CN108997473A - 一种非海参烷型海参皂苷及其制备方法与应用 - Google Patents
一种非海参烷型海参皂苷及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种非海参烷型海参皂苷及其制备方法与应用。所述非海参烷型海参皂苷刺参皂苷A1,结构如下式I所示:式I。本发明首次公开了化合物非海参烷型海参皂苷刺参皂苷A1,与现有已知的海参皂苷相比,其可以在提高化疗效果的同时,提升患者的免疫力,起到综合治疗的目的。
Description
技术领域
本发明涉及一种非海参烷型海参皂苷及其制备方法与应用,属于功能性糖衍生物技术领域。
背景技术
皂苷别称:碱皂体、皂素或皂甙(zao dai)。“皂苷”一词由英文名Saponin意译而来,英文名则源于拉丁语的Sapo,意为肥皂。皂苷是苷元为三萜或螺旋甾烷类化合物的一类糖苷,主要分布于陆地高等植物中,也少量存在于海星和海参等海洋生物中。许多中药如人参、远志、桔梗、甘草、知母和柴胡等的主要有效成分都含有皂苷。有些皂苷还具有抗菌的活性或解热、镇静、抗癌等有价值的特性。
天然产物分子具有超乎人们想象的新颖化学结构,几千年来用来防病治病的中药也是因为其中的各种化学成分起作用,天然产物已成为发现治疗重大疾病的药物或重要先导化合物的主要源泉。
国内外科研机构运用相关技术对海参内脏的生理与生化特性,海参内脏中营养物质、活性物质的分离鉴定以及生物医学作用等进行了不断深入的研究,发现海参内脏中含有海参粘多糖、海参皂苷、活性肽、各种活性酶类等生物活性物质,具有抗肿瘤、抗氧化、降血脂等作用。
繁殖季节海参内脏中皂苷的含量比平时要增加80~200倍以上。皂苷是海参进行化学防御的物质基础,是海参捕食和抵御天敌攻击的重要工具。迄今已阐明了l00余种海参皂苷的结构。海参皂苷的苷元均为羊毛甾烷的衍生物,含有18(20)内酯结构的为海参烷型,含有18(16)位内酯环或无内酯环结构的为非海参烷型。
如中国专利文献CN106636286A(申请号201611236814.0)公开了去糖化的海参次级皂苷及其制备方法,去糖化的海参次级皂苷是海参皂苷HA和海参皂EA的糖链上由于糖苷酶酶解脱去两个糖基后得到的次级皂苷;该次级皂苷的制备方法包括:乙醇浸提、大孔树脂粗提,再利用硅胶柱进行纯化,分别得到HA和EA,最后利用糖苷酶进行酶解,即能够分别得到去糖化的HA和EA次级皂苷。
中国专利文献CN101186633A(申请号200710047331.0)公开了从糙海参中分离的2种皂苷类抗真菌化合物scabraside A和scabraside B,分子式分别为:C54H85O26SNa;C54H85O27SNa。经多种现代光谱分析,特别是综合应用多种先进的二维核磁共振波谱的解析,确定了这些化合物的化学结构和立体构型。体外抗真菌试验表明,这些化合物对白色念珠菌和烟曲霉菌等多种菌株有明显的抑制作用。因此可用于制备抗真菌药物。
中国专利文献CN102060905A(申请号201010541297.4)公开了一种利用鲜海参加工废液制备海参皂苷Holotoxin A1对照品的工艺方法,其特征是它由如下步骤组成:(1)将鲜海参加工废液减压浓缩醇沉,吸取上清液;(2)将所得上清液通过大孔吸附树脂柱,吸附饱和后冲洗并收集洗脱液,将洗脱液减压浓缩得浸膏;(3)将所得浸膏拌硅胶粉,利用由甲醇,二氯甲烷,乙酸乙酯,水组成的溶液体系进行硅胶柱层析;再利用甲醇∶二氯甲烷∶乙酸乙酯∶水=2∶2∶4∶1为展开体系,取下层进行TLC层析,收集硅胶柱层析迁移率为0.2-0.3的组分,将该组分减压浓缩,真空干燥得白色粉末。(4)将白色粉末利用乙腈水溶液溶解,利用HPLC,203nm紫外光吸收,乙腈水溶液为流动相进行纯化,收集主峰,真空干燥得海参皂苷Holotoxin A1对照品。
通过继续研究海参中不同特性的海参皂苷,仍然是目前的研究热点。
发明内容
本发明针对现有技术的不足,提供一种非海参烷型海参皂苷刺参皂苷A1及其制备方法与应用。
一种非海参烷型海参皂苷刺参皂苷A1,结构如下式I所示:
式I所示化合物为18(16)-内酯,(3S,5R,9S,10R,13S,14S,16S,17R,20S)构型非海参烷苷元,糖链依次为:D-葡萄糖-(1→3)-D-葡萄糖-(1→4)-D-喹诺糖-(1→2)-D-葡萄糖。
上述非海参烷型海参皂苷刺参皂苷A1的制备方法,步骤如下:
(1)将海参内脏烘干、粉碎后,无水乙醇提取,回收溶剂提取物;
(2)将步骤(1)制得的溶剂提取物溶于水中,然后用正丁醇萃取,取正丁醇相,去除萃取液,制得粗提物;
(3)将步骤(2)制得的粗提物溶于水中,经大孔吸附树脂吸附后,先经体积百分比浓度为20%的乙醇冲洗去除可溶性多糖、蛋白质,然后用体积百分比浓度为80%的乙醇洗脱,去除洗脱液,制得海参内脏总皂苷;
(4)将步骤(3)制得的海参内脏总皂苷吸附于100~200目硅胶柱,然后用体积比20:1的二氯甲烷/甲醇混合液洗除低极性成分,然后收集体积比为二氯甲烷/甲醇20:(8~16)洗脱液,浓缩,甲醇结晶,制得非海参烷型海参皂苷刺参皂苷A1。
根据本发明优选的,所述步骤(1)中,提取温度为55~65℃。
根据本发明优选的,所述步骤(1)中,提取次数为2~4次。
根据本发明优选的,所述步骤(2)中,提取次数为2~4次。
根据本发明优选的,所述步骤(2)中,水与正丁醇的体积比为4:(3~5)。
根据本发明优选的,所述步骤(3)中,吸附时间为10~14h。
根据本发明优选的,所述步骤(3)中,体积百分比浓度为20%的乙醇用量为3~5倍柱体积。
根据本发明优选的,所述步骤(3)中,体积百分比浓度为80%的乙醇用量为5~7倍柱体积。
上述非海参烷型海参皂苷刺参皂苷A1在制备抗肿瘤、抗高血压和/或免疫调节药物中的应用。
上述非海参烷型海参皂苷刺参皂苷A1在制备抗肿瘤、抗高血压和/或免疫调节保健品中的应用。
有益效果
本发明首次公开了化合物非海参烷型海参皂苷刺参皂苷A1,与现有已知的海参皂苷相比,其可以在提高化疗效果的同时,提升患者的免疫力,起到综合治疗的目的。
附图说明
图1是化合物刺参皂苷A1的氢谱图;
图2是化合物刺参皂苷A1的碳谱图;
图3是化合物刺参皂苷A1的质谱图;
图4是VEGF和bFGF mRNA的表达水平柱状图;
图中,对照:空白对照组;A1:刺参皂苷A1施用组;B:刺参皂苷B施用组;顺铂:顺铂施用组;顺铂+B:顺铂与刺参皂苷B共同施用组;顺铂+A1:顺铂与刺参皂苷A1共同施用组。
具体实施方式
下面结合实施例对本发明的技术方案做进一步阐述,但本发明所保护范围不限于此。
实施例中所述海参为刺参Apostichopus japonicus,购自蓬莱海洋(山东)股份有限公司,普通市售产品。
实施例1刺参皂苷A1的制备
15kg海参内脏烘干、粉碎后,60℃无水乙醇浸泡提取3次,回收溶剂得浸膏864g。将浸膏分散在2000mL水中,正丁醇萃取3次,每次1500mL,合并滤液,蒸干得粗提物557g。粗提物三倍量水悬浮,上样5kg大孔吸附树脂,充分吸附12h;先用4倍柱体积的20%乙醇冲洗,洗去残留的可溶性多糖、蛋白质等杂质成分,然后用6倍柱体积的80%乙醇进行洗脱,收集洗脱液,蒸干即得海参内脏总皂苷381g。取3kg 100~200目硅胶湿法装柱,将总皂苷硅胶吸附后加入装好的柱顶,用二氯甲烷∶甲醇(体积比20∶1)洗去低级性的成分后,逐渐增加甲醇的比例,TLC检测洗脱的组分,当洗脱液成分为二氯甲烷/甲醇体积比为20:(8~16)时,洗脱的组份含有刺参皂苷A1,合并浓缩,甲醇结晶得到纯品27g,为白色无定形粉末。对制得的刺参皂苷A1进行检测,氢谱图如图1所示,碳谱图如图2所示,质谱图如图3所示;具体数据如下:
IR(KBr)νmax cm-1:3299,2924,2856,1748,1377,1065,1033;HR-ESI-MS m/z:1085.5522[M-H-O]-(理论值C54H85O22,1085.5532),1121.5269[M+Cl-O]-(理论值C54H86O22Cl,1121.5299);1H NMR(400MHz,氘代吡啶)δ:1.51(2H,m,H-1),1.94(1H,m,H-2α),2.22(1H,m,H-2β),3.34(1H,dd,J=11.8,3.5Hz,H-3),1.03(1H,m,H-5),2.06(2H,m,H-6),5.66(1H,m,H-7),3.27(1H,brd,J=13.1,H-9),1.56(1H,m,H-11α),1.84(1H,m,H-11β),2.16(1H,m,H-12α),2.67(1H,m,H-12β),1.97(1H,m,H-15α),2.17(1H,m,H-15β),5.05(1H,brs,H-16),2.62(1H,s,H-17),1.07(3H,s,H-19),1.48(3H,s,H-21),1.79(2H,m,H-22),1.81(1H,m,H-23α),2.00(1H,m,H-23β),2.08(2H,m,H-24),4.80(1H,brs,H-26α),4.83(1H,brs,H-26β),1.72(3H,s,H-27),1.16(3H,s,H-30),1.35(3H,s,H-31),1.42(3H,s,H-32),4.81(1H,d,J=7.0Hz,Glc H-1'),5.00(1H,d,J=7.8Hz,Qui H-1”),5.18(1H,d,J=7.5Hz,Glc H-1”'),5.29(1H,d,J=8.0Hz,Glc H-1””).13C NMR(100MHz,氘代吡啶)δ:36.3(C-1),27.4(C-2),89.7(C-3),40.2(C-4),48.4(C-5),24.0(C-6),123.1(C-7),148.5(C-8),46.7(C-9),36.6(C-10),22.6(C-11),21.1(C-12),55.4(C-13),46.5(C-14),45.3(C-15),80.2(C-16),62.7(C-17),182.4(C-18),24.7(C-19),71.8(C-20),27.8(C-21),43.2(C-22),22.9(C-23),39.0(C-24),146.5(C-25),111.0(C-26),22.7(C-27),17.9(C-30),29.3(C-31),35.1(C-32),106.1(C-1'),88.0(C-2'),76.6(C-3'),71.1(C-4'),78.9(C-5'),62.9(C-6'),105.5(C-1”),76.5(C-2”),75.6(C-3”),84.9(C-4”),71.3(C-5”),18.8(C-6”),106.2(C-1”'),75.5(C-2”'),88.5(C-3”'),70.4(C-4”'),78.5(C-5”'),61.3(C-6”'),106.2(C-1””),78.9(C-2””),76.9(C-3””),72.3(C-4””),78.8(C-5””),63.2(C-6””).
实施例2肿瘤细胞增殖抑制活性
1、试验材料
人体肝癌QGY-7703、人体肺癌LAX、人体乳腺癌Bre-04、人体肠癌Col-06肝癌由山东大学齐鲁医院提供;顺铂,购自山东齐鲁制药厂;
刺参皂苷B,按现有技术制备,结构式如下式:
2、试验方法
取对数生长期QGY-7703、肺癌LAX、乳腺癌Bre-04和肠癌Col-06肿瘤细胞,接种到96孔板,每孔100μL(约1×104个),置37℃、5%CO2及饱和湿度的培养箱中培养24h。待细胞完全贴壁后,按分组情况,阴性组加0.1wt%DMSO完全培养液100μL/孔,阳性组加32μg/mL、16μg/mL、8μg/mL、4μg/mL、2μg/mL顺铂溶液100μL/孔,给药组分别加40μg/mL、20μg/mL、10μg/mL、5μg/mL、2.5μg/mL刺参皂苷A1和刺参皂苷B药液100μL/孔,置培养箱中继续培养。在药物作用细胞12h、24h、36h、48h后吸去上清液,并用PBS缓冲液清洗至无颜色残留,每孔加MTT溶液20μL,置培养箱中培养4h后取出,吸去上清液,每孔加DMSO 150μL,震荡10min,用酶标仪在490nm处测定各孔吸光度值,计算药物对肿瘤细胞生长的抑制率。并根据量效和时效关系,通过线性拟合计算半数抑制浓度(IC50)。
3、结果及分析
表2-1刺参皂苷A1肿瘤细胞增殖抑制活性
体外试验结果表明,刺参皂苷A1对人体肝癌QGY-7703细胞具有显著的抑制增殖作用。随着给药剂量的增加和给药时间的延长,测试细胞株的增殖抑制率显著升高,呈现明显的量效和时效关系。由IC50可知,刺参皂苷A1对人肺癌LAX增殖抑制作用强度高于海参烷型刺参皂苷B。海参皂苷可与生物膜上甾醇分子结合形成复合物,在膜上形成单一离子通道和大的水孔导致生物膜溶解,进而发挥抗肿瘤功效。
实施例3对小鼠移植瘤的抑制作用及肿瘤血管生成作用
1、试验材料
ICR小鼠(体重18~22g);小鼠肉瘤S180、小鼠肝癌H22和小鼠宫颈癌U14,均购自ATCC细胞库。刺参皂苷B,按现有技术制备;顺铂,购自山东齐鲁制药厂。
2、试验方法
每批ICR小鼠腋窝皮下分别接种0.1mL小鼠S180、H22和U14细胞悬液(细胞密度1×107个/mL),次日将小鼠随机分为6组,每组10只;一组小鼠腹腔注射阳性药顺铂5mg/kg,每天一次,连续10d;两组小鼠分别灌胃刺参皂苷B(200mg/kg),刺参皂苷A1(200mg/kg),每天一次,连续10d;其余两组联合给药顺铂/刺参皂苷B(5mg/kg,200mg/kg),顺铂/刺参皂苷A1(5mg/kg,200mg/kg),每天一次,连续10d;最后一组小鼠作空白对照组,每天灌胃等体积生理盐水。停药后次日处死各组小鼠,称体重、解剖剥离瘤块并称重,计算抑瘤率,比较各治疗组和阴性组的差异。抑瘤率=(对照组平均瘤重—实验组平均瘤重)/对照组平均瘤重×100%。
选取空白对照组和各用药组小鼠S180肿瘤组织,用Trizol法提取细胞总RNA,反转录成cDNA后参照Promega公司GoTaq qPCR Master Mix(A6001)说明书进行荧光定量PCR扩增,引物序列为:
VEGF上游引物:5’-GAGGAGCAGTTACGGTCTGTG-3’,
VEGF下游引物:5’-TCCTTTCCTTAGCTGACACTTGT-3’;
bFGF上游引物:5’-AAGAGCGACCCTCACATCAA-3’,
bFGF下游引物:5’-CGTTTCAGTGCCACATACCAA-3’;
GAPDH上游引物:5’-GGAGCGAGATCCCTCCAAAAT-3’,
GAPDH下游引物:5’-GGCTGTTGTCATACTTCTCATGG-3’。
荧光定量PCR扩增反应体系如下:模板1μL,上、下游引物各0.4μL,2×GoTaq qPCRMaster Mix荧光染料10μL,ddH2O 8.2μL,总反应体积为20μL。
反应条件:95℃预变性5min;40个热循环(95℃变性3s,60℃延伸34s);溶解曲线生成的反应程序为:95℃15s,60℃1min,95℃15s。以GAPDH作为内参照基因。
3、结果及分析
表3-1刺参皂苷A1对小鼠移植瘤的作用
连续灌胃刺参皂苷A1 200mg/kg 10d,对小鼠肉瘤S180的生长抑制率可达38.1%,对小鼠肝癌H22的生长抑制率可达36.6%,对小鼠U14的生长抑制率可达到50.7%,有显著的抑制肿瘤生长的作用。联合用药试验显示,顺铂/刺参皂苷A1肿瘤增殖抑制效果明显优于其他给药组。本实验例不但说明刺参皂苷A1可通过口服给药的方式在体内发挥优良的抗肿瘤活性,而且其与化疗药协同使用具有更好的治疗效果。
与生理盐水组比较,刺参皂苷A1组VEGF和bFGF mRNA的表达水平显著降低。在测试样本中,顺铂/刺参皂苷A1联合用药组VEGF和bFGF mRNA表达水平下降趋势最为明显。VEGF和bFGF是血管生成的重要促进因子,本实验例说明刺参皂苷A1可通过抑制肿瘤组织中新生血管的生成,达到阻断肿瘤细胞生长和转移的目的,而化疗药与刺参皂苷A1协同使用对肿瘤组织血管生成具有更为优秀的抑制效果,结果如图4所示。
实施例4对化疗荷瘤小鼠的免疫增强作用
1、试验材料
昆明小鼠,雌雄各半,体质量(20±2)g;小鼠肉瘤S180细胞,购自ATCC;刺参皂苷B,按现有技术制备;顺铂,购自山东齐鲁制药厂。
2、试验方法
将腹腔接种小鼠肉瘤S180细胞后7d的昆明种小鼠颈椎脱臼处死,腹部皮肤消毒后抽取腹水,调整细胞浓度为2×107/mL,75%乙醇消毒接种于小鼠的右腋皮下,每只0.2mL。
小鼠随机分为6组,每组10只,即正常对照组、化疗模型组、环磷酰胺/刺参皂苷B(200mg/kg)组及环磷酰胺/刺参皂苷A1低、中、高剂量(50、100、200mg/kg)组。除正常对照组外各组制备S180荷瘤小鼠模型,并每天腹腔注射环磷酰胺20mg/kg;联合给药组灌胃相应药物,化疗模型组灌胃生理盐水。各组于瘤细胞接种24h后开始给药,1次/d连续3d,末次给药后24h处死动物。
末次给药12h后,摘眼球采血,4℃3 000r/min离心30min,取上层血清进行细胞因子IL-2、IL-6、TNF-α含量测定,检测方法均严格按照试剂盒说明书进行。
3、结果及分析
表4-1对化疗小鼠血清IL-2、IL-6、TNF-α含量的影响
与模型组比较,*P<0.05,*P<0.01
与正常对照组比较,化疗模型组小鼠血清IL-2、IL-6、TNF-α含量显著降低。刺参皂苷A1可显著提高免疫抑制小鼠血清IL-2、IL-6、TNF-α含量,与模型组比较,化合物中、高剂量组呈现显著差异(*P<0.05,**P<0.01)。且上述数据显示,在同等剂量下刺参皂苷A1对化疗后机体免疫的恢复效果要强于刺参皂苷B,这可能与其非海参烷型苷元及糖链中罕见的3-O-葡萄糖结构密切相关。
IL-2可引起T细胞增殖和维持T细胞在体外的持续增长,诱导CTL、NK和LAK等多种杀伤细胞的分化和效应功能,并诱导杀伤细胞产生IFN-γ、TNF-α等细胞因子。IL-6能提升固有免疫的免疫应答能力和促进致病菌的清除,刺激活化的B细胞产生更多免疫球蛋白;TNF-α既可作为机体免疫防护的重要介质,又可参与机体的免疫病理损伤,促进B细胞生长分化、激活NK细胞等,还可选择性杀伤肿瘤细胞。本试验结果提示刺参皂苷A1在癌症临床放化疗后机体免疫系统修复方面具有广阔的应用前景。
实施例5对自发性高血压大鼠脏器指数及血清因子的影响
1、试验材料
雄性自发性高血压大鼠(SHR)40只,10周龄,体质量200±10g;雄性同龄Wistar大鼠8只。谷胱甘肽过氧化物酶(GSH-Px)试剂盒、超氧化物歧化酶(SOD)试剂盒、丙二醛(MDA)试剂盒。
2、试验方法
将Wistar大鼠设为正常对照,灌胃蒸馏水1mL/100g,每天1次;40只SHR随机分为5组,即模型对照组、阳性对照组和化合物低、中、高剂量组,模型组灌胃蒸馏水,阳性对照组灌胃50mg·kg-1·d-1卡托普利,化合物低、中、高剂量组分别灌胃50、100、200mg·kg-1·d-1刺参皂苷A1。每天灌胃1次,连续给药8周及停药2周,记录各组大鼠血压值和心率的变化。
末次给药2周后,大鼠禁食不禁水16h,用10%水合氯醛麻醉,腹主动脉取血,4℃1000r·min-1离心15min,吸取上清液置于—20℃保存,按照试剂盒要求测定血清中的GSH-Px、SOD、MDA的含量;迅速取出心脏、肝脏、脾脏、肾脏,并分离左心室,称重计算各组大鼠的脏器重量指数:脏器指数=脏器质量/体质量×100%。
3、结果及分析
表5-1对各组大鼠脏器指数的影响
注:与模型组比较*P<0.05,与卡托普利组比较#P<0.05
与空白组比较,模型组、卡托普利组、化合物低、中、高剂量组的左心室指数、左肾指数、右肾指数均明显升高;随着化合物给药剂量的增加左心室指数逐渐降低,显现出治疗趋势,化合物中、高剂量组与模型组比具有统计学差异(P<0.05)。与卡托普利组比较,化合物对肾脏的相关影响略弱,低剂量状态下左肾指数、右肾指数呈现统计学差异(P<0.05);肝脏指数、脾脏指数于各组中水平相当,无统计学差异(P>0.05)。
左心室肥厚是高血压靶器官损害最常见的表现。研究发现60%-70%的高血压患者伴随着左心室肥厚。左心室肥厚是神经体液因子与血流动力学因素综合作用的结果。它的危害主要有:损害心肌收缩和舒张功能;损害冠脉储备功能,致供血不足;导致心率紊乱;增加高血压合并左心室肥厚患者脑卒中的危险系数。本实验研究发现,刺参皂苷A1能够抑制自发性高血压大鼠左心室重量增加,降低左心室肥厚指数,从而起到保护心脏的作用。
表5-2对血清GSH-Px、MDA、SOD含量的影响
与模型组比较,*P<0.05
与模型组相比,化合物低、中、高组血清GSH-Px含量出现上升,尤其高剂量组GSH-Px含量呈现统计学差异(P<0.05)。相较于模型组,化合物给药组MDA含量下降、SOD含量上升,高浓度时两者均呈现统计学差异。
GSH-Px具有清除自由基和衍生物的作用,抑制脂质过氧化物的形成,增强机体抗氧化损伤的能力。SOD为机体内一种非常重要的自由基清除剂,它能特异性地阻断脂质过氧化反应,保护细胞膜,其含量的降低预示着机体代偿的下降。MDA为过氧化脂质的降解产物,对细胞的多种成分有损伤作用,其含量的多少代表着机体细胞受自由基攻击和脂质过氧化的程度高低。因此,上述实验现象说明刺参皂苷A1能够缓解由高血压及其并发症导致的氧化应激损伤。
实施例6对高血压大鼠钠钾ATP酶活性的作用
1、试验材料
自发性高血压大鼠(SHR)30只,10周龄,体质量200±10g;同龄Wistar大鼠10只;ATP酶测定试剂盒
2、试验方法
Wistar大鼠设为正常对照,灌胃生理盐水;SHR大鼠随机分为3组,每组10只,即模型对照组、阳性对照组和化合物低、高剂量组,模型组灌胃生理盐水,阳性对照组灌胃100mg·kg-1丹酚酸A,化合物低、高剂量组分别灌胃50mg·kg-1和200mg·kg-1的刺参皂苷A1。试验前禁食12h,麻醉前30min灌胃给药1次。
取大脑皮质和海马,加入10倍量预冷生理盐水于冰水浴中匀浆。离心取上清液,按试剂盒要求测定蛋白质浓度和ATP酶活性。ATP酶测定原理:ATP酶分解ATP生成ADP及无机磷(Pi),测定Pi的量可判断ATP酶活性。ATP酶活性单位以每毫克组织蛋白(Pr)每小时新产生的Pi的量为酶活性单位μmol Pi·mg Pr-1·h-1。
3、结果及分析
表6-1对大鼠大脑皮质和海马Na+-K+ATP酶活性的影响
与模型对照组比较*P<0.05,**P<0.01
模型组动物大脑皮质和海马的Na+-K+ATP酶活性均呈现下降趋势。刺参皂苷A1可明显提高自发性高血压大鼠大脑皮质和海马的Na+-K+ATP酶活性;特别是大脑皮质Na+-K+ATP酶的活性,高剂量化合物组与模型组存在极其显著差异(P<0.01)。
高血压病的发生发展与机体多个系统组织器官的机能变化有密切关系。这些组织器官的基本活动都与其细胞内外的跨膜电位和离子代谢有关。Na+-K+ATP酶对维持细胞膜两侧的Na+、K+离子跨膜浓度、保障细胞代谢过程中的离子平衡、维持细胞正常机能状态具有重要意义。本试验例阐述了刺参皂苷A1对中枢Na+-K+ATP酶的促进功能,再次证明其可用于临床高血压症的治疗。
序列表
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Claims (10)
1.一种非海参烷型海参皂苷刺参皂苷A1,其特征在于,结构如下式I所示:
2.权利要求1所述的非海参烷型海参皂苷刺参皂苷A1的制备方法,其特征在于,步骤如下:
(1)将海参内脏烘干、粉碎后,无水乙醇提取,回收溶剂提取物;
(2)将步骤(1)制得的溶剂提取物溶于水中,然后用正丁醇萃取,取正丁醇相,去除萃取液,制得粗提物;
(3)将步骤(2)制得的粗提物溶于水中,经大孔吸附树脂吸附后,先经体积百分比浓度为20%的乙醇冲洗去除可溶性多糖、蛋白质,然后用体积百分比浓度为80%的乙醇洗脱,去除洗脱液,制得海参内脏总皂苷;
(4)将步骤(3)制得的海参内脏总皂苷吸附于100~200目硅胶柱,然后用体积比20:1的二氯甲烷/甲醇混合液洗除低极性成分,然后收集体积比为二氯甲烷/甲醇20:(8~16)洗脱液,浓缩,甲醇结晶,制得非海参烷型海参皂苷刺参皂苷A1。
3.如权利要求2所述的制备方法,其特征在于,所述步骤(1)中,提取温度为55~65℃。
4.如权利要求2所述的制备方法,其特征在于,所述步骤(1)中,提取次数为2~4次;
优选的,所述步骤(2)中,提取次数为2~4次。
5.如权利要求2所述的制备方法,其特征在于,所述步骤(2)中,水与正丁醇的体积比为4:(3~5)。
6.如权利要求2所述的制备方法,其特征在于,所述步骤(3)中,吸附时间为10~14h。
7.如权利要求2所述的制备方法,其特征在于,所述步骤(3)中,体积百分比浓度为20%的乙醇用量为3~5倍柱体积。
8.如权利要求2所述的制备方法,其特征在于,所述步骤(3)中,体积百分比浓度为80%的乙醇用量为5~7倍柱体积。
9.权利要求1所述非海参烷型海参皂苷刺参皂苷A1在制备抗肿瘤、抗高血压和/或免疫调节药物中的应用。
10.权利要求1所述非海参烷型海参皂苷刺参皂苷A1在制备抗肿瘤、抗高血压和/或免疫调节保健品中的应用。
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