CN116139189A - 一种匙羮藤总皂苷的制备方法及其提高肠道产丁酸菌群丰度的用途 - Google Patents
一种匙羮藤总皂苷的制备方法及其提高肠道产丁酸菌群丰度的用途 Download PDFInfo
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Abstract
本发明公开了一种匙羮藤总皂苷的制备方法及将其用于提高肠道产丁酸菌群丰度的用途，该提取物的制备流程主要包括：①取匙羮藤根或叶，加入溶剂后进行亚临界萃取，过滤后减压浓缩，得第一产物；②将上述第一产物分散于水中，加有机溶剂萃取，得第二产物；③将上述第二产物溶解、吸附、层析、洗脱，收集洗脱液并浓缩、干燥成粉，得匙羮藤总皂苷提取物。本发明制备的匙羮藤总皂苷提取物对肠道功能具有良好的调节作用；动物实验研究表明在给药剂量≥125 mg/kg∙d时，肠道产丁酸菌群的丰度有显著提升。
Description
技术领域
本发明属于植物提取物技术领域，具体涉及一种匙羮藤总皂苷的制备方法及将其用于提高肠道产丁酸菌群丰度的用途。
背景技术
短链脂肪酸(SCFAs)由结肠内的厌氧菌酵解肠道中未被消化吸收的碳水化合物产生，主要包括乙酸、丙酸、丁酸等。其中，丁酸是结肠保持完整性和健康的关键调节物质，与增强肠道屏障有关。作为结肠细胞的主要和首选能量底物，丁酸的细胞代谢优先于葡萄糖或谷氨酰胺，占结肠黏膜总能量需求的70％。丁酸能刺激表皮生长因子和抗炎细胞因子的产生，减少炎性细胞因子的产生，并具有抑制NF-κB活化的能力。据报道，消化道中发现的已知丁酸盐生产者大多数属于毛螺菌科和瘤胃球菌科，且大肠产丁酸的菌种主要是梭菌属XIVa和IV族，以及真杆菌属和梭杆菌属。当肠道中产丁酸菌群丰度下降时，丁酸等SCFAs含量降低，有害菌群丰度升高、肠黏膜通透性增加以及血液循环中LPS等炎症因子的增加，肠道黏膜屏障功能下降、炎症和免疫力下降，引发肠道内肠毒素、细胞毒素、病原体等侵入人体产生危害，导致糖尿病(DM)、肥胖、肠炎代谢性疾病的发生。因此产丁酸菌群对宿主的影响受到越来越多的关注，有实力成为防治DM等代谢性疾病的重要潜在靶点。
匙羹藤(Gymnema sylvestre(Retz)Schult)属萝藦科(Asclepiadaceae)植物，是我国南方地区传统的常用药材之一，其根或嫩枝叶均可入药，以“gurmar”(糖分杀手)而闻名。现代研究表明，匙羹藤总皂苷是其主要功效成分，在降糖、降脂和减轻体重方面效果显著，被广泛应用于食品、保健品、化妆品等行业，然而目前对其在通过调节肠道菌群进而缓解DM鲜有报道。
亚临界萃取是利用亚临界流体作为萃取剂，在密闭、无氧、低压的压力容器内，依据有机物相似相溶的原理，通过物料与萃取剂在浸泡过程中的分子扩散过程，使固体物料中目的成分快速转移到液态萃取剂中的一种新型萃取与分离技术。该技术提取速度快、效率高、有效成分提取充分，并且溶媒耗量少、出液系数小、浸出液浓度高，节省溶剂，节省后道工序生产成本，非常适合从复杂中药化学成分中直接大量提取皂苷类成分。
发明内容
针对上述存在的问题，本发明提供了一种匙羹藤总皂苷提取物的制备方法并将其用于改善肠道产丁酸菌群丰度的用途，有助于DM和肥胖的治疗。
为了实现上述目的，本发明采用以下技术方案：
匙羮藤总皂苷提取物的制备方法，包括以下步骤：
①取匙羮藤叶或根干燥后粉碎，加入60％-70％乙醇进行亚临界萃取，萃取液经过滤后减压浓缩，得第一产物；
②将上述第一产物分散于水中，加正丁醇萃取，并将萃取液进行减压浓缩，得第二产物；
③将上述第二产物经乙醇溶解后，加入至D101大孔树脂吸附柱进行层析，然后收集洗脱液，减压浓缩并冷冻干燥，得匙羮藤总皂苷提取物。
优选地，所述步骤①中亚临界萃取时间为1h，萃取2次。
优选地，所述步骤③中洗脱过程为先用水洗脱，再用60％-70％乙醇洗脱。
匙羮藤总皂苷提取物在调节动物肠道中产丁酸菌群丰度中的应用：动物实验研究表明匙羮藤总皂苷提取物可显著升高肠道内产丁酸菌群丰度，在本发明的具体实施例中，给药剂量≥125mg/kg·d时，大鼠肠道中产丁酸菌群的丰度有显著提升。
匙羮藤总皂苷提取物在制备提高肠道中产丁酸菌群丰度的药物或添加剂中的应用。进一步地，产丁酸菌群包括毛螺菌科及瘤胃球菌科的菌种，尤其是梭状芽孢杆菌属、粪杆菌属、粪球菌属与罗氏菌属的菌种。
本发明具有如下优点和有益效果：
(1)本发明采用的亚临界萃取设备，依据有机物相似相溶的原理，通过萃取物料与萃取剂在浸泡过程中的分子扩散，以液化的亚临界溶剂对物料进行逆流萃取，最终得到目的产物，这种新型萃取与分离技术具有无毒、无污染、易于和产物分离等优点。
(2)本发明提供的匙羹藤总皂苷富集流程，结合特征性萃取剂和吸附柱对匙羹藤中特异性功效成分进行两次富集，可最大程度提高匙羹藤提取物中活性成分含量。
(3)丁酸在抑制有害菌群维持肠道微生态平衡、保护肠道黏膜屏障和降低炎性反应中起着重要作用。本发明制备的匙羹藤总皂苷提取物可显著提高动物肠道中产丁酸菌群丰度，因此对DM、肥胖、肠炎代谢性疾病的治疗具有重要意义。
附图说明
图1为本发明的整体步骤流程图。
图2为实施例1中负离子模式下匙羹藤总皂苷提取物的质谱监测图(总离子流图)。
图3为实施例1中负离子模式下匙羹藤总皂苷提取物总离子流图峰8(匙羹藤酸，Gymnemic acid A)的二级碎片谱图。
图4为实施例1中负离子模式下匙羹藤总皂苷提取物总离子流图峰21(羟基积雪草酸，Madecassic acid)的二级碎片谱图。
图5为实施例1中负离子模式下匙羹藤总皂苷提取物总离子流图峰22(Alternoside XVIII)的二级碎片谱图。
图6为实施例3中匙羹藤总皂苷提取物对2型DM大鼠肠道产丁酸毛螺菌科(Lachnospiraceae)丰度的影响。*p<0.05具有显著性差异，**p<0.01具有极显著性差异，以下相同。
图7为实施例3中匙羹藤总皂苷提取物对2型DM大鼠肠道产丁酸瘤胃球菌科(Ruminococcaceae)丰度的影响。
图8为实施例3中匙羹藤总皂苷提取物对2型DM大鼠肠道产丁酸梭状芽孢杆菌属(Clostridium spp.)丰度的影响。
图9为实施例3中匙羹藤总皂苷提取物对2型DM大鼠肠道产丁酸粪杆菌属(Faecalibacterium spp.)丰度的影响。
图10为实施例3中匙羹藤总皂苷提取物对2型DM大鼠肠道产丁酸粪球菌属(Coprococcus spp.)丰度的影响。
图11为实施例3中匙羹藤总皂苷提取物对2型DM大鼠肠道产丁酸罗氏菌属(Roseburia spp.)丰度的影响。
具体实施方式
实施例1：亚临界萃取技术用于匙羮藤总皂苷提取物的制备
匙羮藤总皂苷提取物的制备流程包括如下步骤：
①取匙羮藤(Gymnema sylvestre(Retz)Schult)叶片，晒干后粉碎，加入10倍量的60％乙醇浸泡后进行亚临界萃取2次，每次1h；合并提取液，减压抽滤后经旋转蒸发仪减压浓缩，得第一产物(粗提取物)。
亚临界萃取实验流程如下所示：
a.按上述物料比称重、量取匙羮藤材料和萃取剂乙醇，投入萃取罐中；
b.启动电加热器，设置油温为100℃；
c.启动真空泵，将萃取罐抽到真空状态；
d.关闭萃取罐所有阀门，启动导热油泵，对萃取罐进行循环加热；萃取2次，每次1h；
e.打开冷凝器的冷却水进水阀门，通入冷却水；萃取液在负压下经过过滤器和冷凝器，进入萃取液暂存罐；
②将上述第一产物以体积比1:10分散于水中，并以体积比1:1加有机溶剂正丁醇萃取3次；合并正丁醇萃取液，并经旋转蒸发仪减压浓缩，得第二产物。
③将上述第二产物加5％乙醇溶解，加入至D101大孔吸附树脂层析柱，样品上样体积为30％柱体积；静置6h，首先用3个柱体积的纯水洗脱，然后用3个柱体积的60％乙醇洗脱；合并乙醇洗脱液，并经旋转蒸发仪减压浓缩后得到棕褐色浸膏；经冷冻干燥后，得匙羮藤总皂苷提取物。
④匙羮藤总皂苷提取物的色谱-质谱检测分析
将上述匙羮藤总皂苷提取物依次使用50％甲醇溶解、0.22μm滤膜过滤，进行色谱-质谱检测分析。
(1)高效液相色谱(HPLC)分析条件如下：
高效液相色谱仪：Thermo Accela 600；
色谱柱：Waters SunfireTM RP-C18；规格为4.6×150mm，5μm；
流动相：A-0.1％甲酸-水，B-乙腈；
流速：0.5-0.6mL/min；
进样量：5-10μL；
检测波长：210-230nm；
梯度洗脱程序：0-2min，15％ B；2-20min，15％-55％ B；20-25min，55％-70％B；25-40min，70％-95％ B；40-45min，95％-15％ B。
(2)三重四级杆串联质谱(TSQ-MS)分析条件如下：
质谱仪：TSQ Quantum Access MAX；
离子源：电喷雾(ESI)电离源；
采集模式：负离子模式；
分子量扫描范围：100-1500Da；
二级谱扫描方式：数据依赖型扫描；
喷雾电压：3000V；
锥孔电压：40V；
毛细管温度：350℃；
辅助氮气气压：40psi；
碰撞能量30-45eV
将谱图中各峰的保留时间、母离子和碎片离子信息，与已有参考文献或标准品比较，进行色谱峰化学成分的结构解析与鉴定。
进一步地，匙羮藤总皂苷提取物LC-MS分析的总离子流图如附图2所示。其中，部分代表性功效成分MS/MS质谱信息详见附图3(匙羹藤酸，Gymnemic acid A)、附图4(羟基积雪草酸，Madecassic acid)与附图5(Alternoside XVIII)。
实施例2：DM大鼠模型造模
所述的大鼠为SD大鼠，优选为雄性；本实施例中DM模型造模所用化学药物优选为链脲佐菌素(STZ)。
2型DM大鼠模型造模包括如下步骤：
(1)大鼠与饲养条件：
6周龄SPF级雄性SD大鼠，40只，体重180-200g，购买于北京维通利华动物实验技术有限公司。
将大鼠饲养于SPF级动物房，房间控制条件为：室温22±2℃，湿度50±10％；光照/黑暗交替周期为12h/12h；适应性饲养3d，期间自由进食饮水。
(2)2型DM大鼠模型造模
40只SD大鼠经适应性喂养3d后，随机选取7只作为正常组(NC)。正常对照组大鼠给予常规饲料，其余各组大鼠均给予高脂饲料喂养4周。
高脂饮食诱导造模结束后，大鼠禁食不禁水12h，腹腔注射1％链脲佐菌素(STZ)溶液(35mg/kg，溶于0.1mol/L柠檬酸钠缓冲液中，30min内完成注射)，正常对照组大鼠注射相同剂量的柠檬酸钠缓冲溶液。
于STZ注射72h后，取大鼠尾尖血检测空腹血糖(FBG)，将FBG>11.1mmol/L、随机血糖值>16.7mmol/L确定为2型DM大鼠造模成功。
实施例3：匙羮藤总皂苷提取物对2型DM大鼠肠道产丁酸菌群丰度的提高作用
丁酸具有抑制有害菌群、维持肠道微生态平衡、保护肠道黏膜屏障和降低炎性反应等重要作用。
本实施例动物实验所用阳性对照药物为盐酸二甲双胍(Met)
(1)2型DM大鼠分组与给药
将上述实施例2造模成功的28只2型DM大鼠，依照体重、FBG值随机分为4组(n＝7)，即模型组(DM)、阳性组(Met，150mg/kg)、低剂量组(GS-L，125mg/kg)与高剂量组(GS-H，250mg/kg)。
临用前，将实施例1制得的匙羮藤总皂苷提取物，使用生理盐水溶解，配制成浓度为20mg/mL的溶液(混悬)。
阳性组、匙羮藤总皂苷提取物低/高剂量组大鼠分别按上述剂量灌胃给药30d，1次/d；正常组、模型组大鼠按体重给予灌胃生理盐水。
上述各组大鼠2-3只/笼，自由饮水至给药30d结束。
(2)大鼠粪便中肠道菌群的16S rRNA分析
给药结束前一天，收集各组大鼠粪便，保存于无菌EP管中，标记后于-80℃冰箱保存，尽快送检。
使用Illumina公司的TruSeq Nano DNA LT Library Prep Kit制备测序文库，并采用MiSeq测序仪进行测序，观察各组大鼠肠道菌群的分布情况。
通过质量初筛的原始序列按照index和Barcode信息，进行文库和样本划分，并去除barcode序列。
对各样本(组)在不同物种分类学水平的具体组成进行展示，了解整体概况。在物种分类学组成层面，通过各种非监督、监督的排序、聚类和建模手段，结合相应统计学检验方法，进一步衡量不同样本(组)间的物种丰度组成差异。
(3)统计方法
实验数据采用SPSS 24.0统计软件进行处理分析，计量资料用均数±标准差
表示。多组间比较且方差齐性数据采用单因素方差(One-way ANOVA)分析，并采用LSD法进行两两间多重比较。数据间差异以p<0.05为有统计学意义。
(4)大鼠肠道菌群分析结果
附图6为匙羹藤总皂苷提取物对2型DM大鼠肠道菌群芽孢杆菌科丰度的影响。试验数据表明，匙羹藤总皂苷提取物低剂量组(GS-L，125mg/kg)与高剂量组(GS-H，250mg/kg)均可显著提升2型DM大鼠肠道产丁酸毛螺菌科(附图6)、瘤胃球菌科(附图7)丰度。
进一步地，匙羹藤总皂苷提取物低剂量组(GS-L，125mg/kg)与高剂量组(GS-H，250mg/kg)均可提升2型DM大鼠肠道产丁酸菌群梭状芽孢杆菌属(附图8)、粪杆菌属(附图9)、粪球菌属(附图10)与罗氏菌属(附图11)丰度。
上述实验数据表明，与模型组(DM)大鼠比较，匙羹藤总皂苷提取物可提升2型DM大鼠肠道多种产丁酸菌群(尤其是丁酸毛螺菌科、瘤胃球菌科)丰度，作用效果呈现较好的量效关系，并且在给药浓度为125mg/kg时即可达到显著性差异。
Claims (7)
1.匙羮藤总皂苷提取物的制备方法，其特征在于，包括以下步骤：
①取匙羮藤叶或根干燥后粉碎，加入60%-70%乙醇进行亚临界萃取，萃取液经过滤后减压浓缩，得第一产物；
②将上述第一产物分散于水中，加正丁醇萃取，并将萃取液进行减压浓缩，得第二产物；
③将上述第二产物经乙醇溶解后，加入至D101大孔树脂吸附柱进行层析，然后收集洗脱液，减压浓缩并冷冻干燥，得匙羮藤总皂苷提取物。
2.根据权利要求1所述的匙羮藤总皂苷提取物的制备方法，其特征在于，所述步骤①中亚临界萃取时间为1h，萃取2次。
3.根据权利要求1所述的匙羮藤总皂苷提取物的制备方法，其特征在于，所述步骤③中洗脱过程为先用水洗脱，再用60%-70%乙醇洗脱。
4.匙羮藤总皂苷提取物在调节动物肠道中产丁酸菌群丰度中的应用。
5.匙羮藤总皂苷提取物在制备提高肠道中产丁酸菌群丰度的药物或添加剂中的应用。
6.根据权利要求4或5所述的应用，其特征在于，产丁酸菌群包括毛螺菌科及瘤胃球菌科的菌种。
7.根据权利要求6所述的应用，其特征在于，产丁酸菌群包括梭状芽孢杆菌属、粪杆菌属、粪球菌属与罗氏菌属的菌种。
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| CN1322539A (zh) * | 2000-05-08 | 2001-11-21 | 陈文彬 | 由匙羹藤叶制备治疗糖尿病药物的方法 |
| CN1475222A (zh) * | 2003-07-31 | 2004-02-18 | 凯 曹 | 匙羹藤叶精提物及其制备工艺 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322539A (zh) * | 2000-05-08 | 2001-11-21 | 陈文彬 | 由匙羹藤叶制备治疗糖尿病药物的方法 |
| CN1475222A (zh) * | 2003-07-31 | 2004-02-18 | 凯 曹 | 匙羹藤叶精提物及其制备工艺 |
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