CN113244302A - 一种用于调血脂的大黄游离蒽醌组合物 - Google Patents
一种用于调血脂的大黄游离蒽醌组合物 Download PDFInfo
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- CN113244302A CN113244302A CN202011455696.9A CN202011455696A CN113244302A CN 113244302 A CN113244302 A CN 113244302A CN 202011455696 A CN202011455696 A CN 202011455696A CN 113244302 A CN113244302 A CN 113244302A
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- free anthraquinone
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
本发明涉及一种用于调血脂的大黄游离蒽醌组合物,该组分主要包括芦荟大黄素、大黄酸、大黄素、大黄酚和大黄素甲醚,能够显著降低血液中的胆固醇和低密度脂蛋白胆固醇,平衡甘油三酯水平,也能显著升高高密度脂蛋白胆固醇,达到调节血脂的作用。该组分除了可以作为调脂类药物的主要原料,也可作为辅助成分添加到功能食品和美容外护产品中。
Description
【技术领域】
本发明属于卫生健康技术领域,具体涉及一种用于调血脂的大黄游离蒽醌组合物。
【背景技术】
伴随着我国经济腾飞和城市化的快速推进,人们工作生活节奏的加快、饮食结构的改变,血脂异常已成为一种常见的代谢性疾病。血脂异常会导致多种生理、心理疾病的发病,而且这种代谢性疾病正在向年轻人群中蔓延。尽管调血脂药物或保健品屡见不鲜,但调脂类化学药物的毒副作用大大降低了患者服药的依从性;各种保健品因调脂成分不详、功效不稳定、质量良莠不齐,患者对此类保健品的信任度呈下降趋势。近年来,寻找成分明确的天然源调脂类活性成分越来越被健康领域的研究者重视。
中药大黄蓼科植物掌叶大黄(Rheum palmatum L.)、唐古特大黄(Rheumtanguticum Maxim.ex Balf.)或药用大黄(Rheum officinale Baill.)的干燥根和根茎。作为中医药古籍中的“将军”级药材,是当今中医界用来减肥、美容及防治代谢性疾病的高频使用植物药。譬如文献报道大黄对治疗青春痘、面疱、疮疡痈肿、疔疮、丹毒、痤疮、酒渣鼻等有很好的疗效。大黄减肥汤可促进脂肪动员和利用,调节脂代谢;大黄减肥合剂能抑制大鼠食欲,增加排便次数,并影响肠内容物的吸收,可能具有抑制脂肪合成,减少细胞内脂肪含量的作用;大黄醇或水提取物调节脂质代谢的作用,大黄有效成分黄酮、二苯乙烯苷具有减少胆固醇吸收,降低LDLC,升高HDLC的作用。
大黄调脂活性的文献报道并未明确指出大黄发挥调脂作用的活性组分;其次大黄通过简单的水或醇提或粉碎后直接使用,用量较大,活性组分相对含量低,且因杂质过多存在安全隐患。因此,有必要对大黄不同提取物的调脂作用进行筛选,进而明确发挥调脂作用的活性组分,为以该组分为核心成分的药物或大健康产品的研发及质量控制奠定物质基础。
【发明内容】
本发明的目的在于提供一种用于调血脂的大黄游离蒽醌组合物。
本发明的另一个目的是提供该组合物的制备方法。
为了实现上述目的,本发明所采取的技术方案是:该组合物由5-15%,w/w的芦荟大黄素、 15-25%,w/w的大黄酸、18-28%,w/w的大黄素、23-33%,w/w的大黄酚和5-15%,w/w的大黄素甲醚组成。
本发明中大黄游离蒽醌组合物的提取方法如下:
(1)将大黄根及根茎粉碎,粉末与70%乙醇溶液按照1:8w/v比例混合浸泡过夜,60-70℃加热回流2h,过滤,收集滤液备用;
(2)滤渣与70%乙醇溶液按照1:5w/v加热回流2小时,过滤收集滤液;
(3)合并两次滤液,旋蒸除去乙醇,残留水混悬液用二氯甲烷萃取,真空干燥得大黄游离蒽醌提取物。
本发明所述的大黄游离蒽醌组合物可以在制备治疗高脂血症药物中应用。
本发明所述的大黄游离蒽醌组合物可以在制备辅助调血脂功能食品中应用。本发明所述的大黄游离蒽醌组合物亦可在制备美容外护产品中应用。
本发明所述大黄游离蒽醌组合物均提取自掌叶大黄(Rheum palmatum L.)、唐古特大黄(Rheum tanguticum Maxim.ex Balf.)或药用大黄(Rheum officinale Baill.)。
本发明所指大黄游离蒽醌组合物的调脂活性是经血脂异常模型大鼠和高脂细胞模型的防治试验确认。(1)SD大鼠连续灌胃给予50天的高脂乳剂,大鼠血脂四项,特别是胆固醇和低密度脂蛋白胆固醇含量显著升高时,可认为血脂异常模型建立成功。此后,同时灌胃给予模型大鼠高脂乳剂和大黄游离蒽醌组分制成的混悬液(间隔8h),给药时间约1个月,待血脂四项趋于正常时,停止给药。试验结果显示,大黄游离蒽醌组合物具有显著地降低血脂胆固醇和低密度脂蛋白胆固醇并升高高密度脂蛋白胆固醇,同时对甘油三酯也具有一定的平衡作用;治疗后高脂大鼠模型肝匀浆上清液中的总胆固醇含量下降,而粪便中总胆固醇含量却上升,表明大黄游离蒽醌可能促进胆固醇的代谢和排泄。经ORO染色的高脂大鼠模型肝组织显微观察表明大黄游离蒽醌能平衡肝组织中甘油三酯的水平至正常值。(2)HepG2细胞株经油酸培养基诱导,形成高脂细胞模型。经大黄游离蒽醌干预24h后,ORO染色表明大黄游离蒽醌组合物高剂量组可显著减少高脂细胞中的脂肪滴,表明大黄游离蒽醌组合物能阻止肝细胞对油脂的摄取或促进油脂的代谢。
本发明的有益效果是具有调血脂作用的大黄游离蒽醌组合物的化学组成是明确的,因而其质量也是可控的;其调脂作用是经高脂实验动物和细胞模型实验动物验证的,从而调脂活性的可靠性值得信赖。该组分可用于调脂类药物或大健康产品的研发原料。
【附图说明】
图1大黄游离蒽醌样品高效液相色谱图
A:芦荟大黄素
B:大黄酸
C:大黄素
D:大黄酚
E:大黄素甲醚
图2建模期50天模型组与对照组血脂指标对比
A:建模前血脂水平
B:建模后血脂水平
图3给药末期正常对照组、阴性对照组、阳性药物组和大黄游离蒽醌组血脂水平
A:血清总胆固醇TC
B:低密度脂蛋白胆固醇LDLC
C:高密度脂蛋白胆固醇HDLC
D:血清甘油三酯TG
图4给药末期正常对照组、阴性对照组、阳性药物组和大黄游离蒽醌组血脂水平各组血清低密度脂蛋白胆固醇与血清高密度脂蛋白胆固醇之比(LDLC/HDLC)水平
图5给药末期大黄游离蒽醌对肝脏和粪便中脂质的影响
A:肝脏胆固醇水平
B:粪便胆固醇水平
C:肝脏甘油三酯水平
D:粪便甘油三酯水平
图6给药末期正常对照组、阴性对照组、阳性药物组和大黄游离蒽醌组肝脏H&E染色和ORO 染色结果
A:正常对照组H&E染色结果
B:阴性对照组H&E染色结果
C:大黄游离蒽醌组H&E染色结果
D:正常对照组ORO染色结果
E:阴性对照组ORO染色结果
F:大黄游离蒽醌组ORO染色结果
图7MTT法细胞抗增殖活性检测
图8细胞高脂模型的大黄游离蒽醌调脂活性实验ORO染色结果
A:空白对照blank:10%FBS的高糖DMEM培养基
B:阴性对照OA+Vehicle:10%FBS+200μM OA+0.2%BSA的高糖DMEM培养基
C:阳性对照OA+PC 5μM:10%FBS+200μM OA+0.2%BSA的高糖DMEM培养基+5μM诺伐他汀
D:大黄游离蒽醌组OA+DE:10%FBS+200μM OA+0.2%BSA的高糖DMEM培养基+50μg/mL DE
E:大黄游离蒽醌组OA+DE:10%FBS+200μM OA+0.2%BSA的高糖DMEM培养基+10μg/mL DE
F:大黄游离蒽醌组OA+DE:10%FBS+200μM OA+0.2%BSA的高糖DMEM培养基+5μg/mL DE
【具体实施方式】
下面结合具体实施例,进一步详细阐明本发明。实施例中未注明具体条件的实验方法,通常按照常规条件以及手册中所述的条件,或按照制造厂商所建议的条件;所用的通用设备、材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1大黄游离蒽醌组分的的提取和精制
掌叶大黄根及根茎粉碎,粉末与70%乙醇溶液按照1:8(w/v)比例混合浸泡过夜,60-70℃加热回流2h,过滤,收集滤液备用,滤渣再与70%乙醇溶液按照1:5(w/v)加热回流2小时,过滤收集滤液。合并两次滤液,旋蒸除去乙醇,残留水混悬液用二氯甲烷萃取,真空干燥得大黄游离蒽醌提取物。以大黄游离蒽醌五种单体对照品为参照,反相高效液相色谱外标一点法定量分析,色谱柱为Diamonsil-C18柱(250mm×4.6mm,5μm),流动相为甲醇-0.1%磷酸(75:25,v/v),流速1mL/min,紫外检测波长254nm,柱温40℃,进样体积:20μL。大黄游离蒽醌高效液相色谱(HPLC)图见图1。按面积归一化法计算,大黄游离蒽醌的总含量达到94.56%,各游离蒽醌质量百分比如表1。
表1 5种大黄游离蒽醌含量百分比(n=3,mean±SD)
实施例2高脂模型大鼠的建立
SPF级雄性SD大鼠,体质量为180-200g,由兰州大学基础医学院医学实验动物中心提供,实验动物生产许可证SCXK(甘)2018-0002,动物实验使用许可证SYXK(甘)2018-0002。适应性喂养7天,随机分组,正常对照组均予基础饲料定量喂养,高脂模型组每日(10mL/kg)灌胃高脂乳剂(配方为10%猪油、5%胆固醇、5%蔗糖、1%胆酸钠、0.25%丙基硫氧嘧啶、10%丙二醇、10%吐温-80、58.75%蒸馏水),不限量给水。定期自眼眶静脉丛采血分离血清并用试剂盒检测血脂指标,血清总胆固醇TC(COD-PAP单试剂比色法)、血清甘油三酯TG(GPO-PAP单试剂比色法)、血清低密度脂蛋白胆固醇LDL-C(直接法)和血清高密度脂蛋白胆固醇HDL-C(直接法)四项指标的检测试剂盒均由南京建成生物工程研究所提供,建模期达50天。
高脂模型大鼠的验证:以建模前血脂水平(无差异)作为对比,建模后模型组大鼠的血清总胆固醇(TC)、血清低密度脂蛋白胆固醇(LDL-C)和血清高密度脂蛋白胆固醇(HDL-C)与正常对照组相比显著升高(p<0.01),模型组大鼠的血清甘油三酯(TG)与正常对照组相比显著降低(p<0.01),见图2。图2实验结果表明,高脂模型大鼠建立成功。
实施例3大黄游离蒽醌组合物的调血脂试验
建模完成之后,模型组再随机分组,分为空白对照组(GC)、阳性药物组(PC)、给药组和阴性对照组(NC)。自给药期开始,所有模型大鼠每日继续灌胃高脂乳剂的同时,间隔8小时后再分别按照10mL/kg的标准灌胃生理盐水(GC)、含非诺贝特的混悬液(PC)、含大黄游离蒽醌组分的混悬液(DE)和不含药物的混悬液(NC)。定期自眼眶静脉丛采血分离血清用试剂盒检测血脂指标,给药期达1个月。
灌胃药物混悬液的制备:将用于成人治疗量的非诺贝特,按照体表面积法换算成相应大鼠灌胃量所需的药物浓度,分别配制含阳性药物混悬液。按照40mg/mL的浓度配制含大黄游离蒽醌药物混悬液。另配制一个不含药物的溶媒混悬液。所有灌胃药物混悬液的组成成分如表2。
表2各组试验药物的成分组成
给药末期血脂检测中,阳性药物组(PC)与阴性对照组(NC)相比,血清中总胆固醇(TC)水平明显降低(p<0.05),低密度脂蛋白胆固醇(LDLC)水平明显降低(p<0.01),高密度脂蛋白胆固醇(HDLC)水平明显升高(p<0.05)。大黄游离蒽醌组(DE)与阴性对照组(NC)相比,血清总胆固醇(TC)水平显著降低(p<0.01),低密度脂蛋白胆固醇(LDLC)水平显著降低(p<0.01),高密度脂蛋白胆固醇(HDL-C)水平明显升高(p<0.05)。此外,大黄游离蒽醌组(DE)与阴性对照组(NC)相比,血清总胆固醇(TG)水平显著上升(p<0.01),与正常对照组甘油三酯(TG)水平相比无显著性差异。各组血脂指标,见图3。尤其是LDLC/HDLC 的比值相比NC组显著下降,有利于血脂稳定,见图4。
分别称取每组大鼠的粪便/肝样品1mg,并分别加提取试剂1mL(TC:异丙醇;TG:异丙醇与正庚烷的1:1混合物),于玻璃匀浆器中充分研磨提取,离心以取上清液并进行检测。严格遵循甘油三酸酯(TG)含量测定试剂盒和总胆固醇(TC)含量测定试剂盒(Solarbio)的说明。动物实验后取各组肝脏组织包埋与O.C.T.并用冷冻切片机切片机(Leica)制作7μm冷冻切片用于H&E染色及ORO染色。
由于血脂异常,NC组肝脏中的胆固醇水平高于GC组,DE组肝脏中的胆固醇明显降低,并显著提高了粪便中的胆固醇。这表明大黄游离蒽醌组合物增加了胆固醇排泄。对于甘油三酯,NC组大鼠肝脏中明显减少,而DE组甘油三酯水平明显回升,见图5。组织切片的H&E染色也显示NC组肝脂肪空泡减少,而DE组再次出现。ORO染色的结果也同样证明了,NC组的脂质滴减少,而DE组重新出现,见图6。以上实验结果表明,大黄游离蒽醌组合物具有调血脂作用。
实施例4基于细胞高脂模型的大黄游离蒽醌组合物调脂活性实验
HepG2细胞株虽属于癌细胞,但其保留了正常肝细胞的糖脂代谢等绝大多数生理功能,是文献报道较多的用于调脂活性试验的细胞。将HepG2细胞用含有10%胎牛血清(Sigma)和1×青霉素-链霉素溶液的高糖DMEM培养基中于37℃和5%CO2常规培养,2-3天更换培养基。当细胞达到约80%融合时,经胰酶消化,离心收集细胞。以20w细胞/孔的细胞密度接种在24孔板上,孔中已提前放入细胞爬片。贴壁后,在无血清的培养基中培养12h。在加有200μM油酸(OA,Sigma)的培养基中诱导24h,油酸提前与终浓度0.2%BSA结合。通过MTT检测选取无抗细胞增殖活性的样品浓度进行干预细胞脂质积累模型的实验,避免由于高浓度提取物引起的毒性对细胞生长产生抑制作用。预实验结果表明,100μg/ml以下大黄游离蒽醌提取物浓度对HepG2细胞的生长无影响,见图7。以空白对照作为对比,阴性对照给与溶媒,非诺贝特选取终浓度为5μM,大黄游离蒽醌(DE)选取三个浓度设为梯度进行干预(50μg/mL,10μg/mL,5μg/mL)。24h干预后进行ORO染色观察细胞间质中的脂质区域并进行观察对比。组别如下:
空白对照blank:10%FBS的高糖DMEM培养基
阴性对照OA+Vehicle:10%FBS+200μM OA+0.2%BSA的高糖DMEM培养基阳性对照OA+PC 5μM:10%FBS+200μM OA+0.2%BSA的高糖DMEM培养基+5μM诺伐他汀
大黄游离蒽醌组OA+DE:10%FBS+200μM OA+0.2%BSA的高糖DMEM培养基 +50/10/5μg/mL DE
药物干预24h后,ORO染色表明,经OA诱导过后的阴性对照组细胞间质中有大量红色的脂肪滴。5μM非诺贝特和50μg/mL DE可以显著减少脂肪滴,见图8。结果表明,大黄游离蒽醌组合物对于细胞高脂模型具有调脂作用。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
Claims (7)
1.一种用于调血脂的大黄游离蒽醌组合物,其特征在于,由以下化合物组成:5-15%,w/w的芦荟大黄素、15-25%,w/w的大黄酸、18-28%,w/w的大黄素、23-33%,w/w的大黄酚和5-15%,w/w的大黄素甲醚。
2.根据权利要求1所述的大黄游离蒽醌组合物,其特征在于,该组分的提取方法如下:
(1)将大黄根及根茎粉碎,粉末与70%乙醇溶液按照1:8w/v比例混合浸泡过夜,60-70℃加热回流2h,过滤,收集滤液备用;
(2)滤渣与70%乙醇溶液按照1:5w/v加热回流2小时,过滤收集滤液;
(3)合并两次滤液,旋蒸除去乙醇,残留水混悬液用二氯甲烷萃取,真空干燥得大黄游离蒽醌提取物。
3.根据权利要求1-2任意一项所述的大黄游离蒽醌组合物在制备治疗高脂血症药物中的应用。
4.根据权利要求1-2任意一项所述的的大黄游离蒽醌组合物在制备辅助调血脂功能食品中的应用。
5.根据权利要求1-2任意一项所述的的大黄游离蒽醌组合物在制备美容外护产品中的应用。
6.一种制备调血脂的大黄游离蒽醌组合物的方法,其特征在于,包括以下步骤:
(1)将大黄根及根茎粉碎,粉末与70%乙醇溶液按照1:8w/v比例混合浸泡过夜,60-70℃加热回流2h,过滤,收集滤液备用;
(2)滤渣与70%乙醇溶液按照1:5w/v加热回流2小时,过滤收集滤液;
(3)合并两次滤液,旋蒸除去乙醇,残留水混悬液用二氯甲烷萃取,真空干燥得大黄游离蒽醌提取物。
7.根据权利要求2-6任意一项所述的的大黄游离蒽醌组合物,其特征在于:所述组合物提取自掌叶大黄(Rheum palmatum L.)、唐古特大黄(Rheum tanguticum Maxim.ex Balf.)或药用大黄(Rheum officinale Baill.)。
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