CN116407580A - 素馨花提取物在调节肠道菌群及改善代谢综合征中的应用 - Google Patents
素馨花提取物在调节肠道菌群及改善代谢综合征中的应用 Download PDFInfo
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Abstract
本发明属于医药技术领域，公开了一种素馨花提取物在调节肠道菌群及改善代谢综合征中的应用。本发明将素馨花提取物在制备预防或治疗代谢综合征及肠道菌群紊乱的药物、制备金氏副拟杆菌的特效富集药物的制剂中应用。本发明为素馨花及其活性成分用于防治代谢综合征及肠道健康的调节作用机理的研究提供了基础，可将素馨花提取物作为药用成分及相关药物的开发与应用。
Description
技术领域
本发明涉及医药技术领域，具体涉及一种素馨花提取物在调节肠道菌群及改善代谢综合征中的应用。
背景技术
肥胖是一种以身体脂肪含量过多为重要特征的、多病因的、能够并发多种疾病的慢性病。肥胖者更易患2型糖尿病、非酒精性脂肪肝、心血管疾病、癌症等疾病。
目前，肥胖的治疗方法包括饮食治疗、运动疗法、药物治疗和外科疗法。饮食治疗即限制总热量的摄入；运动疗法即在医师的指导下依据病人运动量、种类、强度、耐受力等多个方面制定方案；饮食治疗、运动疗法均需要个体化治疗，在治疗的过程中也需要依据阶段疗效不断调整治疗方案。药物治疗包括作用于中枢和外周的减肥药，但此类药物都有不同程度的副作用。外科疗法仅适用于内科治疗失败且有严重并发症的患者。现有技术中，中药对肥胖的认识源远流长，治疗方式多样，且多采用“药食同源”资源如黄芪、灵芝、冬虫夏草等名贵中药材及其活性成分，无明显副作用。因此，亟需开发新的中药资源作为治疗肥胖的药用成分及相关药物。
发明内容
本发明的目的在于克服现有技术的不足之处而提供一种素馨花提取物在调节肠道菌群及改善代谢综合征中的应用。
为实现上述目的，本发明采取的技术方案如下：
本发明将素馨花提取物在制备预防或治疗代谢综合征及肠道菌群紊乱的药物、制备金氏副拟杆菌的特效富集药物的制剂中应用。优选的，将素馨花提取物在制备预防或治疗肥胖及相关代谢性疾病的药物中应用。
素馨花为木樨科茉莉属的药食同源植物，具有“舒肝解郁，行气止痛”之功效。发明人发现素馨花提取物及其活性成分橄榄苦苷、羟基酪醇具有良好的改善肥胖、非酒精性脂肪肝、糖尿病等代谢综合征的作用。它们能够高效调节肠道菌群的结构与组成，如选择性地调节某些菌群，包括金氏副拟杆菌(Parabacteroides goldsteinii，PG)。此外，作为“药食同源”资源的中药成分，橄榄苦苷在日用橄榄油中含量高达80mg/g，而且长期食用未发现有毒副作用，因而在医药和功能食品等领域具有高度的可应用性，备受关注。
作为本发明所述的应用的优选实施方式，所述素馨花提取物包含橄榄苦苷。
作为本发明所述的应用的优选实施方式，所述素馨花提取物的制备方法为：
取素馨针干燥花蕾，用乙醇提取得滤液，浓缩后得粗提物；将粗提物水溶解，按照石油醚、乙酸乙酯和正丁醇的顺序对粗提物进行萃取，取正丁醇层提取物上大孔树脂柱洗脱；收集洗脱液，蒸干溶剂即得素馨花提取物。
作为本发明所述的应用的优选实施方式，所述提取采用80％乙醇于常温常压下提取。
作为本发明所述的应用的优选实施方式，所述上大孔树脂柱时，依次用3倍体积的水、10％乙醇、20％乙醇和5倍体积的30％乙醇洗脱；收集30％乙醇洗脱液。
本发明提供了一种预防或治疗代谢综合征或肠道菌群紊乱的药物，包含素馨花提取物。
作为本发明所述的药物的优选实施方式，所述代谢综合征为肥胖及相关代谢性疾病。
与现有技术相比，本发明的有益效果为：
本发明利用16S rDNA扩增子测序技术和生物信息学技术分析发现，富含橄榄苦苷的素馨花提取物有效调节肥胖小鼠肠道菌群的结构与组成，防止高脂饮食引起的菌群紊乱。素馨花提取物能够高效富集肠道副拟杆菌属微生物，尤其是在科、属、种水平上显著富集新一代益生菌--金氏副拟杆菌。金氏副拟杆菌具有潜在的改善代谢综合征功能，素馨花提取物可作为金氏副拟杆菌的特效富集药物开发及应用。
本发明利用“脏笼共享”交叉饲养技术，揭示金氏副拟杆菌可能介导素馨花提取物的抗代谢综合征作用，该菌可作为抗代谢综合征的潜在靶点。
本发明为素馨花及其活性成分用于防治代谢综合征及肠道健康的调节作用机理的研究提供了基础，可将素馨花提取物作为药用成分及相关药物的开发与应用。
附图说明
图1为高效液相色谱(HPLC)检测素馨花粗提物(A)和橄榄苦苷标准溶液(B)的结果图。
图2为素馨花提取物经肠道菌群改善代谢综合征(n＝8-10)；
图中，(A)实验流程；(B)进食量；(C)体重增长量；(D-F)附睾、腹股沟及肾周WAT的重量；(G-I)血浆总胆固醇(CHO)、甘油三酯(TG)及低密度脂蛋白胆固醇(LDL-C)水平。
图3为素馨花提取物显著改善肥胖小鼠肠道菌群的结构与组成(n＝8)；
图中，(A)香农指数；(B)PCoA图；(C)门水平上的菌群组成。
图4为素馨花提取物选择性富集肥胖小鼠肠道金氏副拟杆菌(n＝8)；
图中，(A)LEfSe分析图；(B)差异菌群的LDA图；(C)种水平上s_Parabacteroides_goldsteinii的相对丰度。
图5为“脏笼共享”受体小鼠肠道菌群的结构与组成(n＝10)；
图中，(A)脏笼共享实验示意图；(B)香农指数；(C)PCoA图；(D)门水平上的菌群组成。
图6为素馨花提取物饲养供体小鼠显著增加受体小鼠肠道金氏副拟杆菌的相对丰度(n＝10)；
图中，(A)LEfSe分析图；(B)差异菌群的LDA图；(C)种水平上s_Parabacteroides_goldsteinii的相对丰度。
图7为粪便微生物传递素馨花提取物改善代谢紊乱的作用(n＝10)；
图中，(A)受体小鼠体重增长量；(B)受体小鼠摄食量；(C)受体小鼠附睾白色脂肪(gWAT)含量；(D)受体小鼠皮下白色脂肪(iWAT)含量；(E)受体小鼠肾周白色脂肪组织(rpWAT)含量；(F)受体小鼠血浆甘油三酯(TG)含量；(G)受体小鼠血浆总胆固醇(CHO)含量；(H)受体小鼠低密度脂蛋白胆固醇(LDL-C)含量；(I)受体小鼠血浆谷丙转氨酶(ALT)；(J)受体小鼠血浆谷草转氨酶(AST)。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。本领域技术人员应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
实施例中所用的试验方法如无特殊说明，均为常规方法；所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明利用高脂饮食诱导的肥胖小鼠模型，结合分子生物学和高通量多组学测序等技术，通过系统研究金氏副拟杆菌(PG)与富含橄榄苦苷的素馨花提取物(OJGF)之间的相互作用，进一步阐明挖掘金氏副拟杆菌在素馨花发挥抗肥胖作用过程中扮演的作用。
实施例1：素馨花提取物制备
取素馨针干燥花蕾8kg，用80％乙醇于常温常压下提取3次约72h，合并3次的滤液，旋蒸仪浓缩后得3.6kg粗提物。将其水溶解后，按照石油醚、乙酸乙酯和正丁醇的顺序对提取物进行萃取，依次得到萃取物重量是：30g、280g和1300g。取240g正丁醇层提取物上大孔树脂柱，依次用3倍体积的水、10％乙醇、20％乙醇和5倍体积的30％乙醇洗脱。收集30％乙醇洗脱部分的21到34瓶，旋转蒸发仪蒸干溶剂得到58g样品，即素馨花提取物。
采用高效液相色谱法(HPLC)测得橄榄苦苷含量为69.8％，在280nm波长下检测HPLC图谱如图1所示。
实施例2：素馨花提取物调节肠道菌群及改善代谢综合征的作用
高脂饲料(HFD)喂养的小鼠给予素馨花提取物干预8周，观察作用效果。具体如下：7周龄SPF级C57BL/6J雄性小鼠，实验前适应环境1周(以下其他动物实验与此相同)。所有小鼠按设定时间点自由食用普通饲料(Chow)、HFD(45％脂肪热量)或含富含0.024％、0.12％和0.6wt％素馨花提取物(橄榄苦苷含量为69.8％)HFD饲养8周，同时小鼠饮用含或不含抗生素(1g/L氨苄青霉素、0.5g/L万古霉素、1g/L新霉素和1g/L甲硝唑)混合物(antibioticcocktail，ABX)的无菌水。即实验组别分别为：正常组(Chow)、模型组(HFD)、抗生素组(ABX)、素馨花提取物低剂量组(OL)、中剂量组(OM)、高剂量组(OH)、素馨花提取物高剂量+ABX组(OH-ABX)，具体分组(8只/组)和给药方法详见图1A。观察小鼠体重、体成分、生化指标、肠道菌群结构与组成等指标的变化。
如图2所示：实验结束后，与HFD饮食小鼠比较，素馨花提取物干预小鼠的体重增长量和附睾(gWAT)、皮下(iWAT)及肾周(rpWAT)白色脂肪的重量以及血浆中ALT、AST、CHO、TG和LDL-C水平呈剂量依赖性降低(图2B-I)，提示素馨花提取物具有良好的抗肥胖效应。利用抗生素混合物ABX耗竭肠道菌群后，素馨花提取物干预小鼠的体重增长量和体脂成分含量及血浆中ALT、AST、CHO、TG和LDL-C并未发生显著的变化。这些结果，共同表明素馨花提取物通过肠道菌群发挥抗肥胖作用。
16S rRNA扩增子测序分析各组小鼠粪便肠道菌群多样性与结构。收集小鼠粪便，液氮速冻后，干冰送至北京百迈客生物科技有限公司进行测序分析。简而言之，用QIAGENDNA Mini KIT试剂盒提取粪便DNA，经PCR扩增16S DNA的V3～V4区后，进行琼脂糖凝胶电泳和核酸蛋白定量检测来检测提取的DNA的纯度及浓度。合格DNA于PacBio测序平台进行16SrDNA测序。
分析结果如图3和图4所示，素馨花提取物干预8周可有效改善HFD饮食小鼠肠道菌群结构与组成，增加有益菌的相对丰度，尤其是金氏副拟杆菌(PG)在科、属、种水平上显著富集，提示素馨花提取物在体内能有效地促进金氏副拟杆菌(PG)的生长，有益于其抗代谢综合征作用的发挥。
为了进一步明确金氏副拟杆菌(PG)在素馨花提取物抗肥胖中的作用，利用“脏笼共享”交叉饲养HFD饮食小鼠8周。即上述素馨花提取物饲养小鼠和HFD饲养小鼠的生活脏笼，分别用于饲养HFD饮食小鼠(即HRecip组和ORecip组，10只/组)，每周换笼2次，持续8周，检测受体小鼠肠道菌群结构与组成、体重、体脂成分、血糖血脂、肝功能等指标的变化。
如图5、图6和图7所示，与供体小鼠相似地，素馨花提取物干预的小鼠生活脏笼饲养可有效重塑HFD饮食小鼠的肠道菌群结构与组成，尤其是有益菌金氏副拟杆菌(PG)，在科、属、种水平上显著富集(图5和图6)。与此同时，受体小鼠体重、体重增长量、gWAT、iWAT及rpWAT重量，和血浆中ALT、AST、CHO、TG及LDL-C水平明显降低(图7)，进一步提示金氏副拟杆菌(PG)可能在素馨花提取物改善代谢综合征过程中扮演着重要角色。
肥胖的形成和发展与肠道菌群紊乱密切相关。靶向肠道菌群及其代谢产物调节相关代谢通路逐渐成为代谢性疾病防治的新趋势。本发明通过深度分析了富含橄榄苦苷的素馨花提取物对HFD饮食小鼠的肠道菌群，发现其可特异性富集肥胖小鼠肠道副拟杆菌属微生物，该类菌的富集有利于减弱HFD饮食引起的肥胖。副拟杆菌属微生物可能作为新的防治代谢综合征的作用靶点，而“药食同源”植物则可作为其开发有效的资源库，为临床抗肥胖及相关代谢病的精准干预和新药研发提供新思路和新靶点。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (10)
1.素馨花提取物在制备预防或治疗代谢综合征的药物中的应用。
2.素馨花提取物在制备预防或治疗肠道菌群紊乱的药物中的应用。
3.根据权利要求1所述的应用，其特征在于，所述代谢综合征为肥胖及相关代谢病。
4.素馨花提取物在制备金氏副拟杆菌的特效富集药物的制剂中的应用。
5.根据权利要求1～4任一项所述的应用，其特征在于，所述素馨花提取物包含橄榄苦苷。
6.根据权利要求5所述的应用，其特征在于，所述素馨花提取物的制备方法为：取素馨针干燥花蕾，用乙醇提取得滤液，浓缩后得粗提物；将粗提物水溶解，按照石油醚、乙酸乙酯和正丁醇的顺序对粗提物进行萃取，取正丁醇层提取物上大孔树脂柱洗脱；收集洗脱液，蒸干溶剂即得素馨花提取物。
7.根据权利要求6所述的应用，其特征在于，所述提取采用80％乙醇于常温常压下提取。
8.根据权利要求6所述的应用，其特征在于，所述上大孔树脂柱时，依次用3倍体积的水、10％乙醇、20％乙醇和5倍体积的30％乙醇洗脱；收集30％乙醇洗脱液。
9.一种预防或治疗代谢综合征或肠道菌群紊乱的药物，其特征在于，包含素馨花提取物。
10.根据权利要求8所述的药物，其特征在于，所述代谢综合征为肥胖及相关代谢病。
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