CN112168848A - 一种海蓬子降血糖组分的制备方法及应用 - Google Patents
一种海蓬子降血糖组分的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种海蓬子降血糖组分的制备方法及应用,具体步骤为:首先对海蓬子进行预处理,然后烘干、粉碎、过筛得到海蓬子粉末;再将海蓬子粉末放入容器中,向容器中加入浓度为50%~60%的乙醇溶液,将容器放入温度为40~50℃的水浴锅中,控制超声波功率为150~200W,并持续运行0.5h,最后使用真空泵抽滤取上清液,即得。本发明以海蓬子为原料,采用超声波辅助提取技术制备海蓬子降血糖组分,研制出一种高活性的海蓬子降血糖组分。其制备方法简便易行,生产成本低,降血糖效果显著,降血糖组分来源于药食两用植物资源,资源相当丰富,使用方便、安全,对人体无毒副作用。
Description
技术领域
本发明属于生物技术领域,具体涉及一种海蓬子降血糖组分的制备方法及应用。
背景技术
糖尿病作为一种以高血糖为特征的人体慢性非传染性代谢疾病,会导致人体肾脏、神经、视力等组织发生慢性损害和功能障碍,已经严重影响人们的生活水平。经济的快速发展,使人们的生活水平不断提升,但由于糖尿病等这类慢性病的发生,又导致人们健康水平显著下降。调查研究表明,我国18岁以上的成年人糖尿病的患病率达到11.6%,并且还在以较快的速度增长,根据调查结果推测我国成年人糖尿病的患病人数超过1.14亿人。随着居民生活水平的提高以及人口老龄化的加剧,糖尿病患者的患病人数也将进一步增加,因此带来的高致残致死率,不仅带来严重的健康问题,还带来了严重的医疗经济负担,因此糖尿病预防及治疗对策的研究显得至关重要,需要建立符合我国当前糖尿病防治的手段。
糖尿病患者最大的特点表现为“三多一少”,具体为吃多、喝多、尿多、身体消瘦,还有部分患者出现其他一些症状,如肥胖、乏力等症状。目前糖尿病的治疗途径是以医学证据与指南为依据,主要以口服降糖药和注射胰岛素等联合治疗达到控制血糖的目的,通过药物治疗需要结合药物代谢动力学和患者肾脏功能情况进行综合研判。有超过一半的糖尿病患者会出现冠心病等症状,因此药物治疗糖尿病还需要注意药物对心血管的安全性问题。糖尿病患者前期防治过程中,食物对病情的影响非常显著,因此糖尿病化患者需要根据病情及时调整饮食,通过食疗的方法防止疾病的变化。糖尿病患者的饮食要做到科学合理,满足个性化需求,对于食品的种类也有严格的要求,除了有效控制主食,还需要合理搭配副食,以免带来不良后果。随着科学的发展,越来越多的研究发现草本植物中许多成分可以用于糖尿病患者的治疗,尤其是与西药联合使用,可以显著提高治疗效果。
海蓬子是美国亚利桑那大学学者从1300多种野生盐生植物中筛选出来的的最具经济价值的真盐生植物,属于藜科(Chenopodiaceae)海蓬子属(Salicornia),又称海虫草、海芦笋、海鹿茸、富贵菜,海蓬子可作为无公害蔬菜、动物青饲料和生物能源物质,江苏省盐城市在2001年成功引种海蓬子,已经在该地区海域种植海蓬子超过133.3公顷,海蓬子已经作为高档食材进入酒店餐桌。海蓬子有很强的耐盐能力,耐旱不耐涝,非常适合在亚热带滨海地区生长。其耐盐度可高达5%NaCl浓度,超过海水盐度的20%~40%,内茎含有的可溶性盐分的量达37%(干质量百分比),因此可以使用海水直接进行灌溉,海蓬子还可以改良沿海滩涂盐碱地,对促进我国海涂农业的发展具有重要意义。
《中华本草》记载海蓬子(Salicornia bigelovii Torr.)具有平肝、利尿、降压的功效,主治高血压和头痛。海蓬子富含多种活性物质,研究表明海蓬子含有黄酮类、生物碱类、有机酸类、多糖和三萜皂苷类等多种成分,具有抗氧化、抗肿瘤、抗炎、免疫调节、降血脂等多种作用。同时,海蓬子是公认的绿色有机蔬菜,素有“植物海鲜”和“海人参”的美誉,在欧美地区有着悠久的食用历史。海蓬子籽实可作为提炼高级食用油的原料;作为西方传统减肥草药,其枝梗可用来做减肥茶,具有清热解毒、降脂、减肥的功效;而作为具有极强的集盐功能的天然盐生植物,可用来提炼低钠、富含矿物质和有机碘的生物盐;海蓬子全草可作利尿、抗坏血病的中药,表明海蓬子具有较高的营养价值和良好的生物活性作用。
糖尿病是由基因和环境等多种因素引起的代谢性疾病,患病率高。目前降糖药种类繁多,作用机制各异,决定了其不同的临床特点和不良反应。从天然植物中筛选安全、有效的活性成分成为一条重要的途径。海蓬子在耐盐方面表现出极大的优势,可直接用海水灌溉。在耕地减少、淡水污染等问题日益严重的今天,将我国广大的盐碱地与滩涂地这一宝贵的国土资源利用起来,大力发展海水灌溉农业具有重大意义。巨大的原料生长空间,为以海蓬子作为原料来源的技术提供了保障,既有良好的经济价值,又具有重要的社会价值。但目前尚未检索到利用海蓬子制备降血糖组分的报道。
发明内容
针对现有技术的不足,本发明提供一种海蓬子降血糖组分的制备方法及应用,以海蓬子为原料,采用超声波辅助提取技术制备海蓬子降血糖组分,采用UPLC-ESI-TOF-MS鉴定出海蓬子降血糖基本组成成分和相对比例,验证海蓬子降血糖组分的降血糖效果,研制出一种高活性的海蓬子降血糖组分。其制备方法简便易行,生产成本低,降血糖效果显著,降血糖组分来源于药食两用植物资源,资源相当丰富,使用方便、安全,对人体无毒副作用。
本发明是通过以下技术方案实现的:
一种海蓬子降血糖组分的制备方法,包括以下步骤:
步骤1)材料预处理:收集海蓬子地上部分,洗净泥沙,沥干水分;将处理好的海蓬子材料置于鼓风干燥箱中,55℃进行烘干处理;材料烘干后采用粉碎机将其粉碎,过筛,将得到的海蓬子粉末置于含有干燥剂的干燥器中保存,待用;
步骤2)称取经步骤1)处理的海蓬子粉末放入容器中,再向容器中加入浓度为50%~60%的乙醇溶液,将容器放入温度为40~50℃的水浴锅中,控制超声波功率为150~200W,并持续运行0.5h,最后使用真空泵抽滤取上清液,即得所述海蓬子降血糖组分。
优选地,步骤2)所述海蓬子粉末与乙醇溶液的料液比为1g:15mL。
优选地,步骤2)所述乙醇溶液的浓度为60%;所述温度为50℃;所述超声波功率为180W。
上述方法制得的海蓬子降血糖组分在制备具有降血糖作用的保健食品或食品添加剂中的应用。
上述方法制得的海蓬子降血糖组分在制备降血糖药物中的应用。
一种降血糖药物,包括上述方法制得的海蓬子降血糖组分以及药学上可接受的药物载体。
上述方法制得的海蓬子降血糖组分在制备保护和修复肝脏及胰腺的药物中的应用。
上述方法制得的海蓬子降血糖组分在制备针对因糖尿病造成的肝损伤进行保护和修复的药物中的应用。
上述方法制得的海蓬子降血糖组分在制备针对因糖尿病造成的胰岛损伤进行保护和修复的药物中的应用。
本发明的有益效果如下:
1、本发明采用超声辅助提取技术结合响应面法获得海蓬子降血糖组分,采用UPLC-ESI-TOF-MS鉴定出海蓬子降血糖基本组成成分和相对比例。对获得的组分进行药效学评价研究,明确该组分对链尿佐菌素(STZ)诱导形成Ⅱ型糖尿病小鼠血糖的影响,阐明该组分的降血糖作用。将海蓬子降血糖组分灌胃糖尿病小鼠,通过尾静脉取血测定其空腹血糖,并对小鼠肝脏还原糖及组织病理学分析,发现该组分能显著降低糖尿病小鼠血糖值,且是通过增加肝糖原的合成达到降低血糖值,同时表明海蓬子降血糖组分能够显著改善小鼠肝脏和胰岛细胞的损伤情况。通过服用海蓬子降血糖组分可以起到降低糖尿病小鼠血糖的目的。该发明提供了一种新来源的降血糖组分制备方法,提高了海蓬子的高附加值利用,具有广阔的开发前景。
2、海蓬子原料为药食两用植物资源,生长于我国沿海滩涂盐碱地,我国海岸线较长,滩涂面积非常大,以海蓬子为原料制备降血糖组分,原料资源丰富;产品制备过程简便易行,生产成本低,易于实现机械化操作,能够满足工业化生产需求。海蓬子降血糖组分的降血糖效果显著,使用方便、安全,对人体无毒副作用。
附图说明
图1为实施例3各个对照组中糖尿病小鼠肝脏病理组织的H&E染色切片结果;
图2为实施例3各个对照组中糖尿病小鼠胰腺病理组织的H&E染色切片结果;
图中:A、空白对照组;B、模型组;C、阳性对照组;D、低剂量组;E、中剂量组;F、高剂量组。
具体实施方式
以下结合附图与具体实施例对本发明做进一步详细说明。
实施例1
一种海蓬子降血糖组分的制备方法,具体步骤如下:
(1)材料预处理
收集海蓬子植物的地上部分,用流水洗净泥沙,沥干水分。将处理好的海蓬子材料置于鼓风干燥箱中,55℃进行烘干处理。材料烘干后采用粉碎机将其粉碎,过4号筛子,将得到的海蓬子粉末置于含有干燥剂的干燥器中保存,待用。
(2)单因素研究
单因素研究中,分别对料液比、提取温度以及提取功率进行考察,称取4份1.00g海蓬子粉末放入圆底烧瓶中,配制浓度分别为40%、50%、60%、70%的乙醇溶液后,向圆底烧瓶中分别加入15mL,即料液比为1:15(g/mL),放入温度分别为40℃、50℃、60℃、70℃的水浴锅中,控制超声波功率分别为100W、150W、200W、250W,并持续运行0.5h,最后使用真空泵抽滤取上清液,即得。采用紫外分光光度计测定每个提取物中吸光值,所得提取物采用芦丁含量标准曲线进行计算。
实施例2海蓬子降血糖组分的成分及比例分析
一、响应曲面优化实验设计及结果
由单因素实验结果可知,提取时间和料液比在5种因素(乙醇浓度、提取功率、提取温度、提取时间、料液比)中对提取海蓬子降血糖组分的影响较小。选择乙醇浓度(X1)、提取功率(X2)、提取温度(X3)作为实验因素,采用Box-Behnken设计方案做响应曲面研究,设计三因素三水平的响应面实验如表1所示,以确定最优工艺条件,结果见表2。
表1 Box-Behnken实验因素与水平设计表
表2响应面分析实验设计及结果
二、多元二次响应面回归模型的建立和显著性检验
实验结果(表3)对表2结果进行回归分析,得出二次回归方程:Y=+4.10+0.091X1-0.061X2-0.010X3+0.040X1X2+0.013X1X3-0.027X2X3-0.44X1 2-0.46X2 2-0.42X3 2,经分析得出得率回归方程的方差分析(表3)。
通过表3可得,此模型的显著性水平为P值<0.0001,表明模型显著;失拟误差值P值=0.2149>0.05,失拟不显著,此回归方程和实验结果拟合性较佳。同时表3中的X1 2、X2 2、X3 2项(P值<0.0001)对得率的影响是极其显著的;而各因素对得率的影响强弱顺序为X1>X2>X3。其中R2=0.9835,RAdj2=0.9827,也都可以说明这个模型拟合度较好,该模型可解释98.35%的实验数据,表明了这个方程比较准确,也说明海蓬子总黄酮得率的实际值与预测值之间具有较好的拟合相关性。
表3回归方程模型方差分析及其系数的显著性检验
表3注:*差异不显著(P>0.05),**差异显著(P<0.05),***差异极显著(P<0.01)。
三、海蓬子降血糖组分提取工艺的验证
采用Design-Expert-8.0软件分析得出:将得率作为响应值的各因素相互作用3D图,由回归模型预测得出较佳理论值的各项参数为:乙醇浓度为61.30%,提取功率为178.37W,提取温度为49.71℃,预测总黄酮得率最优值为3.91%。在此条件下,为了判断这个方法是否准确,还有做实验时有一定的误差,所以把工艺条件修正为乙醇浓度为60%,提取功率为180W,提取温度为50℃。在这个参数下进行验证,实际测得海蓬子总黄酮得率更佳,为4.20%,两者的误差只有0.28604%,与理论值相差较近。
四、UPLC-ESI-TOF-MS技术分析海蓬子降血糖组分及比例
岛津LC30A液相条件:色谱柱为ZORBAX Eclipse Plus C18(2.1mm×50mm,5μm),流速:0.2mL/min,流动相:70%甲醇-30%水等梯度洗脱,检测波长280nm,柱温30℃。岛津质谱条件:ESI源,采用负离子模式,气体温度300℃,干燥气流速5L/min,喷雾器30psig,鞘气温度350℃,鞘气流速10L/min,电压帽电压4000V,裂解电压135V。
化合物名称、分子式、离子模式和相对含量如表4所示。海蓬子降血糖组分中共鉴定出34种化合物,其中黄酮类和糖苷类占11种,相对含量为57.52%。脂肪酸类化合物有5种,相对含量为28.58%。甾体类化合物有4种,相对含量为0.90%的。维生素类化合物有3种,相对含量为4.14%,碳水化合物有2种,相对含量为1.72%,氨基酸类化合物有6种,相对含量为7.08%。氨基酸中谷氨酸和天冬氨酸的含量最高,说明海蓬子具有用做调味品的潜力。叶绿素类化合物有三种,相对含量为0.06%的。
表4海蓬子降血糖组分的成分及比例
实验例3海蓬子降血糖组分的降血糖活性研究
一、海蓬子降血糖组分对糖尿病小鼠血糖及肝糖原的影响
(1)糖尿病小鼠的造模及分组
糖尿病小鼠的造模方法参考文献进行,小鼠购买后适应性饲养2周,然后随机抽取部分小鼠作为空白试验对照组,喂食普通饲料和饮用水。其余小鼠腹腔注射链尿佐菌素(STZ)并配合灌喂高脂饲料,诱导复制Ⅱ型糖尿病小鼠模型,以空腹血糖值≥11.1mmol/L为Ⅱ型糖尿病造模成功的标准。并将这些造模成功的小鼠随机分成模型组、阳性对照组(盐酸二甲双胍32mg/kg)、海蓬子降血糖低剂量组(100mg/kg)、海蓬子降血糖中剂量组(200mg/kg)和海蓬子降血糖高剂量组(400mg/kg),海蓬子给药剂量参考文献设定,给药时间为四周。
(2)小鼠血糖值及肝糖原含量的测定
从给药开始,采用剪尾法每3天测量一次小鼠空腹血糖值,以罗氏血糖仪和血糖试纸进行测定。给药结束后,采用二氧化碳窒息法将小鼠安乐死,立即取出小鼠肝脏,吸水纸吸干肝脏表面水分及血液,称取小鼠肝脏湿重为0.1g,加入5mL磷酸缓冲液,采用电动匀浆器将肝脏组织制成匀浆,加入4%磺基水杨酸0.05mL,混匀后5000rpm离心20min。空白对照组加入蒸馏水0.2mL和蒽酮试剂4mL;标准液(模型组)中加入1mg/mL葡萄糖溶液0.2mL和蒽酮试剂4mL;实验组(阳性对照组、低剂量组、中剂量组、高剂量组)加入组织提取液0.2mL和蒽酮试剂4mL。沸水浴15min,在620nm下测定吸光度值。
由表5结果可知,模型组及实验组小鼠血糖显著高于空白组,表明Ⅱ型糖尿病小鼠模型构建成功。给予小鼠灌喂海蓬子降血糖组分,结果表明海蓬子降血糖组分对糖尿病小鼠的血糖值有一定的影响,特别是高剂量组能显著降低糖尿病小鼠的血糖值(P<0.05),中剂量组有影响但不显著。模型组与空白组相对比,模型组小鼠肝脏合成肝糖原的能力不显著。阳性对照组和高剂量组与模型组相比,肝糖原含量均显著升高,中剂量组小鼠肝糖原含量有所升高但不显著性,表明海蓬子降血糖组分能促进小鼠肝脏合成肝糖原。因此,推测海蓬子降血糖组分是通过增加肝糖原的合成而达到降血糖的作用。
表5海蓬子对糖尿病小鼠空腹血糖的影响(n=6)
表5注:与模型组比较,*差异显著(P<0.05)。
二、海蓬子降血糖组分对糖尿病小鼠肝脏及胰腺组织的影响
(1)小鼠器官组织切片分析
小鼠安乐死后,取剩余肝脏置于10%的甲醛溶液中进行脱水固定,蜡块进行包埋,切片机切成5μm厚度,用光学显微镜进行分析,评价海蓬子提取物对小鼠肝脏和胰腺组织的保护和修复作用。结果如图1、图2所示。
(2)海蓬子降血糖组分对糖尿病小鼠肝脏组织的影响
糖尿病小鼠肝脏组织病理学切片H&E染色结果如图1所示,图1A空白对照组小鼠肝脏组织切片显示,中央静脉周边,肝索放射状排列明显,肝窦较宽,干细胞形态正常。图1B所示,糖尿病模型组小鼠肝脏组织中出现肝细胞水肿明显,肝脏门静脉周边肝窦变窄明显,因肝细胞水肿导致的肝索放射状排列不明显,部分细胞核周边出现空泡化,表明肝脏组织出现损伤。图1C所示,阳性对照组小鼠肝脏组织出现水肿现象,症状表现与模型组一致,但其损伤程度较模型组小鼠轻。图1D、图1E和图1F结果显示,小鼠肝脏组织均出现水肿现象,其中高剂量组小鼠症状轻。因此,该结果表明海蓬子降血糖组分对糖尿病造成的肝损伤有一定保护作用。
(3)海蓬子降血糖组分对糖尿病小鼠胰腺组织的影响
糖尿病小鼠胰腺组织病理学切片H&E染色结果如图2所示,从图2A空白对照组可以看出,小鼠胰岛组织面积较大,边缘圆润,形状规则,内部颗粒饱满。图2B模型组小鼠胰岛面积出现萎缩,呈现不规则形状,中间颗粒减少,出现空泡化,部分细胞出现变形,边界模糊不清。图2C中,阳性对照组胰岛损伤情况改善较明显,虽有少许空泡,较模型组已有较大改善。图2D、图2E和图2F中,给予海蓬子降血糖组分后,糖尿病小鼠胰岛组织损伤情况均有所改善,胰岛边较缘清晰,空泡化减轻,且高剂量组小鼠胰腺组织改善效果更显著。
Claims (9)
1.一种海蓬子降血糖组分的制备方法,其特征在于,包括以下步骤:
步骤1)材料预处理:收集海蓬子地上部分,洗净泥沙,沥干水分;将处理好的海蓬子材料置于鼓风干燥箱中,55℃进行烘干处理;材料烘干后采用粉碎机将其粉碎,过筛,将得到的海蓬子粉末置于含有干燥剂的干燥器中保存,待用;
步骤2)称取经步骤1)处理的海蓬子粉末放入容器中,再向容器中加入浓度为50%~60%的乙醇溶液,将容器放入温度为40~50℃的水浴锅中,控制超声波功率为150~200W,并持续运行0.5h,最后使用真空泵抽滤取上清液,即得所述海蓬子降血糖组分。
2.根据权利要求1所述的一种海蓬子降血糖组分的制备方法,其特征在于,步骤2)所述海蓬子粉末与乙醇溶液的料液比为1g:15mL。
3.根据权利要求1所述的一种海蓬子降血糖组分的制备方法,其特征在于,步骤2)所述乙醇溶液的浓度为60%;所述温度为50℃;所述超声波功率为180W。
4.权利要求1~3任一项制得的海蓬子降血糖组分在制备具有降血糖作用的保健食品或食品添加剂中的应用。
5.权利要求1~3任一项制得的海蓬子降血糖组分在制备降血糖药物中的应用。
6.一种降血糖药物,其特征在于,包括权利要求1~3任一项制得的海蓬子降血糖组分以及药学上可接受的药物载体。
7.权利要求1~3任一项制得的海蓬子降血糖组分在制备保护和修复肝脏及胰腺的药物中的应用。
8.权利要求1~3任一项制得的海蓬子降血糖组分在制备针对因糖尿病造成的肝损伤进行保护和修复的药物中的应用。
9.权利要求1~3任一项制得的海蓬子降血糖组分在制备针对因糖尿病造成的胰岛损伤进行保护和修复的药物中的应用。
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