CN113896752B - 青龙衣中一种支链醇苷化合物的制备方法和抗菌用途 - Google Patents
青龙衣中一种支链醇苷化合物的制备方法和抗菌用途 Download PDFInfo
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Abstract
本发明公开了一种支链醇苷化合物的制备方法和在抗菌领域的应用。属于医药技术领域,其特征是以核桃青皮为原料,经过硅胶柱层析、Sephadex LH‑20凝胶柱层析分离,再结合制备型HPLC纯化得到了一种新的支链醇苷化合物。同时,本发明化合物经药效学实验证实对金黄色葡萄球菌、大肠杆菌和铜绿假单胞菌等显示出较强的抑制作用,有具有良好的应用开发前景。
Description
技术领域
本发明属于医药技术领域,具体涉及一种具有抗菌作用的新化合物的制备方法和在制备治疗肿瘤药物中的应用。
背景技术
抗菌药物研究一直是人类健康面临的重大挑战,目前临床上所用的抗菌消炎药物主要有青霉素类、头孢菌素类、氮基糖苷类,这类药物在人体内发挥抗菌作用的同时也带来了许多副反应,并且由于长期服用这类药物,导致细菌的耐药性不断出现,从而增加了治疗难度;因此要求广大科研工作者不断开发新型的抗菌药物应用于临床。中药是我国巨大的资源宝库,在长期的临床实践中已经积累了许多成功的经验和独特的优势,从中药中寻找效果显著的抗菌活性成分已成为近年来新药研究的重要趋势,核桃青皮,又名青龙衣,是未成熟核桃外面的肉质果皮,现代研究表明,青龙衣不同极性提取部位对许多革兰氏阳性菌和阴性菌均有抑制作用,有较广的抗菌谱,亟待开展其药效物质基础的深入挖掘工作;同时,我国核桃分布广泛,资源十分丰富,但是在食用核桃时外层果皮随意丢弃会造成大量资源浪费和生态环境污染,因此充分开发和综合利用核桃青皮,对于抗菌药物的开发和资源的合理再利用均具有非常重要的意义。
本发明采用现代提取分离技术和波谱分析方法,从青龙衣中提分离得到一种支链醇苷类化合物,并且为一种新化合物,实验证明该化合物具有非常明显的抗菌活性,有望开发成新的抗菌药物。
发明内容
本发明的目的是为了提供一种新的醇苷类化合物及其在制备抗菌药物中的应用,用这种方法制备的该化合物具有显著抗菌作用,可扩大抗菌药物的资源、来源。
为了达到上述目的,本发明提供了一种具有抗菌作用的新化合物青龙衣支链醇苷I,其特征为:3R-乙基-4-甲基戊醇-1-O-α-L-呋喃阿拉伯糖基(1→6)-β-D-吡喃葡萄糖苷,其结构式如下:
本发明还提供了3R-乙基-4-甲基戊醇-1-O-α-L-呋喃阿拉伯糖基(1→6)-β-D-吡喃葡萄糖苷的制备方法:以核桃青皮为原料,依次通过醇提、柱层析制备得到。
上述柱层析依次包括正相硅胶柱、Sephadex-LH20凝胶柱和制备型HPLC。
本发明3R-乙基-4-甲基戊醇-1-O-α-L-呋喃阿拉伯糖基(1→6)-β-D-吡喃葡萄糖苷具体制备步骤如下:
(1)醇提:将核桃青皮(来源为核桃楸Juglans mandshurica Maxim的青果皮)鲜品低温40℃烘干,得到的干燥品为原料,采用乙醇回流提取3次,分别为,第1次用60L 70%乙醇提取3h,第2次40L 70%乙醇提取2h,第3次用40L 70%乙醇提取2h,过滤,合并3次滤液,减压回收溶剂,干燥,得干浸膏状提取物;
(2)正相硅胶柱层析分离:将上述步骤(1)中所得干浸膏经过硅胶柱层析分离,分别用30:1、8:1、3:1的二氯甲烷-甲醇梯度淋洗,将3:1的二氯甲烷-甲醇洗脱部分合并,减压回收溶剂得到分离产物;
(3)Sephadex LH-20凝胶柱层析分离:取经上述步骤(2)分离后的产物,通过葡聚糖凝胶柱,以甲醇:水=1:1.5(V/V)洗脱4个柱体积,弃去第1个柱体积洗脱液。收集后面3个柱体积洗脱液,回收溶剂,得粗品;
(4)制备型HPLC分离:将上述步骤(3)中所得粗品采用甲醇溶解进入制备型HPLC,以流动相甲醇:水=32:68(V/V)的混合溶液洗脱,洗脱流速为2.5 mL/min,保留时间tR=25.5min~25.8min阶段内收集馏分后,回收干燥即得。
本发明还提供了3R-乙基-4-甲基戊醇-1-O-α-L-呋喃阿拉伯糖基(1→6)-β-D-吡喃葡萄糖苷在制备抗菌药物方面的应用。优选为在制备抑制金黄色葡萄球菌、大肠杆菌和铜绿甲单胞菌方面的应用。
迄今为止以上研究成果尚未有专利或文献报道。
本发明的有益效果和意义在于:采用的原料为核桃外壳的青衣,通常被当做废物抛弃,以此为原料进行化合物提取,能充分利用该植物资源;此外,本研究对核桃青皮进行深入成分研究开发,寻找到具有独特化学结构且活性较强的化合物,制备方法简单易行,为后续临床研究提供了新型抗菌药物来源。
附图说明
图1为本发明化合物的化学结构式;
图2为本发明化合物的正性HR-ESI-MS谱图;
图3为本发明化合物的1H-NMR谱图;
图4为本发明化合物的13C-NMR谱图;
图5为本发明化合物的DEPT谱图;
图6为本发明化合物的HSQC谱图;
图7为本发明化合物的HMBC谱图。
具体实施方式
根据本发明所公开的技术内容,本领域技术人员很清楚本发明的其他实施方案,下述实施方案仅作示例。在不违反本发明主旨及范围的情况下,可对本发明进行各种调整和改进。这些变化应在本发明的保护范围内。下面结合具体实施例对本发明进行详细说明。
实施例1 本发明化合物的制备方法:
(1)醇提:将核桃青皮(来源为核桃楸Juglans mandshurica Maxim的青果皮)鲜品低温40℃烘干,得到的干燥品6kg为原料,采用乙醇回流提取3次,分别为,第1次用60L 70%乙醇提取3h,第2次40L 70%乙醇提取2h,第3次用40L 70%乙醇提取2h,过滤,合并3次滤液,减压回收溶剂,干燥,得干浸膏状提取物558g;
(2)正相硅胶柱层析分离:将上述步骤(1)中所得干浸膏经过硅胶柱层析分离(色谱柱内径10cm长1.8m,其中硅胶有效高度1.2m),依次采用二氯甲烷-甲醇(30:1,V/V,3.5柱体积)→二氯甲烷-甲醇(8:1,V/V,2.5柱体积)→二氯甲烷-甲醇(3:1,V/V,3柱体积)进行梯度洗脱,将3:1的二氯甲烷-甲醇洗脱部分合并,减压回收溶剂至干,称重4.12g;
(3)Sephadex LH-20凝胶柱层析分离:取经上述步骤(2)分离后的产物,通过葡聚糖凝胶柱(内径2cm,柱长1.5m),以甲醇:水=1:1.5(V/V)洗脱4个柱体积,弃去第1个柱体积洗脱液。收集后面3个柱体积洗脱液,回收溶剂,得粗品0.32g;
(4)制备型HPLC分离:将上述步骤(3)中所得粗品采用甲醇溶解进入制备型HPLC(Waters 515-2414,SunFire TM Prep C18,250 mm×10 mm i.d.,5μm),以流动相甲醇:水=32:68的混合溶液洗脱,洗脱流速为2.5 mL/min,保留时间tR=25.5min~25.8min阶段内收集馏分后,回收干燥即得纯品32.6 mg。
实施例2 本发明化合物的表征
本发明化合物为淡黄色无定形粉末,易溶于甲醇。在UV光谱(MeOH)中呈现紫外末端吸收。正性HR-ESI-MS谱中,在m/z 425.2287处可见[M+H]+离子峰,表明该新化合物的分子量为424。结合1H-NMR、13C-NMR及DEPT谱等推测其分子式为C19H36O10,计算其不饱和度为2。
在1H-NMR (CD3OD, 400MHz)谱中,δ0.85 (3H, d, J=7.0 Hz)、0.86 (3H, d, J=7.0 Hz)和0.88 (3H, t, J=7.0 Hz)处为三个甲基的质子信号。在δ4.24 (1H, d, J=7.8Hz)处为葡萄糖端基质子信号,根据其偶合常数判断其糖苷键为β构型;δ4.95 (1H, d, J=1.1 Hz)处为阿拉伯糖端基质子信号,根据其偶合常数判断其糖苷键为α构型。在δ3.88(1H, ddd, J=9.2, 9.2, 5.8 Hz)、3.53 (1H, ddd, J=8.8, 8.8, 6.5 Hz)、4.01 (1H,dd, J=11.3, 2.3 Hz)、3.60 (1H, dd, J=10.9, 5.8 Hz)、3.73 (1H, dd, J=11.8, 3.2Hz)和3.63 (1H, dd, J=11.2 ,5.3 Hz)处为三个氧代亚甲基质子信号。13C-NMR (CD3OD,100MHz)谱及DEPT谱,显示有19个碳信号,其中δ104.5 (CH)、75.1 (CH)、78.1 (CH)、72.0(CH)、76.7 (CH)和68.1 (CH2)为葡萄糖的碳信号;δ110.0 (CH)、85.9 (CH)、78.9 (CH)、83.2 (CH)和63.1 (CH2)为阿拉伯糖的碳信号。在δ19.8、19.8、12.3处为三个甲基的碳信号;δ31.2、70.2和24.3处为三个亚甲基的碳信号;δ43.5和30.3处为两个次甲基的碳信号。
在该化合物的HMBC谱中,可明显观察到H-1 (δ3.88, 3.53)与C-3 (δ43.5)有远程相关;H-2 (δ1.48, 1.64)与C-3 (δ43.5)、C-4 (δ30.3)、C-7 (δ24.3)有远程相关;H-3 (δ1.19) 与C-1 (δ70.2)、C-5 (δ19.3)、C-7 (δ24.3)有远程相关;H-6 (δ0.86) 与C-3 (δ43.5)有远程相关;H-7 (δ1.27, 1.35)与C-8 (δ12.3)有远程相关;H-8 (δ0.88)与C-2 (δ31.2)、C-3 (δ43.5)有远程相关,其中C-3的手性碳原子的绝对构型通过Mosher方法确定[用(R)-(-)-MTPA-Cl和(S)-(+)-MTPA-Cl分别和本化合物反应,得到了(S)-Mosher酯和(R)-Mosher酯,通过比较两个产物间的化学位移的改变发现C-1,2向低场位移,C-5,6向高场位移,可以确定C-3为R构型],综上分析此化合物存在3R-乙基-4-甲基戊醇结构。H-6′ (δ4.01, 3.60)与C-1′′ (δ110.0)有远程相关;H-1′′ (δ4.95)与C-6′ (δ68.1)有远程相关;H-1 (δ3.88, 3.53)与C-1′ (δ104.5)有远程相关,说明阿拉伯糖连接在葡萄糖的C-6′上并且两个糖连接在3R-乙氧基-4-甲基戊醇结构的C-1位上。
综上所述,对DEPT、HSQC和HMBC等综合解析,鉴定化合物为3R-乙基-4-甲基戊醇-1-O-α-L-呋喃阿拉伯糖基(1→6)-β-D-吡喃葡萄糖苷,命名为青龙衣支链醇苷I,其具体波谱数据见表1。
实施例3 抗菌活性测试实验
(1)菌种选择:对实施例1所得的化合物进行抑制金黄色葡萄球菌、大肠杆菌和铜绿假单胞菌活性测试;
(2)实验具体操作方法:菌种分别接种在培养基上,经37℃培养24h后,从培养基平板中刮取菌落,以无菌生理盐水稀释,配置成1×106CFU/mL的稀释液,用麦士比浊管测定细菌浓度。从各细菌稀释管中分别取0.05mL菌液,将一系列递减浓度的本发明化合物、对照药物及不含本化合物的空白样品种于各平板中,再经过37℃培养24h后观察并记录化合物的MIC值,并重复进行3次;
(3)实验结果:化合物对上述三种细菌均有较显著的抑制作用,其中对金黄色葡萄球菌的抑制效果最为显著,MIC值为0.28μg/mL该化合物及对照药物(头孢曲松钠)的抑菌效果MIC值见表2。
综上所述,本发明所述的从青龙衣中分离得到的支链醇苷化合物青龙衣支链醇苷I具有开发成为抗菌药物的前景。
Claims (3)
2.权利要求1所述化合物的制备方法,其特征在于,包括如下步骤:
(1)醇提:将来源为核桃楸Juglans mandshurica Maxim的核桃青皮鲜品低温40℃烘干,得到的干燥品为原料,采用乙醇回流提取3次,分别为,第1次用60L 70%乙醇提取3h,第2次40L 70%乙醇提取2h,第3次用40L 70%乙醇提取2h,过滤,合并3次滤液,减压回收溶剂,干燥,得干浸膏状提取物;
(2)正相硅胶柱层析分离:将上述步骤(1)中所得干浸膏经过硅胶柱层析分离,分别用体积比30:1、8:1、3:1的二氯甲烷-甲醇梯度淋洗,将3:1的二氯甲烷-甲醇洗脱部分合并,减压回收溶剂得到分离产物;
(3)Sephadex LH-20凝胶柱层析分离:取经上述步骤(2)分离后的产物,通过葡聚糖凝胶柱,以甲醇:水=1:1.5(V/V)洗脱4个柱体积,弃去第1个柱体积洗脱液,收集后面3个柱体积洗脱液,回收溶剂,得粗品;
(4)制备型HPLC分离:将上述步骤(3)中所得粗品采用甲醇溶解进入制备型HPLC,以流动相甲醇:水=32:68(V/V)的混合溶液洗脱,洗脱流速为2.5 mL/min,保留时间tR=25.5min~25.8min阶段内收集馏分后,回收干燥即得。
3.根据权利要求1所述的青龙衣支链醇苷I在制备抗菌药物中的应用,其特征在于:所述菌为金黄色葡萄球菌、大肠杆菌和铜绿甲单胞菌。
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