中两个烯基质子信号(4.92, s, 1H)和(4.74, s, 1H)],五个次甲基[其中一个连氧次甲基(4.22, t,J= 10.8 Hz, H-1a),一个连羟基次甲基(3.95, td,J= 10.6, 4.0 Hz, H-4)]。碳谱、HSQC 和 Dept 135表明化合物含有十七个碳信号,其中包括四个季碳(可能含有两个羰基碳,其中一个酯羰基碳),五个次甲基[一个连氧次甲基(4.22, t,J= 10.8 Hz, H-1a,79.0),一个连羟基次甲基(3.95, td, J = 10.6, 4.0 Hz, H-4, 68.4)],六个亚甲基[其中两个烯基质子信号(4.92, s, 1H, 109.1)和(4.74, s, 1H, 109.1)]和两个甲基δc22.6 和19.4,与氢谱相呼应,猜测结构可能为倍半萜酰胺类衍生物。结合阳离子HR-MS m/z308.1886 [M+H]+ (C17H26NO4),330.1743 [M+Na]+ (C17H25NO4Na),因此该化合物分子式为C17H25NO4,含有六个不饱和度,其中除了三个不饱和度(两个羰基、一个双键)外,剩余三个不饱和度可能归于三个环。再结合COSY和HMBC谱图确定化合物的平面结构:H-6/H-7/H-8的COSY相关信号,H-15/C-8, C-9, C-9a、H-6/C-9a、H-8/C-9a、H-14/C-6、H-14/C-9a的HMBC相关信号证实了9,5a二取代甲基环己烯的存在,并C-9和C-15间形成双键;类似地,H-9a/H-1a/H-3a/H-4/H-5的COSY相关信号,H-14/C-5、H-5/C-9a、H-5/C-3a、H-4/C-1a、H-3a/C-9a的HMBC相关信号证实了二取代环己烷的存在,并共用5a、9a两个位点,H-3a/H-3、H-3/H-10的COSY相关信号,H-3/C-1a、H-3a/C-10、H-3/C-11、H-10/C-2、H-13/C-11的HMBC相关信号,结合氢碳位移,证实了五元内酯环的存在,并在位点3有酰胺片段。将新化合物命名为Borneolactone B。
Borneolactone B(Bl-B)的空间构型的确定:在NOESY谱图中,发现H-1a、H-4均与H-14存在明显相关信号,表明H-1a、H-4、H-14位于分子的同一侧(β)。发现H-9a与H-3a、H-6、H-5b存在明显相关信号,表明H-9a、H-3a、H-6、H-5b位于分子的另一侧(α)。然而,并未发现H-9a与H-14存在相关信号,表明两个环通过反式稠合形成了反式十氢化萘结构。H-1a裂分为t峰,耦合常数为10.8 Hz,表明该质子与H-9a、H-3a均为反偶。即H-9a、H-1a、H-3a均位于直立键a(二面角为180°)。再根据耦合常数判断,H-10偕偶产生耦合常数14.0 Hz,H-3与H-10耦合产生耦合常数12.4 Hz。同时未发现H-3与H-3a存在相关信号,表明H-3与H-3a不在同一平面。综上所述,该化合物的空间结构确定为:
;
新化合物Borneolactone B的氢碳数据如表1,化合物的化学结构式如下:
;
化合物主要的COSY和HMBC相关如图1,化合物的氢谱、碳谱、DEPT、HSQC、COSY、HMBC、HR-MS谱、NOESY谱如图2-图9。
;
化合物Bl-B的抗菌活性测试:抗菌实验通过微量稀释完成,二倍稀释测定最小抑菌浓度(MIC),供试菌株为四个致病细菌铜绿假单孢菌(Pseudomonas aeruginosa)、耐甲氧西林金黄色葡萄球菌(Methicillin-resistantStaphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、大肠杆菌(Escherichia coli)和一个致病真菌白色念珠菌(Candida albicans),作为细菌测试:环丙沙星(Ciprofloxacin)作为阳性对照;真菌测试:两性霉素 B(Amphotericin B)作为阳性对照。在超净工作台上,把冷藏的致病细菌与真菌活化,分别用铁勺取一小块加入事先配制好新鲜并灭过菌的牛肉膏蛋白胨液体培养基和PDA 液体培养基。将 250mL 锥形瓶在摇床上固定好,180rmp/min,真菌 28℃培养,一般为两天,细菌 37℃培养,一般为一天,待浑浊。把上述活化好供试菌,取 100μL 放于 100mL新鲜的培养基,充分摇匀,致5×106cfu/mL菌液,倒入加液槽,用DMSO把样品溶解,配制成浓度为20mg/ml,在96孔板上完成滴加样品液和菌稀释液。使用移液枪第一行加入4μL待测样品和176μL菌稀释液,充分混合,取 90μL,加到下一行,再补加 110μL 菌稀释液达到每往下一行,呈现待测样品浓度减半的效果,并保证都在灭菌无污染情况下执行。滴加好了之后,96 孔板在摇床上固定好,180rmp/min,真菌 28℃培养,一般为两天,细菌 37℃培养,一般为一天,再观察每行的清澈程度,若 D 行呈现清澈,E 行呈现混浊,则视D行时对应的样品浓度作为MIC值,也可在这个基础上再进行二倍稀释,来得到更为精确的 MIC 值,每次实验随机重复三次及以上。实验结果表明:化合物Bl-B对四个致病细菌铜绿假单孢菌、耐甲氧西林金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌和一个致病真菌白色念珠菌表现广谱抗菌活性,活性与阳性对照环丙沙星和两性霉素相当,其中对大肠杆菌表现比阳性对照环丙沙星更强的抑制作用,MIC值为0.3125μg/mL。
;
Ciprofloxacin(环丙沙星)a作为细菌阳性对照; Amphotericin B (两性霉素B)b作为真菌阳性对照。
最后应说明的是:以上所述仅为本发明的优选实例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种倍半萜酰胺衍生物,其特征是,化学结构式如下:
。
2.一种从龙脑樟叶中制备权利要求1所述的倍半萜酰胺衍生物的方法,其特征是,将提取完冰片的龙脑樟叶干燥后粉碎,取粉碎后的龙脑樟叶,用95%的乙醇浸泡搅拌提取,减压浓缩后得到龙脑樟叶有机溶剂粗提物,将粗提物用温水打散,加入等量二氯甲烷萃取,之后进行旋转蒸发、减压浓缩得到萃取部位浸膏,用活性炭对萃取部位浸膏进行脱色处理,之后冷冻干燥,得到脱色样品,通过硅胶柱层析,用二氯甲烷和甲醇按体积比100:0、100:2、100:4、10:1、5:1、3:1梯度洗脱得到5个组分,取其中的第3个组分过硅胶柱,用石油醚和乙酸乙酯按体积比9:1、8:2梯度洗脱得到4个亚组份,第2个亚组份再经C18柱, 60%甲醇洗脱得到单体化合物,即为倍半萜酰胺衍生物。
3.根据权利要求 1 所述的倍半萜酰胺衍生物在制备抗菌药物中的应用。
4.根据权利要求1所述的倍半萜酰胺衍生物在制备抗铜绿假单孢菌、耐甲氧西林金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌、白色念珠菌中一种或多种的药物中的应用。
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