CN111437310A - 一种高活性白花蛇舌草总黄酮的制备方法及其在过氧化肝损伤中的应用 - Google Patents
一种高活性白花蛇舌草总黄酮的制备方法及其在过氧化肝损伤中的应用 Download PDFInfo
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- CN111437310A CN111437310A CN202010283762.2A CN202010283762A CN111437310A CN 111437310 A CN111437310 A CN 111437310A CN 202010283762 A CN202010283762 A CN 202010283762A CN 111437310 A CN111437310 A CN 111437310A
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Abstract
本发明涉及中药材提取技术及医药技术领域,具体涉及一种高活性白花蛇舌草总黄酮的制备方法,即直接采用少量纤维素酶提取白花蛇舌草总黄酮;相较于其它提取方法,本发明方法操作简单,能耗小,可行性更强、提取的总黄酮(异黄酮类成分含量较高)的生物活性较高;且采用本法制备白花蛇舌草总黄酮可获得其他提取方法中未提取到的3个成分,分别是二氢山奈酚、芹黄素及白杨素;且获得了含量显著增高(上调2倍以上)的6个成分,分别是柚皮苷、牡荆素、异牡荆素、染料木素、木犀草素及7‑O‑葡萄糖苷木犀草素;此9个化合物均具有抗炎、抗氧化、抗肿瘤及肝保护等多种生物活性;本发明制备的高活性白花蛇舌草总黄酮提取物可用于过氧化肝损伤的预防和治疗及可发挥辅助降血脂的作用。
Description
技术领域
本发明涉及中药材提取技术及医药技术领域,具体涉及一种高活性白花蛇舌草总黄酮的制备方法及其在过氧化肝损伤中的应用。
背景技术
白花蛇舌草为茜草科植物白花蛇舌草Hedyotis diffusa Willd的干燥全草,味苦、淡,无毒,归胃、大肠、小肠经。始见于《广西中药志》,别名蛇舌草、蛇刺草等。白花蛇舌草为一年生草本植物,全草均可入药。主产于中国浙江、江西、湖南、湖北、广东、广西、福建等地;云南及贵州也有大量分布,主要为野生型;河南地区近年来也有栽培,是一种分布广泛的药用植物。其具有多种活性成分包括黄酮类、蒽醌类、萜类、甾醇类、多糖类、有机酸类、烷烃类、微量元素及挥发油成分。其具有多种药理活性如清热解毒、消痈散结、利尿除湿、抗肿瘤、肝保护、抗炎、抗氧化、调节免疫的功效。临床上被广泛用于治疗各种炎症和肿瘤。包括肝炎、扁桃腺炎、咽喉痛、阑尾炎、尿道感染等。肝癌、肺癌、胃癌等多种癌症,其中黄酮类成分是白花蛇舌草发挥多种药理作用的活性成分之一。常见的植物黄酮提取方法有溶剂浸提法、加热回流法、超声波辅助提取法、微波辅助提取法、超临界CO2流体萃取法。现有的提取方法存在方法复杂、耗能高、提取的总黄酮活性较低的技术问题,为了充分开发利用白花蛇舌草总黄酮,一种简单高效、绿色环保的提取方法有待于进一步开发。
肝损伤是临床常见的危害人类健康的疾病,是一种由多因素介导的复杂的生物学过程。肝脏作为人体重要解毒器官,最容易受到毒性物质损害,临床上可见肝功能异常,严重时可发生肝功能衰竭,引起肝细胞化学损伤的机制有氧自由基、肝细胞内钙超载、脂代谢异常、胆汁淤积及微量元素代谢障碍等。肝损伤可分为免疫性和非免疫性肝损伤两大类,常见的病毒性肝炎(如乙型、丙型病毒性肝炎)、自身免疫性肝炎及部分药物性肝损伤中都是免疫介导的肝细胞损伤。非免疫性肝损伤主要见于由化学性物质如括酒精、化学毒物和药物等引起的过氧化肝损伤。据调查,脂肪性肝病成为仅次于病毒性肝炎的第二大肝病,正在严重威胁国人的健康。研究表明引起脂肪肝的原因很多,临床根据患者有无长期过量饮酒史,将脂肪肝大致分为酒精性脂肪肝和非酒精性脂肪肝或者二者并存,其中脂肪肝主要为非酒精性脂肪肝。随着经济发展社会进步,在中国经济发达区域,非酒精性脂肪肝发病率达到15%。
非酒精性脂肪肝发病机制十分复杂,早在1998年Day和Janmes提出的“二次打击”学说已成为解释非酒精性脂肪肝发生机制的主要理论,其初次打击主要指胰岛素抵抗和脂质代谢紊乱所导致的的肝细胞脂肪变性,继而肝细胞活力相对不足;二次打击主要指各种原因所致的氧化应激及脂质过氧化损伤,引起脂肪变性的肝细胞发生炎症、坏死甚至纤维化。活性氧簇ROS在二次打击中起到了非常重要的作用。非酒精性脂肪肝属于过氧化型肝损伤。
发明内容
本发明为解决现有的植物黄酮提取方法操作复杂、耗能高,所提取的总黄酮生物活性较低的技术问题,提供一种高活性白花蛇舌草总黄酮的制备方法。
为解决上述技术问题,本发明所采用的技术方案为:一种高活性白花蛇舌草总黄酮的制备方法,包括以下步骤:a、样品前处理,将白花蛇舌草药材干燥粉碎,过60目筛(孔径0.25mm),用石油醚超声处理,弃去石油醚,滤渣挥干石油醚,重复此操作1次,干燥后冷却备用;
b、取步骤a经过前处理的白花蛇舌草样品加入纤维素酶,以50~70%乙醇为溶剂,提取总黄酮,减压抽滤,合并滤液,即得白花蛇舌草总黄酮粗提液;
c、白花蛇舌草总黄酮粗提液用粒度为20-60目的弱极性大孔吸附树脂纯化,蒸馏水洗去水溶性杂质后,用70%乙醇洗脱,收集洗脱液,减压浓缩,冷冻干燥,粉碎,即得精制白花蛇舌草总黄酮。
白花蛇舌草总黄酮在对由化学性物质包括酒精、化学物质和药物引起的过氧化肝损伤的预防中的应用。
进一步的,步骤b中所述纤维素酶添加量为经过前处理的白花蛇舌草样品质量的0.5-1.0%;料液比为1:30~1:50g/mL。
进一步的,所述步骤b粗提液的pH为4.0~5.5。
进一步的,步骤b中的酶解温度为50~60℃。
进一步的,步骤b中的酶解时间为1~1.5h。
通过本发明制得的精制白花蛇舌草总黄酮经UPLC-MS分析,发现了6种含量显著上调的成分(上调2倍以上),分别是柚皮苷、牡荆素、异牡荆素、染料木素、木犀草素及7-O-葡萄糖苷木犀草素,以及3个仅在本发明方法中获得的成分,分别是二氢山奈酚、芹黄素及白杨素。此9个成分均具有抗炎、抗氧化、抗肿瘤及肝保护作用。由本发明制得的白花蛇舌草总黄酮的生物活性显著增强。
本发明制得的精制白花蛇舌草总黄酮经体外细胞实验结果表明,其可著降低由H2O2诱导的HL-02细胞损伤所致的ALT、AST、LDH及ALP含量的升高,对过氧化肝损伤具有保护作用;且作用显著优于其他方法制备的白花蛇舌草总黄酮。
本发明制得的精制白花蛇舌草总黄酮经体内动物实验结果表明,其安全低毒,对非酒精性脂肪肝具有治疗作用,且在发挥肝保护作用的同时还可降低体内胆固醇和甘油三酯水平发挥降血脂作用。
同时本发明还提供一种采用上述制备方法所制备的高活性白花蛇舌草总黄酮在过氧化肝损伤治疗中的应用。
一种采用上述的制备方法所制备的高活性白花蛇舌草总黄酮在预防由化学性物质包括酒精、化学物质和药物引起的过氧化肝损伤中的应用。
一种采用上述制备方法所制备的高活性白花蛇舌草总黄酮在预防H2O2诱导的HL-02细胞损伤中的应用,在降低ALT、AST、LDH及ALP含量肝损伤指标中的应用。
一种采用上述制备方法所制备的高活性白花蛇舌草总黄酮在脂肪性肝病导致的肝损伤的治疗与预防中的的应用。
一种采用上述制备方法所制备的高活性白花蛇舌草总黄酮在非酒精性脂肪性肝病发挥降血脂作用中的应用。
与现有技术相比本发明具有以下有益效果:
本发明提供了一种可获得高活性白花蛇舌草总黄酮的制备方法,可提取出其他方法中未见的有效成分且可提高具有生物活性的有效成分的含量。采用本发明提供的酶解法提取白花蛇舌草总黄酮可获得高活性低毒的白花蛇舌草总黄酮。对过氧化肝损伤具有预防和治疗作用,同时还可发挥一定降血脂作用。这为白花蛇舌草的进一步开发利用提供了参考,可开发成新的抗肝损伤的药物及保健食品;且本发明提供的精制白花蛇舌草总黄酮可以制备成临床上常用剂型,方便临床应用。
与传统的回流法、超声法、酶解超声法等提取方法相比,本发明方法制备的白花蛇舌草总黄酮中的柚皮苷、牡荆素、异牡荆素、染料木素、木犀草素、7-O-葡萄糖苷木犀草素的含量显著提高(p<0.05)(上调2倍以上),并且增加了三种成分,分别是芹黄素、白杨素及二氢山奈酚。本发明方法制备的总黄酮与其他制备方法相比,酶解法制备的总黄酮含有含量更高的芹黄素、白杨素、染料木素、柚皮苷、牡荆素、异牡荆素等(3.5倍以上)具有肝保护作用的有效成分,从而使本发明方法制备的总黄酮具有更高效的肝损伤保护活性。
附图说明
图1为实施例2中QC样本的RSD分布图;1:芦丁;2:柚皮苷;3:黄豆素;4:黄豆苷;5:异甘草素;6:染料木苷;7:柚皮素;8:木犀草素;9:山奈酚;10:二氢槲皮素;11:杨梅素;12:槲皮苷;13:牡荆素;14:异牡荆素;15:异槲皮苷;16:甘草素;17:黄豆苷元;18:染料木素;19:圣草酚;20:槲皮素;21:芹黄素;22:白杨素;23:异鼠李素;24:7-O-葡萄糖苷木犀草素。
图2为实施例2中采用t检验和差异倍数(FC)分析方法对所有数据进行比较分析并绘制的火山图;A表示酶解法;B表示回流法;C表示超声法;D表示酶解超声法;A vs B,A vsC,A vs D,B vs C,B vsD,C vs D分别表示四种制备方法两两比较。
图3为实施例3中体外细胞肝保护活性筛选实验结果图;图3(a)为过氧化氢造模浓度筛选结果;*(p<0.05),**(p<0.01)表示与对照组有显著性差异;图3(b)为不同制备方法下白花蛇舌草总黄酮肝保护活性筛选结果,其中A为酶解法、B为加热回流法、C为超声提取法、D为超声酶解法。阳性药为5mg/mL联苯双酯,*(p<0.05),表示与A组酶解法相比具有显著性差异;图3(c)为6种黄酮化合物对HL-02细胞存活率的影响,*(p<0.05),**(p<0.01)表示与模型组比有显著性差异;##(p<0.01)表示与对照组相比具有显著性差异。
图4为实施例3中AST、ALT、LDH及ALP指标测定结果图;##(p<0.01)表示与对照组相比有显著性差异;a*(p<0.05),a**(p<0.01)表示与模型组相比有显著性差异;b*(p<0.05)表示与酶解法相比具有显著性差异;A酶解法;B回流法;C超声法;D酶解超声法;联苯双酯:5mg/mL。
图5为实施例3中HL-02细胞形态图;图5(a):正常HL-02;图5(b):H2O2损伤后的HL-02;图5(c):联苯双酯预处理后的HL-02;图5(d):低剂量白花蛇舌草总黄酮预处理后的HL-02;图5(e):中剂量白花蛇舌草总黄酮预处理后的HL-02;图5(f):高剂量白花蛇舌草总黄酮预处理后的HL-02;所有照片均为10×镜下拍摄。
具体实施方式
以下结合具体实施例对本发明作进一步说明。
实施例1
一种高活性白花蛇舌草总黄酮的酶解提取方法
1.实验方法
一种具有预防和治疗非免疫性肝损伤的高活性白花蛇舌草总黄酮,其通过以下制备方法得到:样品前处理:将白花蛇舌草药材干燥粉碎,过60目筛(孔径0.25mm),用石油醚超声处理,弃去石油醚,滤渣挥干石油醚,重复此操作1次,干燥后冷却备用。此步骤是为除去白花蛇舌草中的叶绿素及脂溶性成分,便于提取过程中黄酮类物质的溶出。
取经前处理后的白花蛇舌草粉末适量加入0.5-1.0%的纤维素酶,加入30~50倍体积的50%乙醇溶液,调节pH至4.0~5.5后于50~60℃下恒温酶解提取1~1.5h,酶解结束后于沸水中灭活5min,趁热抽滤,合并滤液即为白花蛇舌草总黄酮粗提取液。
最终白花蛇舌草总黄酮粗提液用AB-8型大孔吸附树脂纯化,蒸馏水洗去水溶性杂质后,用70%乙醇洗脱,收集洗脱液,减压浓缩,冷冻干燥,粉碎,即得精制白花蛇舌草总黄酮。
以相同技术参数,分别采用加热回流法(不加纤维素酶)、超声提取法(不加纤维素酶)、酶解联用法(以酶解联用超声为例)分别制备4种白花蛇舌草总黄酮样品,用于后续成分与活性对比分析。
2.实验结果
本实验比较了不同制备方法下白花蛇舌草总黄酮的提取率、总黄酮得率及总黄酮含量。具体结果见表1,从结果可知,采用酶解法及酶解联用法相较于加热回流法和超声提取法而言提取率高,总黄酮含量更高。酶解法及酶解超声法未见显著性差异,但和酶解超声法相比,采用直接酶解法制备的白花蛇舌草总黄酮中异黄酮类成分含量(4.3±0.9μg/g)显著提高(p<0.05,见表4),肝损伤保护活性更高(见图3(b)),总黄酮中的柚皮苷、牡荆素、异牡荆素、染料木素、木犀草素、7-O-葡萄糖苷木犀草素的含量显著提高(p<0.05)(上调2倍以上),并且增加了芹黄素、白杨素及二氢山奈酚三种成分,其中芹黄素、白杨素、染料木素、柚皮苷、牡荆素、异牡荆素等(3.5倍以上)具有肝保护作用的有效成分(见表5、图3(c)和图4),并且操作更为简单、耗能更少,更适合实际工业生产的需求。
表1不同白花蛇舌草总黄酮制备方法的比较(n=3)
*(p<0.05),**(p<0.01)表示与酶解法相比有显著性差异。
实施例2
不同方法制备的白花蛇舌草总黄酮成分组成分析
1.实验方法
采用UPLC-MS对实施例1中不同方法制备的白花蛇舌草总黄酮组成成分进行分析,对黄酮类物质进行含量测定,旨在发现本发明所采用的纤维素酶提取法与其他提取方法的差异性成分。
采用Waters色谱柱(HSS T3 2.5μm,2.1mm×150mm column);流动相为A相水(0.1%甲酸):B相乙腈(0.1%甲酸);流速为1mL/min;柱温为4℃;进样量为5μL。梯度洗脱程序见表2。采用Sciex 5500 QTRAP质谱仪,正负离子切换模式下进行MRM质谱分析。正离子模式离子源参数如下:Source temperature,550℃;Gas 1,55;Gas 2,50;CRU,30;ISVF,5500V。负离子模式离子源参数如下:Source temperature,550℃;Gas 1,55;Gas 2,50;CRU,30;ISVF,-4500V。
表2梯度洗脱程序
2.实验结果
将所有样品等量混合制备成为QC样本,采用QC样本对检测过程仪器的精密度及稳定性进行考察。QC样本RSD%结果如图1所示,各待测物RSD%<30%,说明实验数据稳定可靠,仪器精密度良好。
本实验测定了各待测物的工作曲线及线性范围。由结果可知各待测物在线性范围内线性良好,相关系数均大于0.99。具体结果见表3(仅列出部分)。
表3工作曲线及线性范围(部分)
本实验分别测定了不同方法制备的白花蛇舌草总黄酮黄酮类物质的含量,A为采用本发明所提供的酶解法制得的白花蛇舌草总黄酮,B为加热回流法,C为超声提取法,D是酶解与超声联用法。含量测定结果见表4。
表4含量测定结果
表5 9种黄酮类化合物含量测定结果
*(p<0.05),**(p<0.01)表示与酶解法相比含量有显著性差异。
四种制备方法中均含有黄酮苷类、异黄酮类、黄烷类及黄酮类化合物,其中,酶解法中异黄酮类成分含量(4.3±0.9μg/g)显著高于其在回流法、超声法及酶解超声法中的含量(p<0.05),其他类型黄酮含量(黄酮苷类、黄烷类及黄酮类)在四种制备方法中均无显著性差异。常见的异黄酮类物质有染料木素、染料木苷、黄豆素、黄豆苷及葛根素等具有抗氧化活性的有效成分,具有减轻肝脏损伤、减轻肝纤维化病变程度发挥肝保护的作用。因此相较于回流法、超声法及酶解超声法,采用本发明酶解法制备白花蛇舌草总黄酮可有效提高异黄酮类物质含量从而提高抗氧化及肝保护能力。
采用t检验和差异倍数(FC)分析方法对所有数据进行比较分析并绘制火山图,见图2。横坐标为log2(FC),纵坐标为-log10(P-value),一个点代表一个变量。平行于Y轴的两条线分别是X=1和X=-1,在X=-1左侧的点是下调2倍以上的成分,在X=1右侧的点是上调2倍以上的成分;平行于X轴有一条虚线Y=1.30,即-log10(0.05),在虚线以上的点表示显著性小于0.05的成分。把虚线Y=1.30以上,X=1右侧和X=-1左侧的成分记为具有显著差异性的成分。A与B、A与C、A与D相比较有具有显著性上调的成分,共有6个,分别是牡荆素、异牡荆素、柚皮苷、染料木素、木犀草素、7-O-葡萄糖苷木犀草素。B、C、D三个样品中未见具有显著性差异变化的成分。
如表5所示,与传统回流法、超声法、酶解超声法等提取方法相比,本发明酶解法制备的白花蛇舌草总黄酮中不仅异黄酮类成分含量高,其中的肝损伤保护活性成分柚皮苷、牡荆素、异牡荆素、染料木素、木犀草素、7-O-葡萄糖苷木犀草素的含量显著提高(p<0.05),并且增加了三种成分,分别是芹黄素、白杨素及二氢山奈酚。综上所述,最终从酶解法制备的白花蛇舌草总黄酮中筛选出上述9个差异成分。
实施例3
不同制备方法下白花蛇舌草总黄酮对过氧化氢诱导的肝损伤的保护作用
1.实验方法
将浓度为50、100、150、200、400、600、800μmol/L的H2O2作用于HL-02细胞4h,MTT法测定OD值,计算H2O2对细胞的抑制率,根据抑制率确定最佳造模浓度。
将待测HL-02细胞分为药物组、模型组、对照组,阳性药物组,每组至少设三个复孔,进行肝保护活性筛选实验。对照组:不做任何处理。模型组:用最佳造模浓度的H2O2培养基培养4h。药物组:预先加药物(实施例1中不同制备方法下的白花蛇舌草总黄酮)作用12h,用最佳造模浓度的H2O2培养基继续培养4h。阳性药物组:预先加联苯双酯作用12h,用最佳造模浓的H2O2培养基继续培养4h。MTT法测定OD值,计算药物的保护率。
从实例2中筛选得到的9个化合物中,进一步挑选出差异倍数大于3.5的4个成分(牡荆素、异牡荆素、柚皮苷、染料木素)、和新增的成分(二氢山奈酚、芹黄素、白杨素)中进一步挑选含量较高的2个活性成分(芹黄素、白杨素),用于活性筛选实验,探究其是否具有肝保护活性。
以实施例1不同方法制备的白花蛇舌草总黄酮给药后取细胞上清液,按照试剂盒说明书操作步骤,测定肝损伤指标AST、ALT、LDH、ALP。
实验数据结果用平均值±标准差表示,t检验分析结果的统计性差异;p<0.05时,认为具有统计学差异,所有实验平行重复3次。
2.实验结果
正常HL-02、过氧化氢损伤HL-02及药物预处理后HL-02的细胞形态图,如图5所示。
根据图3(a)结果可知,随着H2O2浓度的增加,HL-02细胞的存活率逐渐降低。当H2O2浓度为200μmol/L,细胞抑制率约为60%。因此造模浓度选择200μmol/LH2O2。
样品A(本发明酶解法)、B(回流法)、C(超声法)和D(酶解超声法)在31.25、62.5、125μg/mL时的保护活性如图3(b)所示。随着不同方法制备的总黄酮浓度增加,肝保护率逐渐升高且呈剂量依赖性,在125μg/mL时肝保护率最高,分别为59.30±0.81%、39.26±6.18%、27.26±0.75%、42.44±6.35%;本发明酶解法制备的总黄酮肝保护活性显著优于同等浓度下经回流法、超声法和超声辅助酶解法制备的总黄酮(p<0.05)。
根据图3(c)结果可知,预先分别给予4、8、16μmol/L柚皮苷、牡荆素、异牡荆素、染料木素、芹黄素、白杨素及阳性对照药联苯双酯处理,可发现细胞存活率升高且呈剂量依赖性。其中8、16μmol/L给药组细胞存活率较模型组均显著升高(p<0.05,p<0.01),表明柚皮苷、牡荆素、异牡荆素、染料木素、芹黄素、白杨素对H2O2所致HL-02损伤具有保护作用,提高细胞存活率。且柚皮苷、牡荆素、异牡荆素、染料木素、芹黄素、白杨素与同浓度下阳性对照药联苯双酯的细胞存活率无显著性差异(p>0.05),表明上述6种化合物均对H2O2所致HL-02损伤具有保护作用,且肝保护作用与联苯双酯相当。
AST、ALT、LDH及ALP测定结果见图4,与对照组相比,模型组细胞上清液中ALT、AST、LDH及ALP含量显著升高(p<0.01),说明H2O2处理后可致HL-02细胞损伤。相较于模型组,62.5、125μg/mL的白花蛇舌草总黄酮样品A降低细胞上清液中AST、LDH含量的作用显著优于样品B、C、D(p<0.05),见图4(a)和(c);而在降低ALT及ALP含量的作用上未见显著性差异(p>0.05),见图4(b)和(d)。
综上所述,本发明酶解法制得的总黄酮降低AST、LDH含量的作用显著高于回流法、超声法及酶解超声法(p<0.05),其对H2O2诱导的肝损伤的保护作用亦显著高于回流法、超声法及酶解超声法(p<0.05)。与其他制备方法相比,本发明酶解法制备的总黄酮含有含量更高的芹黄素、白杨素、染料木素、柚皮苷、牡荆素、异牡荆素等(3.5倍以上)具有肝保护作用的有效成分,从而使本发明酶解法制备的总黄酮具有更高效的肝损伤保护活性。
实施例4
本发明酶解法制备的白花蛇舌草总黄酮急性毒性实验研究
1.实验方法
预实验:取ICR小鼠20只,雌雄各半,适应性饲养一周后,随机分成4组,每组5只,分别为空白对照组、高剂量(100mg/mL)、中剂量(50mg/mL)及低剂量组(25mg/mL)。给药前12h禁食不禁水,以最大给药剂量0.4mL/10g灌胃给药,对照组给予等量蒸馏水。给药后连续观察七天,期间正常饲养并记录小鼠状况。若可找到小鼠全死或全不死的给药剂量,则设计正式实验找出其半数致死量LD50值;若高剂量组小鼠出现死亡,则可降低高剂量组浓度进一步设计实验测定最大耐受量MTD值;若高剂量组未出现小鼠死亡,按照《中药新药药理毒理研究技术要求》,可进行最大给药量试验。
取ICR小白鼠20只,随机分成2组,分别为给药组与对照组,每组10只。给药组以白花蛇舌草总黄酮可供灌胃的最大浓度及最大给药剂量0.4mL/10g一日单次灌胃给药,对照组给予等量蒸馏水,灌胃前12h禁食不禁水。连续观察14天,期间正常饲养并记录小鼠状况。
2.实验结果
在预实验中,高中低剂量给药组的小鼠7天内均未见死亡;其体重外观、行为能力、精神状态、饮食排泄均与对照组相比均未见明显异常。无法测出LD50和最大耐受量MTD,故进行最大给药量实验。
按照最大给药浓度及最大给药体积一日单次给药,给药初期,对照组与给药组小鼠均现出现萎靡、活动减少、眯眼的现象,但无竖毛、抽搐、翻倒等异常毒性反应,且上述症状在给药后1小时逐渐恢复。连续观察14天,未见小鼠死亡,且体重外观、行为能力、精神状态、饮食排泄均与对照组相比均未见显著异常。体重变化结果见表6。
表6小鼠体重变化(n=10)
白花蛇舌草总黄酮给药组小鼠体重与空白对照组相比无显著性差异(p>0.05)。故可认为本发明酶解法制得的白花蛇舌草总黄酮的最大给药量为4g-1·kg-1·d-1,折合成生药量计算白花蛇舌草总给药量为80.65g-1·kg-1·d-1(由实施例1结果可知,本发明酶解法制备白花蛇舌草总黄酮得率为4.96±0.02%,故白花蛇舌草总给药量(g)=黄酮给药量(g)/总黄酮得率),按白花蛇舌草临床日用量计算(60kg成人每日口服生药量为30g)相当于临床成人日用量的161.3倍,故可认为白花蛇草总黄酮急性毒性较小,常规剂量服用是安全、可靠的。
实施例5
本发明酶解法制备的白花蛇舌草总黄酮对非酒精性脂肪肝的作用研究
1.实验方法
60只SPF级SD雄性大鼠,体重170-220g,山西医科大学动物实验中心提供。将60只大鼠随机分为6组,每组10只,分别为对照组、模型组、阳性对照组(30mg/kg非诺贝特)及白花蛇舌草总黄酮低中高剂量组(60mg/kg、90mg/kg、120mg/kg)。对照组给予普通维持饲料,其余各组均给予高脂饲料(45%脂肪供能),对照组和高脂饮食组分别用各自的饲料喂养3周。3周后,大鼠眼眶采血,分离血清,测定血清中TC、TG水平,判断造模是否成功。造模成功后,阳性对照组给予非诺贝特(30mg/kg),给药组给予实施例1中本发明方法制备的白花蛇舌草总黄酮低中高剂量(60mg/kg、90mg/kg、120mg/kg)。
每天上午10:00进行灌胃给药。将非诺贝特和白花蛇舌草总黄酮总黄酮分别溶解在0.9%的生理盐水中,配成混悬液,按照各自相应的剂量进行灌胃给药。每天给药1次,连续给药2周,定期称量体重并做记录。
样品采集及指标测定:大鼠眼眶采血,3000r/min离心15min后取上清液置于-20℃保存待测,用于测定脂肪肝及肝损伤相关生化指标TG、TC、γ-GT、GSH、SOD、MDA、LDH、AST、ALT及ALP含量,具体测定方法按照相应试剂盒说明书测定。采血完成,将大鼠断颈处死,迅速分离心脏心脏、肝脏、脾脏及肾脏。生理盐水清洗,滤纸擦干后称重,计算脏器系数,脏器系数(mg/g)=脏器湿重/大鼠体重。
2.实验结果
对照组的肝组织形态正常,颜色鲜红,表面光滑,边缘锐利,质地红润柔软。模型组肝脏肿大,重量也比正常对照组显著增加,颜色呈偏棕黄色,且有黄色带状物,边缘变圆钝,粗糙,质地变脆。
大鼠体重变化差值、肝脏系数情况见表7,与空白对照组相比,模型组大鼠体重增长量与肝指数显著增大(p<0.01);与模型组相比,白花蛇舌草总黄酮给药组体重增长量及肝脏系数显著低于模型组(p<0.01)。结果表明,白花蛇舌草总黄酮可以显著控制高脂饮食大鼠体重的增长(p<0.01),可预防其因体重过度增长所致的肥胖及肝脏脂肪沉积。中高剂量组大鼠体重增加量及肝脏系数显著低于阳性对照组(p<0.01),说明白花蛇舌草总黄酮在控制体重增长上的作用优于非诺贝特。
表7大鼠体重变化差值、肝湿重、肝脏系数情况
#表示与对照组相比,p<0.05;##表示与对照组相比,p<0.01;*表示与模型组相比,p<0.05,**表示与模型组相比,p<0.01;◆◆表示与阳性对照组相比,p<0.01。
白花蛇舌草总黄酮对大鼠血脂指标的影响见表8,模型组大鼠血清中总胆固醇(TC)、甘油三酯(TG)显著高于空白对照组(p<0.05);给予90、120mg/kg白花蛇舌草总黄酮治疗后,TC、TG水平相较于模型组显著下降(p<0.05),结果表明白花蛇舌草总黄酮可能是通过调节体内脂质代谢,减少脂质在肝脏中过量沉积从而发挥对非酒精性脂肪肝的治疗作用。给药组与阳性对照组比较,TC和TG含量未见显著性差异(p>0.05),表明白花蛇舌草总黄酮降低TC、TG含量的作用与阳性药非诺贝特相当。
表8白花蛇舌草总黄酮对大鼠血脂指标的影响(n=10)
#表示与对照组相比,p<0.05;*表示与模型组相比,p<0.05。
白花蛇舌草总黄酮对肝损伤指标影响如表9所示,与空白对照组相比,模型组ALT、AST、LDH、ALP及γ-GT含量均显著增高(p<0.05,p<0.01);给予白花蛇舌草总黄酮治疗后,低中高剂量组的ALT、LDH、γ-GT含量与模型组相比具有统计学差异(p<0.05,p<0.01);中高剂量组AST及ALP含量与模型组相比具有统计学差异(p<0.05,p<0.01),表明白花蛇舌草总黄酮对高脂饮食诱导的非酒精性脂肪肝肝损伤具有一定保护作用,可减轻肝脏损伤。与阳性对照组相比,高剂量组白花蛇舌草总黄酮降低ALT、AST含量作用显著优于非诺贝特(p<0.05),对LDH含量改善作用与非诺贝特相当(p<0.05),对于改善ALP及γ-GT含量的作用低于非诺贝特(p<0.05)。
表9白花蛇舌草总黄酮对大鼠肝损伤指标的影响(n=10)
#表示与对照组相比,p<0.05;##表示与对照组相比,p<0.01;*表示与模型组相比,p<0.05,**表示与模型组相比,p<0.01;低中高剂量组为分别为60、90、120mg/kg白花蛇舌草总黄酮;阳性对照组为30mg/kg非诺贝特;◆表示与阳性对照组相比,p<0.05。
白花蛇舌草总黄酮对氧化应激指标影响如表10所示,与空白对照组相比,模型组MDA(丙二醛)含量显著升高(p<0.05),SOD(超氧化物岐化酶)活力及GSH(谷胱甘肽)含量显著降低(p<0.05,p<0.01)。给予白花蛇舌草总黄酮治疗后,SOD酶活力显著升高且中高剂量组与模型组相比具有统计学差异(p<0.05);给药后GSH含量升高,高剂量组与模型组相比,具有统计学差异(p<0.05);给药后MDA含量与模型组相比显著降低(p<0.01)。表明在非酒精性脂肪肝中大鼠中,机体抗氧化能力及自由基清除率下降,导致脂质过氧化,造成肝脏损伤,而白花蛇舌草总黄酮可以通过调节机体自由基使其处于代谢平衡状态,抑制机体脂质过氧化而发挥治疗作用。白花蛇舌草总黄酮在降低MDA含量上与非诺贝特无显著性差异(p>0.05),而提高SOD酶活力及GSH含量的作用显著优于非诺贝特(p<0.05)。
表10白花蛇舌草总黄酮对大鼠氧化应激指标的影响(n=10)
##表示与对照组相比,p<0.01;*表示与模型组相比,p<0.05,**表示与模型组相比,p<0.01;◆表示与阳性对照组相比,p<0.05。
经本发明酶解法制备的白花蛇舌草总黄酮小鼠急性毒性实验及对非酒精性脂肪肝的作用研究,可知白花蛇舌草总黄酮是一种安全低毒,可发挥调节血脂、抗氧化及肝保护作用,对非酒精性脂肪肝具有治疗作用的天然植物提取物。
综上所述,本发明酶解法制备白花蛇舌草总黄酮对过氧化肝损伤具有治疗作用,具有良好的开发利用前景。
Claims (10)
1.一种高活性白花蛇舌草总黄酮的制备方法,其特征在于,包括以下步骤:a、样品前处理,将白花蛇舌草药材干燥粉碎,过60目筛, 用石油醚超声处理,弃去石油醚,滤渣挥干石油醚,重复此操作1次,干燥后冷却备用;
b、取步骤a经过前处理的白花蛇舌草样品加入纤维素酶,以50~70%乙醇为溶剂,提取总黄酮,减压抽滤,合并滤液,即得白花蛇舌草总黄酮粗提液;
c、白花蛇舌草总黄酮粗提液用粒度为20-60目的弱极性大孔吸附树脂纯化,蒸馏水洗去水溶性杂质后,用70%乙醇洗脱,收集洗脱液,减压浓缩,冷冻干燥,粉碎,即得精制白花蛇舌草总黄酮。
2.根据权利要求1所述的一种高活性白花蛇舌草总黄酮的制备方法,其特征在于,步骤b中所述纤维素酶添加量为经过前处理的白花蛇舌草样品质量的0.5-1.0%;料液比为1:30~1:50g/mL。
3.根据权利要求1或2所述的一种高活性白花蛇舌草总黄酮的制备方法,其特征在于,所述步骤b 粗提液的pH为4.0~5.5。
4.根据权利要求3所述的一种高活性白花蛇舌草总黄酮的制备方法,其特征在于,步骤b中的酶解温度为50~60℃。
5.根据权利要求3所述的一种高活性白花蛇舌草总黄酮的制备方法,其特征在于,步骤b中的酶解时间为1~1.5h。
6.一种采用如权利要求1所述的制备方法所制备的高活性白花蛇舌草总黄酮在过氧化肝损伤治疗中的应用。
7.一种采用如权利要求1所述的制备方法所制备的高活性白花蛇舌草总黄酮在预防由化学性物质包括酒精、化学物质和药物引起的过氧化肝损伤中的应用。
8.一种采用如权利要求1所述的制备方法所制备的高活性白花蛇舌草总黄酮在预防H2O2诱导的HL-02细胞损伤中的应用,在降低ALT、AST、LDH及ALP含量肝损伤指标中的应用。
9.一种采用如权利要求1所述的制备方法所制备的高活性白花蛇舌草总黄酮在脂肪性肝病导致的肝损伤的治疗与预防中的的应用。
10.一种采用如权利要求1所述的制备方法所制备的高活性白花蛇舌草总黄酮在非酒精性脂肪性肝病发挥降血脂作用中的应用。
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CN113248483A (zh) * | 2021-04-29 | 2021-08-13 | 广西医科大学 | 天胡荽黄酮苷类单体异牡荆素的制备方法及应用 |
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CN114989124A (zh) * | 2022-06-15 | 2022-09-02 | 华中科技大学同济医学院附属协和医院 | 一种从白花蛇舌草中提取槲皮素的方法及其脂质体的制备方法和脂质体产品 |
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