CN117064906A - 一种龙眼肉提取物的提取方法和应用 - Google Patents
一种龙眼肉提取物的提取方法和应用 Download PDFInfo
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Abstract
本发明属于植物提取技术领域,提供了一种龙眼肉提取物的提取方法和应用,所述提取物以龙眼肉为原料,依次经过粉碎、蒸煮、过滤,沉淀离心、洗涤、干燥后得到龙眼肉提取物龙眼肉多糖。所述提取物经动物实验表明,能改善AD小鼠的学习记忆,能调节肠道菌群,减少有害菌,增加有益菌,能调节肠道菌群代谢产物短链脂肪酸的水平,能调节小胶质细胞代谢相关基因表达;经细胞实验表明,能促进Aβ耐受的小胶质细胞的增殖,降低促炎因子的分泌,增加抗炎因子的分泌,提高线粒体膜电位的水平,增强对Aβ蛋白的吞噬功能。因此,本发明所述的龙眼肉提取物能够用于制备AD免疫治疗药物;制备改善AD患者肠道菌群的药物。
Description
技术领域
本发明属于植物提取技术领域,具体地说是一种龙眼肉提取物的提取方法和应用。
背景技术
阿尔兹海默症(Alzheimer'sdisease,AD)是一种常见的中枢神经系统退行性疾病,俗称老年痴呆。AD患者多出现认知和行为障碍,随着时间的推移,逐渐丧失身体机能,直至死亡,给家庭乃至社会造成重大负担。作为一种隐匿的进行性神经系统疾病,AD病理机制复杂,尽管近年来对AD的认识和了解有了很大进步,但目前临床治疗药物仅能一定程度上缓解轻、中度患者症状,无法有效防止AD的进展,且存在毒副作用大等缺点。
龙眼肉是我国传统中药,功能补益心脾、养血安神,其用于痴呆的论述早有记载。名中医张锡纯用调气养神汤,重用龙眼肉治疗思虑过度,伤其神明,知觉错乱,是非不分诸症(《医学衷中参西录》),效果良好。现代研究发现,龙眼肉石油醚相和水相能提高机体抗氧化能力、抑制脑组织Aβ表达,提高AD小鼠学习记忆;龙眼肉多糖促进淋巴细胞和巨噬细胞增殖,促进巨噬细胞的吞噬作用和炎症因子释放水平,并且具有明显的促益生菌增殖作用,作为药食两用的药物,龙眼资源丰富、价格便宜、食用安全,但因目前研究数量有限,尚未得到很好的开发利用。
为此,本领域技术人员提出了一种龙眼肉提取物的提取方法和应用来解决背景技术提出的问题。
发明内容
为了解决上述技术问题,本发明提供一种龙眼肉提取物的提取方法和应用,本发明的龙眼肉提取物经动物实验表明,能改善AD小鼠的学习记忆,能调节肠道菌群,减少有害菌,增加有益菌,能调节肠道菌群代谢产物短链脂肪酸的水平,能够用于制备AD免疫治疗药物及调节AD肠道菌群的药物。
为了实现上述目的,本发明提供了如下技术方案。
本发明提供了一种龙眼肉提取物在制备调节肠道菌群的药物中的应用。
本发明还提供了一种龙眼肉提取物在制备能够恢复小胶质细胞功能的药物中的应用。
本发明还提供了一种龙眼肉提取物在制备抑制老年痴呆病的药物中的应用。
进一步地,上述任一项所述应用中所述龙眼肉提取物的制备具体包括以下步骤:
S1、准备新鲜的龙眼热风干燥12-25h后,去皮去核得到龙眼果肉;
S2、将龙眼果肉放入破碎机内破碎,得到龙眼肉液混合物;
S3、向龙眼肉液混合物内加入清水浸泡;
S4、对龙眼混合物和水进行煎煮并进行搅拌;
S5、将煎煮后的龙眼肉水分离过滤,得到液A,将残留的龙眼肉渣进行压榨,继续对肉水分离过滤,得到液B,将液B倒入液A中得到提取液;
S6、在提取液内加入适量的乙醇进行加热,加热过程中进行搅拌,使得提取液中的多糖溶解在醇中;
S7、将混合液冷却至室温,利用超声波对提取液进行震动;
S8、将提取液沉淀离心、洗涤、干燥后得到多糖。
更进一步地,在步骤S2中,所述破碎时间控制在20-25min,从而达到龙眼肉完全被破碎。
更进一步地,在步骤S3中,所述清水为去离子水,所述清水和龙眼肉液混合物的比例为10∶1。
更进一步地,在步骤S4中,所述龙眼混合物和水煮至沸腾后,改用小火进行煎煮25-30min,煎煮过程中,对龙眼混合物和水进行搅拌,避免煮焦。
更进一步地,在步骤S5中,所述对龙眼肉水分离采用200目的纱布进行过滤。
更进一步地,在步骤S6中,所述乙醇选用质量分数为95%的乙醇溶液,所用乙醇量为提取液的4倍。
与现有技术相比本发明的有益效果。
本发明的龙眼肉提取物经动物实验表明,能改善AD小鼠的学习记忆,能调节肠道菌群,减少有害菌,增加有益菌,能调节肠道菌群代谢产物短链脂肪酸的水平,调节PI3K、AKT、mTOR基因表达。经细胞实验表明,能促进Aβ耐受的小胶质细胞的增殖,降低促炎因子的分泌,增加抗炎因子的分泌,提高线粒体膜电位的水平,增强对Aβ蛋白的吞噬功能,作用机制与调节糖酵解代谢和PI3K/AKT/mTOR/HIF-1信号通路相关。因此,本发明所述的龙眼肉提取物能够用于制备AD免疫治疗药物;制备改善AD肠道菌群的药物。
附图说明
图1为本发明的龙眼肉多糖单糖组成和分子量检测数据图。其中,A为龙眼肉多糖单糖组成检测数据图;B为龙眼肉多糖单糖分子量检测数据图。
图2为本发明的水迷宫实验中小鼠逃避潜伏期及进入原平台象限次数数据图。其中,A为小鼠逃避潜伏期数据图;B为小鼠进入原平台象限次数数据图。
图3为本发明的16S-rDNA肠道微生物多样性分析图。其中,A为三组样本OUTs分布花瓣图;B为各组样本在不同分类水平(界、门、纲、目、科、属、种)下注释OTUs的总tags数;C为α多样性物种累积曲线;D为等级聚类曲线;E为β多样性PCoA分析图;F为小鼠肠道菌群中厚壁菌门与拟杆菌门相对丰度比较图;G为小鼠肠道菌群结构属水平分析图;H为拟普雷沃菌属相对丰度比较图;I为瘤胃球菌科_UCG-014属相对丰度比较图;J为普雷沃氏菌科UCG-001属相对丰度比较图;K为理研菌科RC9_gut_group属相对丰度比较图;L为瘤胃梭菌属相对丰度比较图;M为瘤胃球菌属1相对丰度比较图;N为Muribaculum属相对丰度比较图;O为毛螺菌科UCG-001属相对丰度比较图。
图4为本发明的GC-MS检测小鼠粪便中短链脂肪酸水平数据图。其中,A为小鼠粪便中乙酸含量;B为小鼠粪便中正丁酸含量;C为小鼠粪便中正戊酸含量;D为小鼠粪便中异丁酸含量;E为小鼠粪便中异戊酸含量;F为小鼠粪便中乳酸含量。
图5为本发明的细胞荧光吞噬代表性图片、细胞增殖率及相对平均荧光强度图。其中,A为Aβ诱导耐受的BV2细胞增殖数据图;B为不同浓度龙眼肉多糖对Aβ诱导耐受的BV2细胞增殖影响的统计图;C为不同浓度龙眼肉多糖对Aβ诱导耐受的BV2细胞吞噬中性红能力影响的统计图;D为各组细胞形态显微镜观察的代表性图片;E为龙眼肉多糖(0.8、1.6 mg/mL)对Aβ诱导耐受的BV2细胞增殖影响的验证性实验数据图;F为各组BV2细胞对荧光标记Aβ吞噬情况的代表性显微镜观察图;G为各组BV2细胞对荧光标记Aβ吞噬能力的统计结果。
图6为本发明的细胞线粒体膜电位检测荧光代表性图片及红绿荧光强度分析图。其中,A为JC-1法检测细胞线粒体膜电位水平的荧光观察图;B~D分别为荧光强度统计结果。
图7为本发明的细胞乳酸分泌水平及促炎因子、抗炎因子分泌水平的检测图。其中,A为乳酸含量分析图;B促炎因子(TNF-α)含量分析图;C为抗炎因子(IL-10)含量分析图;D为TNF-α与IL-10含量比值的分析图。
图8为本发明的龙眼肉多糖改善动物记忆及调控基因与蛋白表达的代表性数据图。其中,A为水迷宫实验小鼠轨迹图;B为脑室注射墨汁验证定位图;C为各组小鼠逃避潜伏期对比图;D~G为qPCR检测GAPDH、PI3K、AKT、mTOR的溶解曲线图;H~K为qPCR检测PI3K、AKT、mTOR、HIF-1α的基因相对表达图。
图9为加入各种抑制剂干预后细胞相对平均荧光强度及荧光吞噬的代表性图。其中,A为Western blot检测蛋白表达图;B为PI3K蛋白表达图;C~E为Akt、p-Akt和HIF-1α蛋白表达图;F~I为荧光吞噬结果。
图10为本发明的提取物提取方法流程图。
具体实施方式
下面结合附图和实施例对本发明的实施方式作进一步详细描述。以下实施例用于说明本发明,但不能用来限制本发明的范围。
实施例1。
一种龙眼肉提取物,以龙眼肉为原料,依次经过粉碎、蒸煮、过滤,沉淀离心、洗涤、干燥后得到龙眼肉提取物龙眼肉多糖,具体包括以下步骤(如图10):
S1、准备新鲜的龙眼热风干燥12-25h后,去皮去核得到龙眼果肉;
S2、将龙眼果肉放入破碎机内破碎,得到龙眼肉液混合物;所述破碎时间控制在20-25min,从而达到龙眼肉完全被破碎;
S3、向龙眼肉液混合物内加入清水浸泡;所述清水为去离子水,所述清水和龙眼肉液混合物的比例为10∶1;
S4、对龙眼混合物和水进行煎煮并进行搅拌;所述龙眼混合物和水煮至沸腾后,改用小火进行煎煮25-30min,煎煮过程中,对龙眼混合物和水进行搅拌,避免煮焦;
S5、将煎煮后的龙眼肉水分离过滤,得到液A,将残留的龙眼肉渣进行压榨,继续对肉水分离过滤,得到液B,将液B倒入液A中得到提取液;所述对龙眼肉水分离采用200目的纱布进行过滤;
S6、在提取液内加入适量的乙醇进行加热,加热过程中进行搅拌,使得提取液中的多糖溶解在醇中;所述乙醇选用质量分数为95%的乙醇溶液,所用乙醇量为提取液的4倍;
S7、将混合液冷却至室温,利用超声波对提取液进行震动;
S8、将提取液沉淀离心、洗涤、干燥后得到多糖(如图1),所得多糖采用HPLC法检测单糖组成(表1),采用HPGPC法测定分子量范围(表2)。
表1 提取物HPLC法检测单糖组及含量
。
表2 HPGPC法测定提取物分子量范围
。
实施例2。
30只昆明小鼠(体重22g-24g),随机分为正常组,模型组和龙眼肉组,每组15只。除正常组外,各组采用皮下注射D-半乳糖、腹腔注射AlCl3联合节制饮食法建立小鼠AD模型,每日一次,连续60d,龙眼肉组于造模第30d开始每日灌胃龙眼肉提取物2g/kg,Morris水迷宫实验检测各组小鼠学习记忆。如图2所示,龙眼肉水提取物显著降低了AD模型组小鼠的逃避潜伏期(图2A),增加了模型组小鼠进入原平台象限的次数(图2B),表明龙眼肉水提取物可改善AD小鼠的学习记忆能力。
实施例3。
龙眼肉水提取物改善AD小鼠肠道微生物组成。实施例2中所述行为学测试完成后的小鼠,无菌收集正常组(Con)、模型组(AD)和龙眼肉组(LA)粪便,进行16S-rDNA测序分析。按照97%的相似度进行OTU分类,基于OTU序列相似性,分析各组样本α多样性和Beta多样性,并进行群落分布与结构分析。如图3所示,三组样本OUTs数(图3A)及各组样本在不同分类水平(界、门、纲、目、科、属、种)下注释OTUs的总tags数目均不同(图3B)。α多样性物种累积曲线(图3C)趋于平缓,等级聚类曲线(图3D)宽而平坦,表明样本抽样充分,物种组成丰富且均匀度良好。由β多样性PCoA分析图(图3E)可见,三组间物种存在显著性差异,表明龙眼肉提取物改变了AD小鼠肠道微生物的组成。在群落分布分析中,以门水平为例,各组小鼠肠道菌群主要包括拟杆菌门(Bacteroidetes),厚壁菌门(Firmicutes),变形菌门(Proteobacteria)等,但其相对丰度发生显著变化,与正常组相比,AD模型组组小鼠肠道菌群中厚壁菌门与拟杆菌门的比值明显升高(图3F),龙眼肉明显降低了AD组厚壁菌门与拟杆菌门的比值。群落结构分析结果表明(图3G~O),以属水平为例,龙眼肉使异常升高的拟普雷沃菌属(Alloprevotella)、瘤胃球菌科_UCG-014属(Ruminococcaceae_UCG-014)、普雷沃氏菌科UCG-001属(Prevotellaceae_UCG-001)、理研菌科RC9_gut_group属(Rikenellaceae_RC9_gut_group)、瘤胃梭菌属(Ruminiclostridium)和瘤胃球菌属1(Ruminococcus_1)丰度显著降低,并使Muribaculum和毛螺菌科UCG-001属(Lachnospiraceae_UCG-001)丰度显著升高。
实施例4。
龙眼肉水提取物改善AD小鼠菌群代谢物短链脂肪酸的水平并降低乳酸含量。行为学测试完成后,无菌收集正常组、模型组和龙眼肉组小鼠粪便(同实施例3中所述粪便),采用GC-MS技术对各组小鼠粪便中的短链脂肪酸水平及乳酸含量进行检测。如图4所示,龙眼肉显著降低了AD模型小鼠粪便中乙酸(图4A)、正丁酸(图4B)、正戊酸水平(图4C),显著升高了异丁酸(图4D)和异戊酸(图4E)水平,并显著降低了异常升高的乳酸(图4F)含量。
实施例5。
龙眼肉水提取物有效成分龙眼肉多糖(LAPs)提高了小胶质细胞(BV2)对Aβ的吞噬功能。如图5所示,低浓度Aβ的长时间处理(4μM Aβ处理细胞48h后常规培养24h)导致BV2细胞免疫耐受,细胞增殖达最大水平(图5A),经不同浓度LAPs处理后,细胞增殖与吞噬能力呈现不同程度的变化,其中0.1mg/mL LAPs对细胞增殖(图5B)及中性红吞噬能力(图5C)具有抑制作用,0.2mg/mL及以上浓度有促进细胞增殖与吞噬能力的趋势,且以0.8、1.6mg\mL作用最显著(图5B、5C)。选取0.8、1.6mg\mL LAPs做进一步增强与吞噬实验验证,通过细胞形态观察发现,LAPs组细胞形态接近正常组(图5D),细胞增殖率较模型组显著提高(图5E),细胞胞内荧光Aβ增多(图5F),细胞吞噬能力增强(图5G)。结果表明适当浓度的龙眼肉多糖可以显著提高Aβ耐受细胞的增殖及吞噬能力。
实施例6。
龙眼肉提取物有效成分LAPs增强了Aβ耐受BV2细胞线粒体膜电位水平。JC-1法(图6A)是检测细胞线粒体膜电位水平常用的检测方法,红/绿荧光的比值越大,代表线粒体膜电位越高。如图6所示,LAPs组与模型组比较,线粒体膜电位红/绿荧光比显著增强(图6B~D),提示LAPs可以增强Aβ耐受细胞的线粒体膜电位,改善细胞的线粒体功能,抑制细胞早期凋亡。
实施例7。
龙眼肉提取物有效成分LAPs抑制了Aβ耐受BV2细胞乳酸及炎症因子释放水平。采用试剂盒检测细胞培养上清液中乳酸、促炎因子TNF-α及抗炎因子IL-10水平,评价LAPs对BV2细胞乳酸分泌及炎症因子水平的影响,如图7所示,较模型组,LAPs组细胞的乳酸分泌量(图7A)及TNF-α水平(图7B)显著降低,IL-10水平(图7C)显著升高,并且TNF-α与IL-10的比值显著降低(图7C),提示LAPs可以降低Aβ耐受BV2细胞乳酸分泌水平,调节免疫炎症反应。
实施例8。
龙眼肉提取物有效成分LAPs改善学习记忆并影响PI3K/Akt/mTOR/HIF-1α信号通路相关基因表达。昆明种小鼠(20-24g)随机分为假手术组、模型组和LAPs 0.7、1.4g/kg(含生药量)组,每组15只。除假手术组外,各组采用侧脑室(图8B)注射Aβ诱导建立小鼠AD模型,采用LAPs进行灌胃治疗,每日一次,连续30d。Morris水迷宫检测小鼠学习记忆,qPCR检测PI3K、AKT、mTOR、HIF-1α等基因表达,以GAPDH为内参。水迷宫实验结果(图8A)表明,与假手术组比较,模型组从定位航行实验第2d开始逃避潜伏期明显延长;与模型组比较,LAPs组(以1.4g/kg组为例)自定位航行实验第3d开始逃避潜伏期明显缩短,提示侧脑室注射Aβ导致小鼠学习记忆障碍,LAPs可明显改善小鼠学习记忆能力(图8C)。qPCR检测结果表明(图8D~K),较假手术组,模型组PI3K、AKT、mTOR基因相对表达均降低,HIF-1α基因相对表达升高,LAPs能够逆转模型组小鼠的上述各基因变化,提示LAPs的抗AD作用可能与调节PI3K/AKT/mTOR信号通路相关。
实施例9。
龙眼肉提取物有效成分LAPs的抗AD作用与调控糖酵解代谢和激活PI3K/Akt/mTOR/HIF-1α信号通路相关。采用4μM Aβ1-42(48h+24h)诱导建立BV2细胞耐受模型,采用龙眼肉多糖(LAPs)0.8、1.6mg/mL进行处理,并加入糖酵解抑制剂及mTOR通路相关各抑制剂,包括糖酵解抑制剂2-脱氧-D-葡萄糖(2-Deoxy-D-glucose,2-DG)、mTOR抑制剂抑制剂雷帕霉素(Rapamycin,Rapa)和HIF-1抑制剂BAY87-2243(BAY)进行干预,Western blot检测蛋白表达,倒置荧光显微观察细胞对荧光标记Aβ(AF488-Aβ)的吞噬情况。如图9所示,Westernblot结果(图9A)表明,与正常组比较,模型组即免疫耐受的BV2细胞除PI3K蛋白表达减少外(图9B),Akt、p-Akt和HIF-1α蛋白表达均增加(图9C~E),提示在BV2吞噬Aβ过程中伴有PI3K/Akt/mTOR/HIF-1α信号通路的激活;与模型组比较,LAPs(0.8、1.6mg/mL)组的PI3K、Akt、p-Akt和HIF-1α蛋白的表达水平均提高,提示LAPs可能通过激活PI3K/Akt/mTOR/HIF-1α信号通路恢复免疫耐受的BV2细胞吞噬功能。荧光吞噬结果(图9F~I)表明,免疫耐受的BV2细胞吞噬能力下降,LAPs能提高其对AF488-Aβ的吞噬能力,并且其作用可被抑制剂2-DG、Rapa和BAY完全或部分阻断,说明LAPs可能通过调控糖酵解代谢和PI3K/Akt/mTOR/HIF-1α信号通路提高Aβ耐受BV2细胞的吞噬能力进而抑制AD进程。
由上可知,本发明制备的龙眼肉提取物能够调节肠道微生物多样性,恢复小胶质细胞相关功能,进而抑制AD的自然病程。
本发明的实施例是为了示例和描述起见而给出的,尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (9)
1.一种龙眼肉提取物在制备调节肠道菌群的药物中的应用。
2.一种龙眼肉提取物在制备能够恢复小胶质细胞功能的药物中的应用。
3.一种龙眼肉提取物在制备抑制老年痴呆病的药物中的应用。
4.根据权利要求1~3任一所述的应用,其特征在于,所述龙眼肉提取物的制备具体包括以下步骤:
S1、准备新鲜的龙眼,热风干燥12-25h后,去皮去核得到龙眼果肉;
S2、将龙眼果肉放入破碎机内破碎,得到龙眼肉液混合物;
S3、向龙眼肉液混合物内加入清水浸泡;
S4、对龙眼混合物和水进行煎煮并进行搅拌;
S5、将煎煮后的龙眼肉水分离过滤,得到液A,将残留的龙眼肉渣进行压榨,继续对肉水分离过滤,得到液B,将液B倒入液A中得到提取液;
S6、在提取液内加入适量的乙醇进行加热,加热过程中进行搅拌,使得提取液中的多糖溶解在醇中;
S7、将混合液冷却至室温,利用超声波对提取液进行震动;
S8、将提取液沉淀离心、洗涤、干燥后得到多糖。
5.如权利要求4所述的应用,其特征在于:在步骤S2中,所述破碎时间控制在20-25min,从而达到龙眼肉完全被破碎。
6.如权利要求4所述的应用,其特征在于:在步骤S3中,所述清水为去离子水,所述清水和龙眼肉液混合物的比例为10∶1。
7.如权利要求4所述的应用,其特征在于:在步骤S4中,所述龙眼混合物和水煮至沸腾后,改用小火进行煎煮25-30min,煎煮过程中,对龙眼混合物和水进行搅拌,避免煮焦。
8.如权利要求4所述的应用,其特征在于:在步骤S5中,所述对龙眼肉水分离采用200目的纱布进行过滤。
9.如权利要求4所述的应用,其特征在于:在步骤S6中,所述乙醇选用质量分数为95%的乙醇溶液,所用乙醇量为提取液的4倍。
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