CN111777657B - 一种皂苷化合物及其制备方法与应用 - Google Patents
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Abstract
本发明公开了一种皂苷化合物及其制备方法与应用;所述皂苷化合物为hirsutuside A。该化合物的提取方法为:将蓬蘽置于有机溶剂中浸提,得到浸提液,将浸提液浓缩后加水制备水混悬液;对水混悬液进行萃取,将萃取液浓缩,获得浸膏;从浸膏中分离纯化得到所述皂苷化合物;本发明从蓬蘽中分离鉴定了一种具有新颖结构的皂苷化合物,具有广泛的药理活性,能用于制备抗肿瘤药物或肿瘤预防保健食品、抗酪氨酸酶药物或美白化妆品。
Description
技术领域
本发明属于药物活性成分技术领域,特别涉及一种皂苷化合物及其制备方法与应用。
背景技术
蓬蘽(Rubus hirsutus Thunb.)是蔷薇科(Rosaceae)悬钩子属(Rubus L.)植物,又名野杜利、三月泡,为多年生落叶灌木,具有较强的根蘗苗能力,分布广、资源量大。蓬蘽在民间应用广泛,全草可入药,果实部位含有酚类、黄酮类及花色苷等化学成分,具有祛风除湿、清热止血的功能,目前一般将之用于风湿性关节痛、急性黄胆型疸炎等疾病。研究发现,蓬蘽果实中至少含有16种氨基酸,其中6种为人体必需氨基酸,另外含有Fe、Mn、Zn等丰富的矿质元素;目前国内外对蓬蘽的研究还相对较少,针对其单体成分,仅仅有人对其花色苷进行了分离和鉴定,其他成分方面尚未涉及。
发明内容
本发明的目的是提供一种皂苷化合物及其制备方法与应用,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:
一种皂苷化合物,所述化合物为皂苷hirsutuside A,所述的皂苷化合物(皂苷hirsutuside A)的结构式为:
所述的皂苷化合物的制备方法,包括以下步骤:
(1)将蓬蘽置于有机溶剂中,通过冷浸法或热回流提取法进行浸提,得到浸提液;
(2)将浸提液浓缩后加水制备水混悬液,对水混悬液进行萃取,将萃取液浓缩,获得浸膏;
所述冷浸法是将蓬蘽放入密封的容器中,按配方加入一定体积的有机溶剂,浸泡约2周;期间经常摇动,待蓬蘽体内的活性成分充分溶解到有机溶剂中后,过滤即可得到浸提液;
所述热回流提取法是应用有机溶剂加热提取,需采用回流加热装置,以免溶剂挥发损失。热回流提取法比冷浸法操作繁琐,但提取效率高;
(3)采用正相硅胶柱层析对浸膏进行分离纯化,得到所述皂苷化合物。
在步骤(1)之前,还包括对所述蓬蘽进行粉碎的粉碎步骤。
在步骤(1)中,所述蓬蘽与有机溶剂的重量体积比(kg/L)为1:3~3:1。
在步骤(1)中,所述蓬蘽与有机溶剂的重量体积比为1:2~2:1。
所述的有机溶剂为甲醇、乙醇、氯仿、乙酸乙酯和丙酮中的一种或多种。
优选的,所述有机溶剂为甲醇、乙醇或它们的混合液;甲醇和乙醇的极性相对较高,利用甲醇或乙醇进行浸提能够获得更多中低极性的活性成分。
萃取时,应选用与浸提时采用的有机溶剂互不相溶的溶剂;当用甲醇浸提时,采用乙酸乙酯进行萃取;当用乙醇浸提时,采用氯仿进行萃取;并且,为提高萃取得率,优选先将浸提液浓缩后加水制备水混悬液,再对水混悬液进行萃取。
步骤(3)中的洗脱剂为石油醚和乙酸乙酯混合液;石油醚和乙酸乙酯依次按照体积比为9:1、4:1、3:1、1:1、1:3、1:9进行梯度洗脱,收集石油醚与乙酸乙酯体积比为4:1时洗脱出的馏分,对该馏分进行旋转蒸干,即获得所述皂苷化合物;石油醚和乙酸乙酯的极性均较低,能对目标化合物进行充分层析,去除全部或大多数杂质;与其他有机溶剂相比,石油醚和乙酸乙酯毒性较低,有利于研究人员身体健康。
在对所述馏分进行旋转蒸干前,还包括对洗脱出的馏分依次进行的高效液相色谱分离和重结晶步骤。
在所述的高效液相色谱分离步骤前,还包括反向硅胶柱层析步骤,所述反向硅胶柱层析步骤中所使用的洗脱剂为甲醇和水的混合液;甲醇和水依次按照体积比为9:1、4:1、1:1、1:3、1:9进行梯度洗脱,每个梯度洗脱1L,TLC检测各馏分,合并、浓缩洗脱比例为4:1的馏分,用丙酮重结晶,即得到目标化合物。
所述皂苷化合物在制备抗肿瘤药物、肿瘤预防保健食品、抗酪氨酸酶药物、美白化妆品、食品保鲜剂之一中的应用。
本发明相对于现有技术的有益技术效果是,
(1)本发明利用皂苷类物质的极性差异从蓬蘽中提取并分离获得了一种具有新颖结构的皂苷化合物,该方法操作简便、提取得率高、产物纯度高,适合规模化生产。
(2)本发明提供的皂苷化合物具有广泛的药理活性,能用于制备抗肿瘤药物或肿瘤预防保健食品、抗酪氨酸酶药物或美白化妆品。
附图说明
图1为皂苷化合物hirsutuside A的1H-NMR图谱(500MHz)。
图2为皂苷化合物hirsutuside A的13C-NMR图谱(125MHz)。
图3为皂苷hirsutuside A的结构式。
具体实施方式
下面结合附图及具体实施例对本发明作进一步的说明。
所述皂苷化合物的制备:
实施例1
取10kg蓬蘽,用10L甲醇浸提2周,甲醇浸提液经浓缩后用1L蒸馏水混悬,水混悬液用1L乙酸乙酯萃取3次,乙酸乙酯萃取液经浓缩得到浸膏23g;用硅胶(100目,100g)拌样,进行正相硅胶柱层析(200-300目,1kg;硅胶柱尺寸L 500mm,),依次以体积比9:1、4:1、3:1、1:1、1:3、1:9的石油醚和乙酸乙酯混合液进行梯度洗脱,每个梯度洗脱5L;TLC检测馏分,收集洗脱比例4:1的馏分合并、浓缩,再用丙酮重结晶(室温),即得到目标化合物。
实施例2
取10kg蓬蘽,用5L乙醇回流提取,乙醇浸提液经浓缩后用1L蒸馏水混悬,水混悬液用1L氯仿萃取3次,氯仿萃取液经浓缩后得到浸膏234g;用硅胶(100目,100g)拌样,进行正相硅胶柱层析(200-300目,1kg;硅胶柱尺寸L 500mm,),依次以体积比9:1、4:1、3:1、1:1、1:3、1:9的石油醚和乙酸乙酯混合液进行梯度洗脱,每个梯度洗脱5L;TLC检测馏分,收集洗脱比例4:1的馏分合并、浓缩,再用丙酮重结晶(室温),即得到目标化合物。
实施例3
取10kg粉碎后的蓬蘽,用5L甲醇和乙醇混合液提取,甲醇/乙醇浸提液浓缩得浸膏366g,用100g硅藻土拌样,5L氯仿热浸3次,进行正相硅胶柱层析(200-300目,1kg;硅胶柱尺寸L 500mm,),依次以体积比9:1、4:1、3:1、1:1、1:3、1:9的石油醚和乙酸乙酯混合液进行梯度洗脱,每个梯度洗脱5L;TLC检测馏分,收集洗脱比例4:1的馏分合并、浓缩,再用丙酮/正己烷(体积比1:1)重结晶(室温),即得到目标化合物。
实施例4
取10kg粉碎后的蓬蘽,用5L甲醇和乙醇混合液浸提,浸提液浓缩,得浸膏350g,用100g硅藻土拌样,5L氯仿热浸3次,进行正相硅胶柱层析(200-300目,1kg;硅胶柱尺寸L500mm,),依次以体积比9:1、4:1、3:1、1:1、1:3、1:9的氯仿和甲醇的混合液进行梯度洗脱,每个梯度洗脱5L;TLC检测馏分,收集洗脱比例4:1的馏分合并、浓缩;浓缩后的馏分进行反相硅胶柱层析,洗脱剂依次为体积比9:1、4:1、1:1、1:3、1:9的甲醇与水的混合液,每个梯度洗脱1L,TLC检测各馏分,合并、浓缩洗脱比例为4:1的馏分,用丙酮重结晶,即得到目标化合物。
经正相硅胶柱层析获得的目标馏分,经浓缩后也可利用高效液相色谱进行纯化;高效液相色谱的检测波长为254nm,洗脱剂依次为30%-100%甲醇,收集保留时间为21-22min的洗脱峰,将洗脱液合并浓缩,用丙酮重结晶,即得到目标化合物。
皂苷化合物的结构鉴定:
采用HPLC对实施例1-4所得的目标化合物进行纯度鉴定,纯度大于98%的目标化合物运用质谱和核磁共振技术进行结构鉴定,核磁共振用Bruker AVANCE DRX-500NMRSectrometer测定,TMS作内标;高分辨质谱FTICRMS用Bruker Apex Spectrometer测定;电喷雾质谱ESI-MS用Bruker Esquire 3000plus Spectrometer测定。
该化合物的13C-NMR图谱如图2所示,NMR数据如表1所示。
表1化合物的NMR数据
ESI-TOF-MS显示分子离子峰m/z767,结合质谱数据并对该化合物的NMR图谱和进行分析可知,该化合物为新皂苷化合物,命名为hirsutuside A,分子式为C41H66O13,结构如附图3所示。
皂苷化合物抗肿瘤活性分析:
Hela细胞培养于含10%小牛血清、青霉素100IU/mL及链霉素100g/mL的RP-MI1640培养基中,每3d换液1次,每5d传代1次。细胞均置于37℃。取对数生长期细胞,以RPMI1640培养基稀释成5×104/mL单细胞悬液,接种于96孔细胞培养板,每个浓度复种3孔,每孔180μL。置培养箱温育12h后,药物组每孔加不同浓度供试液20μL,平行设空白对照组(用等体积的RPMI 1640培养基代替受试药物),共培养48h;每孔加入1mg/mL MTT溶液50μL,继续培养4h后,吸尽上清液,每孔加入二甲基亚砜(DMSO)150μL,充分溶解MTT还原产物;置酶标仪上于492nm波长处测定各药物组和空白组的光密度(D),按公式计算得到药物对肿瘤细胞生长的抑制率(IR,%)以及半数抑制浓度(IC50),并对药效进行初步的评价。
IR(%)=(1-加药组平均D值/对照组平均D值)×100%。
由实验结果可知,该化合物的IC50=31μM(Hela细胞),表明该化合物具有较好的抗肿瘤作用。
所述抗肿瘤药物为抗宫颈癌药物,抗肿瘤药物以本发明的皂苷化合物为主要活性成分,添加药剂学上可接受的辅料制成,可按照药剂学上记载的制剂制备方法制成制剂;所述的制剂可以为注射液、滴注液、粉针剂、颗粒剂、片剂、冲剂、散剂、口服液、糖衣片剂、薄膜衣片剂、肠溶衣片剂、口含剂、颗粒剂、丸剂、膏剂、丹剂、喷雾剂、滴丸剂、崩解剂、口崩片、微丸等。
所述肿瘤预防保健食品以本发明的皂苷化合物为主要活性成分,添加可接受的保健食品辅料制成。
皂苷化合物对酪氨酸酶抑制活性分析:
将R-3-甲基-6,8-二羟基-7-甲基-3,4-二氢皂苷-1-酮溶解在甲醇中,使终浓度为2.5%。25℃条件下,酪氨酸酶(28nM)在50nM Na-phosphate buffer(钠磷酸盐缓冲液,pH6.8)与化合物预孵育10min。然后加入LDOPA(左旋多巴,0.5mM),在波长475nm(37℃)检测。
化合物对酪氨酸酶的抑制活性计算公式如下:
抑制率(%)=[(B–S)/B]×100%
其中,B为空白吸收,S为样品吸收。
由实验结果可知,该化合物的IC50=28μM,表明该化合物对酪氨酸酶抑制活性较好。
所述抗酪氨酸酶药物以本发明的皂苷化合物为主要活性成分,添加药剂学上可接受的辅料制成,可按照药剂学上记载的制剂制备方法制成制剂。所述的制剂可以为注射液、滴注液、粉针剂、颗粒剂、片剂、冲剂、散剂、口服液、糖衣片剂、薄膜衣片剂、肠溶衣片剂、口含剂、颗粒剂、丸剂、膏剂、丹剂、喷雾剂、滴丸剂、崩解剂、口崩片、微丸、膏剂、贴膜剂、气雾剂酊剂、栓剂、洗剂、滴鼻剂等。
含皂苷化合物滴丸制剂的制备方法如下:
取0.5g皂苷化合物R-3-甲基-6,8-二羟基-7-甲基-3,4-二氢皂苷-1-酮与10.5g聚乙二醇-6000混合均匀,加热熔融,化料后移至滴丸滴灌中,药液滴至6~8℃液体石蜡中,除油,制得滴丸300粒。
含皂苷化合物冻干粉针剂的制备方法如下:
取皂苷化合物R-3-甲基-6,8-二羟基-7-甲基-3,4-二氢皂苷-1-酮0.5g、葡萄糖4.5g、硫代硫酸钠0.9g和蒸馏水1000mL,上述组分混合均匀后,冷冻干燥,分装400支,即得。
另外,由于酪氨酸酶还参与果蔬的褐变反应,因此利用本发明皂苷化合物作为活性成分,还可用于食品保鲜剂。
以上所述仅为本发明的较佳实施例,并不用于限制本发明,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,均应包括在本发明的保护范围之内。
Claims (3)
1.一种皂苷化合物的制备方法,其特征在于,所述的皂苷化合物的结构式为:
包括以下步骤:
(1)将蓬蘽置于有机溶剂中,通过冷浸法或热回流提取法进行浸提,得到浸提液;
(2)将浸提液浓缩后加水制备水混悬液,对水混悬液进行萃取,将萃取液浓缩,获得浸膏;
(3)采用正相硅胶柱层析对浸膏进行分离纯化,得到所述皂苷化合物;
在步骤(1)之前,还包括对所述蓬蘽进行粉碎的粉碎步骤;
在步骤(1)中,所述蓬蘽与有机溶剂的重量体积比为1:3~3:1;
在步骤(1)中,所述蓬蘽与有机溶剂的重量体积比为1:2~2:1;
所述的有机溶剂为甲醇、乙醇、氯仿、乙酸乙酯和丙酮中的一种或多种;
步骤(3)中的洗脱剂为石油醚和乙酸乙酯混合液;石油醚和乙酸乙酯依次按照体积比为9:1、4:1、3:1、1:1、1:3、1:9进行梯度洗脱,收集石油醚与乙酸乙酯体积比为4:1时洗脱出的馏分,对该馏分进行旋转蒸干,即获得所述皂苷化合物。
2.如权利要求1所述的皂苷化合物的制备方法,其特征在于,在对所述馏分进行旋转蒸干前,还包括对洗脱出的馏分依次进行的高效液相色谱分离和重结晶步骤。
3.如权利要求2所述的皂苷化合物的制备方法,其特征在于,在所述的高效液相色谱分离步骤前,还包括反向硅胶柱层析步骤,所述反向硅胶柱层析步骤中所使用的洗脱剂为甲醇和水的混合液;甲醇和水依次按照体积比为9:1、4:1、1:1、1:3、1:9进行梯度洗脱,每个梯度洗脱1L,TLC检测各馏分,合并、浓缩洗脱比例为4:1的馏分,用丙酮重结晶,即得到目标化合物。
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