CN109602759B - 罗汉松实多糖的用途 - Google Patents
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Abstract
本发明涉及一种罗汉松实多糖在制备预防和治疗肝癌的药物或保健食品中的用途。罗汉松实多糖是以罗汉松科罗汉松属植物罗汉松或短叶罗汉松的果实为原料,经水提取,脱除蛋白,柱层析分离纯化后获得。本发明所述的罗汉松实多糖对肝癌荷瘤小鼠肿瘤生长有显著抑制作用。罗汉松实多糖按一定比例添加适当的药用辅料后,可制成胶囊、口服液或颗粒剂。罗汉松实多糖应用于肝癌的预防和治疗,没有细胞毒类抗肿瘤药物常见的恶心、呕吐、脱发和骨髓抑制等毒副作用,是一种纯天然的安全有效的植物多糖。
Description
技术领域
本发明属于医药技术和保健食品领域,具体涉及罗汉松实多糖在制备预防和治疗肝癌的药物或保健食品中的用途。
背景技术
罗汉松实多糖是一种来源于天然植物的生物活性多糖,主要从罗汉松科罗汉松属植物罗汉松Podocarpus macrophyllus(Thunb.)D.Don或短叶罗汉松Podocarpusmacrophyllus(Thunb.)D.Don var.maki(Sieb.)Endl.的果实中提取分离纯化获得。罗汉松主要分布在我国广西、江苏、福建、台湾、浙江等地,在广西北海市已有较大规模种植,种植面积超过1万亩,每年产出大量的罗汉松实。罗汉松实是罗汉松的果实,由花托和种子构成。在罗汉松种植地,罗汉松花托可当成水果直接食用,或制成果酒饮用;种子主要用于育种。目前,有关罗汉松实化学成分和生物活性的研究报道较少。有研究表明,罗汉松实药用氨基酸含量较高,且富含钙、镁矿质元素;罗汉松实中含有多糖,鲜品中的含量约为0.5%;罗汉松花托95%乙醇提取物具有降血脂、抗氧化和保肝作用。
国内关于罗汉松的发明专利,目前较多的是集中在罗汉松种植技术方面。与罗汉松实提取物有关的发明专利,国内公开的有3件:
黄增琼(CN201610460262)公开了一种从罗汉松种子高效提取分离高纯度川芎嗪的方法。该方法以罗汉松种子为原料,采用水蒸气蒸馏法进行提取,再以石油醚萃取馏出液,回收石油醚后干燥,得川芎嗪粗品,粗品用水重结晶得纯度大于98%的川芎嗪精品。
张明洞(CN201110095608)公开了一种罗汉松实酒的制备方法。该方法以成熟罗汉松实鲜品为原料,与一定比例的葡萄糖酒酵母和食用糖混合发酵一定时间制得。该发明仅对罗汉松实酒的制备方法进行了专利保护,没有涉及罗汉松实酒的用途。
张明洞(CN201110095609)公开了一种罗汉松实提取物及制品的生产方法。该方法以成熟罗汉松实鲜品为原料,用40%-80%浓度的乙醇提取,提取液浓缩后与辅料制成颗粒、片或胶囊;或者将提取液浓缩后添加入酒中制成果酒,添加入食品基质中制成果汁、饼干、糕点或面条类食品。该专利申请保护了罗汉松实乙醇提取浓缩液的制备方法以及提取浓缩液在食品领域的应用,没有涉及罗汉松实多糖的功能。
除此之外,还有部分以罗汉松实为复方组分之一,用于各种疾病治疗的中药复方专利申请。以往的这些报道,均没有涉及罗汉松实多糖的生物活性,也没有涉及到其在制备预防和治疗肝癌的药物或保健食品中的用途。
来源于植物的多糖已报道的生物活性有:免疫调节、抗肿瘤、降血脂、治疗动脉粥样硬化、治疗阿尔茨海默病等。然而,由于多糖的来源植物不同,提取分离纯化方法不同,所获得多糖的纯度、分子量、化学结构、单糖组成及各单糖所占比例也不相同。这些差异使不同的多糖具有不同的生物活性或不同强度的生物活性。因此,罗汉松实多糖是否具有抗肝癌作用,其作用效果如何,本领域专业人员是无法预知的。
发明内容
本发明的目的在于提供罗汉松实多糖的新用途。
本发明提供了一种罗汉松实多糖在制备预防和治疗肝癌的药品或保健食品中的用途。
本发明所述的罗汉松实多糖是从罗汉松科罗汉松属植物罗汉松Podocarpusmacrophyllus(Thunb.)D.Don或短叶罗汉松Podocarpus macrophyllus(Thunb.)D.Donvar.maki(Sieb.)Endl.的果实中提取分离纯化获得。
所述的罗汉松实多糖的制备方法包括下列步骤:
(1)提取:取罗汉松实鲜品或经60℃烘干的干品,加入鲜品或者干品质量6~10倍量的水,浸泡30分钟,80~100℃水浴加热提取1~2小时,过滤,取滤液,滤渣同法再提取1次,合并两次提取的滤液,记录初始体积,于80℃减压浓缩至剩余体积小于等于初始体积的10%,在浓缩液中加入无水乙醇至终末浓度为80~90%,沉淀24~48小时,用离心机以3000~4000转/分钟的速度离心30分钟,去除上清液,取沉淀;
(2)分离纯化:取沉淀,加入沉淀质量5~8倍量的水溶解,置于分液漏斗中,加入与水等体积的由正丁醇和氯仿按体积比1∶5组成的混合溶剂,振摇萃取,取水层,同法再萃取4次,取水层,记录初始体积,于80℃减压浓缩至剩余体积小于等于初始体积的5%,浓缩液用DEAE-52纤维素柱层析进行分离,以0.5mol/L氯化钠溶液为洗脱剂进行洗脱,收集洗脱液至体积为层析柱体积的8倍,洗脱液于80℃减压浓缩,浓缩液冷冻干燥或烘干即得。
所述的罗汉松实多糖质量百分含量按葡萄糖计不得低于70%。
本发明所述的预防和治疗肝癌的药品或保健食品,是以罗汉松实多糖为活性成分,加入可接受的辅料制备而成。其中,罗汉松实多糖的用量,以辅料重量计,占10~15%。
本发明通过实验证实,罗汉松实多糖对小鼠H22肝癌移植瘤肿瘤的生长具有显著的抑制作用(见说明书附图1),能显著缩小瘤体。本发明所述的药品或保健食品,其活性成分为罗汉松实多糖。罗汉松实多糖安全性好,应用于肝癌的预防和治疗,没有细胞毒类抗肿瘤药物常见的恶心、呕吐、脱发和骨髓抑制等毒副作用,是一种纯天然的安全有效的植物多糖。
下面将结合具体实施例对本发明的上述内容再作进一步的详细说明。但不应该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罗汉松实多糖对小鼠H22肝癌移植瘤肿瘤生长的影响
具体实施方式
实施例1
取罗汉松实干品100g,投入圆底烧瓶中,加入罗汉松实质量10倍量的水,浸泡30分钟,80℃水浴加热提取2小时,过滤,取滤液,滤渣再加入罗汉松实质量8倍量的水,80℃水浴加热提取1小时,过滤,合并两次提取的滤液,记录体积,于80℃减压浓缩至剩余体积为初始体积的5%,在浓缩液中加入无水乙醇至终末浓度为90%,沉淀24小时,用离心机以4000转/分钟的速度离心30分钟,去除上清液,取沉淀,加入沉淀质量5倍量的水溶解,置于分液漏斗中,以正丁醇和氯仿按体积比1∶5组成的混合溶剂为萃取剂萃取5次,每次萃取剂用量与水等体积,末次萃取后,取水层,记录初始体积,于80℃减压浓缩至剩余体积为初始体积的4%,浓缩液加入DEAE-52纤维素层析柱,用层析柱体积5倍量的去离子水洗脱,弃去,再以0.5mol/L氯化钠溶液为洗脱剂洗脱,收集洗脱液至体积为层析柱体积的8倍,洗脱液于80℃减压浓缩,冷冻干燥即得罗汉松实多糖。经苯酚硫酸法测定含量,所得罗汉松实多糖质量百分含量按葡萄糖计为85%。
实施例2
取罗汉松实鲜品300g,投入圆底烧瓶中,加入罗汉松实质量7倍量的水,80℃水浴加热提取1小时,过滤,取滤液,滤渣再加入罗汉松实质量6倍量的水,80℃水浴加热提取1小时,过滤,合并两次提取的滤液,记录体积,于80℃减压浓缩至剩余体积为初始体积的10%,在浓缩液中加入无水乙醇至终末浓度为85%,沉淀48小时,用离心机以3500转/分钟的速度离心30分钟,去除上清液,取沉淀,加入沉淀质量7倍量的水溶解,置于分液漏斗中,以正丁醇和氯仿按体积比1∶5组成的混合溶剂为萃取剂萃取5次,每次萃取剂用量与水等体积,末次萃取后,取水层,记录初始体积,于80℃减压浓缩至剩余体积为初始体积的5%,浓缩液用DEAE-52纤维素层析柱分离,以0.5mol/L氯化钠溶液为洗脱剂洗脱,收集洗脱液至体积为层析柱体积的8倍,洗脱液于80℃减压浓缩,浓缩液烘干即得罗汉松实多糖。经苯酚硫酸法测定含量,所得罗汉松实多糖质量百分含量按葡萄糖计为80%。
实施例3
取罗汉松实干品100g,投入圆底烧瓶中,加入罗汉松实质量8倍量的水,浸泡30分钟,100℃水浴加热提取1.5小时,过滤,取滤液,滤渣再加入罗汉松实质量6倍量的水,100℃水浴加热提取1小时,过滤,合并两次提取的滤液,记录体积,于80℃减压浓缩至剩余体积为初始体积的8%,在浓缩液中加入无水乙醇至终术浓度为90%,沉淀48小时,用离心机以3000转/分钟的速度离心30分钟,去除上清液,取沉淀,加入沉淀质量8倍量的水溶解,置于分液漏斗中,以正丁醇和氯仿按体积比1∶5组成的混合溶剂为萃取剂萃取5次,每次萃取剂用量与水等体积,末次萃取后,取水层,记录初始体积,于80℃减压浓缩至剩余体积为初始体积的5%,浓缩液加入DEAE-52纤维素层析柱,以0.5mol/L氯化钠溶液为洗脱剂洗脱,收集洗脱液至体积为层析柱体积的8倍,洗脱液于80℃减压浓缩,冷冻干燥即得罗汉松实多糖。经苯酚硫酸法测定含量,所得罗汉松实多糖质量百分含量按葡萄糖计为90%。
实施例4
罗汉松实多糖对小鼠H22肝癌移植瘤的抗肿瘤作用实验
(1)实验材料
动物:昆明种小鼠,SPF级,雄性,体质量20±2g,购自广西医科大学实验动物中心。
药物:罗汉松实多糖按实施例1的方法制备。环磷酰胺,批号:2017011325,江苏盛迪医药有限公司。
(2)实验方法
取接种H22瘤种9天、腹部隆起明显的腹水瘤小鼠以脱颈法处死,无菌条件下从腹腔抽出瘤液,用无菌生理盐水稀释后,800rmp·min-1离心5min,去除上清液得到肿瘤细胞,并加入生理盐水调整肿瘤细胞浓度至1×107个/mL,采用该浓度肿瘤细胞悬液对小鼠右腋皮下接种,接种量为0.2mL/只(正常组除外)。接种后24h,将50只小鼠随机分为正常组,模型组,环磷酰胺组(20mg/kg),罗汉松实多糖高(150mg/kg)、低剂量(100mg/kg)组。除正常组和模型组外,其余各组按10mL/kg灌胃给药,正常组和模型组给予等体积去离子水。各组小鼠每天给药1次,连续给药8天。末次给药后24小时,各组小鼠迅速摘取肿瘤,称重,计算肿瘤抑制率。肿瘤抑制率=(模型组平均瘤重-给药组平均瘤重)/模型组平均瘤重×100%。
(3)结果
结果见表1。结果表明,罗汉松实多糖高、低剂量均对小鼠H22肝癌移植瘤肿瘤的生长有显著的抑制作用,其中,罗汉松实多糖高剂量组对H22肝癌移植瘤的抑瘤率达到45.89%,效果较显著,说明本发明所述的罗汉松实多糖对实验性肝癌有明显的抑制作用(见说明书附图1)。
表1罗汉松实多糖对小鼠肝癌移植瘤的瘤重和抑瘤率影响
注:与模型组比较,1)P<0.05,2)P<0.01。
实施例5
罗汉松实多糖胶囊
按实施例1~3任一方法制备罗汉松实多糖。取药用辅料淀粉和糊精适量,加入辅料重量10%的罗汉松实多糖,混匀,加70%乙醇做润湿剂,制粒,烘干,整粒,加入硬脂酸镁或滑石粉作润滑剂,混匀,装入硬胶囊,即得。
实施例6
罗汉松实多糖口服液
按实施例1~3任一方法制备罗汉松实多糖。取蔗糖200g,加入蔗糖重量15%的罗汉松实多糖,加1000mL水加热溶解,混匀,灭菌30分钟,即得。
实施例7
罗汉松实多糖颗粒
按实施例1~3任一方法制备罗汉松实多糖。取药用辅料可溶性淀粉和蔗糖粉适量,加入辅料重量13%的罗汉松实多糖,混匀,用水或70%乙醇做润湿剂,制粒,烘干,整粒,分装,即得。
Claims (5)
1.罗汉松实多糖在制备预防和治疗肝癌的药品或保健食品中的用途。
2.根据权利要求1所述的用途,其特征在于所述的罗汉松实多糖的制备方法包括下列步骤:
(1)提取:取罗汉松实鲜品或经60℃烘干的干品,加入鲜品或者干品质量6~10倍量的水,浸泡30分钟,80~100℃水浴加热提取1~2小时,过滤,取滤液,滤渣同法再提取1次,合并两次提取的滤液,记录初始体积,于80℃减压浓缩至剩余体积小于等于初始体积的10%,在浓缩液中加入无水乙醇至终末浓度为80~90%,沉淀24~48小时,用离心机以3000~4000转/分钟的速度离心30分钟,去除上清液,取沉淀;
(2)分离纯化:取沉淀,加入沉淀质量5~8倍量的水溶解,置于分液漏斗中,加入与水等体积的由正丁醇和氯仿按体积比1∶5组成的混合溶剂,振摇萃取,取水层,同法再萃取4次,取水层,记录初始体积,于80℃减压浓缩至剩余体积小于等于初始体积的5%,浓缩液用DEAE-52纤维素柱层析进行分离,以0.5mol/L氯化钠溶液为洗脱剂进行洗脱,收集洗脱液至体积为层析柱体积的8倍,洗脱液于80℃减压浓缩,浓缩液冷冻干燥或烘干即得。
3.根据权利要求1所述的用途,其特征在于所述的罗汉松实多糖质量百分含量按葡萄糖计不得低于70%。
4.根据权利要求1所述的用途,其特征在于所述的药品或保健食品是以罗汉松实多糖为活性成分,加入可接受的辅料制备而成。
5.根据权利要求4所述的用途,其特征在于所述的药品或保健食品中罗汉松实多糖的用量,以辅料重量计,占10~15%。
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