CN114699459B - 柳芽总黄酮提取物及其制备方法和应用 - Google Patents
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Abstract
本发明属于医药技术领域,涉及柳芽总黄酮提取物及其制备方法和应用。具体涉及柳芽总黄酮提取物及其制备方法和在制备抗氧化、抑菌及抗炎镇痛药物中的应用。所述的柳芽总黄酮通过如下方法制备:将采摘柳芽烘干制成粉状,用乙醇溶液超声辅助提取,乙醇溶液的浓度为30‑70%,液料比为40‑70:1,超声提取2‑3次,提取时间每次15‑35min,过滤,合并滤液,浓缩至尽干,备用。制备得到的总黄酮含量最高可达7.55%。枊芽总黄酮的成分主要包括:黄酮醇(槲皮素、异槲皮素、山奈酚、异鼠李素、芦丁、黄芪苷)、二氢黄酮(柚皮素)、黄烷醇(儿茶素),酚苷类化合物(水杨苷)和生物碱(葫芦巴碱)。
Description
技术领域:
本发明属于医药技术领域,涉及柳芽总黄酮提取物及其制备方法和应用。具体涉及柳芽总黄酮提取物及其制备方法和在制备抗氧化、抑菌及抗炎镇痛药物中的应用。
背景技术:
柳属(Salix L.)植物是杨柳科(Saliaceae)木本植物,已超过4属,500余种,全世界广泛分布。《本草纲目》中记载:柳树作为药用主要以垂柳(Salix babylonica.L)的根、枝、皮、叶和花等入药。柳芽(Willow Sprouts)指的是垂柳的新芽,具有较高的营养价值和药用价值,该药材性寒,味微苦,无毒。作为一种可食用的野菜,柳芽中含有丰富的碘元素,常用于缺碘类疾病的辅助治疗。现代化学成分研究表明,垂柳根、皮及叶种含有丰富的黄酮类、甾体类、生物碱类、有机酸类等。然而,目前国内外对柳芽生物成分及生理作用的研究却很少。越来越多的现代化提取技术被应用于实践实验中,与传统提取方法相比,现代化提取工艺具有提取效率高,提取物纯度高,操作简单,无溶剂残留,产品质量高等优点。目前常用的提取方法有超临界流体萃取法、超微粉碎技术、超声提取法、旋流提取法、大孔树脂吸附法等。超声提取法因其简便,不需加热,提取时间短,适用于固体制剂中测定组分的提取,应用日益广泛。
响应面设计方法(Response Surface Methodoogy,RSM),是利用合理的试验设计方法并通过实验得到一定数据,采用多元二次回归方程来拟合因素与响应值之间的函数关系,通过对回归方程的分析来寻求最优工艺参数,解决多变量问题的一种统计方法。响应面方法不仅可以提高药物成分提取率,而且会减少实验时间及成本。
一直以来细菌和过量的自由基是导致机体患病、衰老、患癌以及其它各类疾病的主要原因。因此,通过抑菌以及抗氧化作用的研究寻找有效物质成为目前医药卫生、保健品、化妆品领域研究的热点。植物一直以来都是膳食抗氧化剂的来源,因为人们发现大多数植物都具有出色的抗氧化潜能。
炎症(inflammation,INFL)是一个指损伤因子带来的对机体的危害过程,其中具有血管系统的活体组织对这个过程所发生的防御反应,它是一种感染和许多疾病发生和发展的最基本的病理过程。炎症发生时,一方面组织和细胞被损伤因子通过某些渠道破坏;另一方面以稀释、杀伤的方式通过炎症充血和渗出反应包围引起危害的损伤因子;所受损的组织很快就修复和愈合的。炎症反应可以分为两类即急性炎症和慢性炎症。有些急性症状以血管系统的反应为主所发生的炎症,比如发红、肿胀、疼痛等。当前,非甾体抗炎药和甾体抗炎药是临床上常用的抗炎药物,虽然这类药物临床效果良好但是在药物治疗时副作用较大。因此低毒高效的新型抗炎药在临床药物研究中有很大的意义,是一重要领域。
镇痛药一向也是药物研究的热门点,临床上常用的一类是镇痛效果很好,但有成瘾性的阿片受体类镇痛药。另一类是抗炎作用显著的抑制前列腺素(Prostaglandin,PG)生成的非甾体类抗炎药,不过肠道里毒副作用明显。因此开发高效低毒的天然药物,具有应用价值。经过很多实验研究发现,从银杏、青果、映山红、桑白皮、皱皮木瓜、棉团铁线莲、蜂胶等植物中提取的黄酮类化合物都有镇痛抗炎作用。
因此,在医药研究领域,发现新的高效低毒的具有抗氧化、抑菌及抗炎镇痛效果的中药成分是医药研究人员一直努力的方向。
现有技术没有柳芽总黄酮的提取方法和在制备抗氧化、抑菌及抗炎镇痛药物方面的相关报道。
发明内容:
为了克服现有技术的缺陷,本发明提供柳芽总黄酮,其总黄酮的含量在7%以上。
本发明通过如下技术方案实现:
所述的柳芽总黄酮通过如下方法制备:将采摘柳芽烘干制成粉状,用乙醇溶液超声辅助提取,乙醇溶液的浓度为30-70%,液料比为40-70:1,超声提取2-3次,提取时间每次15-35min,过滤,合并滤液,浓缩至尽干,备用。
进一步,所述的乙醇浓度为30-50%,料液比为50-70:1,超声提取2-3次,提取时间每次15-35min。
按照本发明方法制备得到的总黄酮含量最高可达7.55%。
所述的枊芽总黄酮的成分主要包括:黄酮醇(槲皮素、异槲皮素、山奈酚、异鼠李素、芦丁、黄芪苷)、二氢黄酮(柚皮素)、黄烷醇(儿茶素),酚苷类化合物(水杨苷)和生物碱(葫芦巴碱)。
本发明提供了一种药物组合物,包含本发明的柳芽总黄酮和药学上可接受的载体或赋形剂。
本发明还提供了柳芽总黄酮和包含柳芽总黄酮的药物组合物在制备抗氧化药物中的应用。
本发明还提供了柳芽总黄酮和包含柳芽总黄酮的药物组合物在制备抑菌药物中的应用。
所述的抑菌药物为抑制细菌的药物。
进一步地,所述的抑菌药物为抑制革兰氏阳性菌或革兰氏阴性菌的药物。
具体地,本发明提供了柳芽总黄酮和包含柳芽总黄酮的药物组合物在制备抑制大肠杆菌、链球菌、金黄色葡萄球菌或沙门氏菌药物中的应用。
本发明还提供了柳芽总黄酮和包含柳芽总黄酮的药物组合物在制备抗炎药或镇痛药物中的应用。
且急性毒性试验结果表明:小鼠未见相关毒性反应及死亡现象,在临床剂量下安全可靠。
附图说明:
图1为乙醇溶液浓度、超声提取时间、液料比对柳芽总黄酮提取率的影响;
乙醇溶液浓度:X1(%) 超声提取时间:X2(min) 液料比:X3(mL/g)
图2为柳芽总黄酮提取的响应面优化曲线;
图3为柳芽总黄酮的LC-MS色谱图谱;
图4为实施例10中解剖后小鼠各脏器剖检结果;
a心脏;b肝脏;c脾脏;d肺脏;e肾脏;f胸腺;
图5为实施例10中MTD试验雌、雄性小鼠体质量变化图;
图6为小鼠肝脏和肾脏病理染色结果(×100);
a、b、c、d为小鼠肝脏组织染色结果;e、f、g、h为小鼠肾脏组织染色结果。
具体实施方式:
实施例1
柳芽总黄酮提取物的提取条件筛选:
考察乙醇溶液浓度、超声提取时间、料液比对柳芽总黄酮含量的影响。
柳芽总黄酮三个单因素的试验结果见图1。
本试验对柳芽中黄酮类化合物的提取工艺进行优化,分别为从乙醇浓度(30%、40%、50%、60%和70%)、超声波提取时间(15、20、25、30和35min)、溶剂和材料的比例(液料比)(30:1、40:1、50:1、60:1和70:1mL/g)进行优化。
因素 | 乙醇浓度(%) | 提取时间(min) | 液料比(mL/g) |
1 | 30 | 15 | 30:1 |
2 | 40 | 20 | 40:1 |
3 | 50 | 25 | 50:1 |
4 | 60 | 30 | 60:1 |
5 | 70 | 35 | 70:1 |
(1)乙醇体积分数对总黄酮得率的影响
固定液料比50:1(mL/g)和提取时间25min,探讨乙醇体积分数(30%、40%、50%、60%、70%)对黄酮得率的影响,试验结果见图1。如图1所示,随着乙醇溶液浓度(X1,%)的增加,呈先上升后下降的趋势,在乙醇体积分数为40%时,黄酮得率达到最大值。X1折线图表明,不同体积分数的乙醇极性不同,黄酮类化合物具有较高的极性,根据相似相溶原理,柳芽黄酮在乙醇体积分数为40%时溶出效果较好。因此,选择乙醇体积分数为40%较为合适。
(2)提取时间对总黄酮得率的影响
固定乙醇的体积分数50%和液料比50:1(mL/g),探讨提取时间(15、20、25、30、35min)对黄酮得率的影响,试验结果见图1。从图1的X2折线图可以看出,随着超声提取时间(X2,min)的变化,黄酮得率呈先增加后降低的趋势,在30min时达到最大值,然后总黄酮提取率下降。提取时间过短,黄酮提取不完全;提取时间过长,能耗增加,同时黄酮类物质的稳定性将变差,并会随着提取溶剂的不断蒸发而损失或分解,从而导致得率降低。因此,提取时间选择为30min较为适宜。
(3)液料比对总黄酮得率的影响
固定乙醇体积分数50%和提取时间25min,探讨液料比(30:1、40:1、50:1、60:1、70:1mL/g)对黄酮得率的影响,试验结果见图1。从图1的X3折线图可以看出,总黄酮得率随着溶剂料比(X3,mL/g)的增加而增加,在70:1mL/g时达到峰值(6.5%)。但由于浓缩难度和实验成本的限制,综合考虑,选取液料比为70:1(mL/g)较为合适。
根据柳芽总黄酮提取物单因素实验的结果,将三个层次的三个因素,即乙醇溶液的浓度(30、40、50%)、超声提取时间(25、30、35min)和液料比(50:1、60:1、70:1mL/g)应用于响应面设计中。
实施例2
响应面优化提取条件
模型拟合与统计分析
如表1所示,以黄酮类化合物得率为指标,优化了柳芽总黄酮的提取工艺。Interms of coded factors,采用Design Expert软件(version 8.0.6)进行多元回归分析,对柳芽总黄酮的最终方程进行统计拟合,最终方程为:Y=5.84+0.58X1-0.21X2+1.34X3+0.42X1X2+0.28X1X3+0.10X2X3-0.42X12-0.095X22-0.24X32,该模型R2=0.9436,R2>0.9,说明该模型能解释94.36%响应值的变化,表明该模型与试验结果拟合较好,可以替代试验真实点对实验结果进行分析。
表1 Box-Behnken实验设计及对柳芽总黄酮提取率的研究结果(n=3).
注:X1:乙醇浓度(%);X2:提取时间(min);X3:料液比(mL/g)
P水平是检验回归系数的标准,当p<0.05时表明回归方程显著;当p<0.01时表明回归方程极显著;当p>0.05时表明回归方程不显著。对柳芽总黄酮的响应面分析方案和结果数据进行方差分析,结果为表2。
表2柳芽总黄酮BBD试验结果的方差分析
从回归模型方差分析表可知,由试验数据所得模型F值为13.02,p值为0.0014表示该模型极显著,表明该模型具有统计学意义。各因素中X1、X3项对响应值达到了极显著水平。3个因数乙醇溶液浓度(X1)、提取时间(X2)、液料比(X3)对柳芽总黄酮的浸出率的影响为:液料比>乙醇溶液浓度>提取时间。
失拟检验的F值为2.10,失拟检验p值为0.2428(>0.05),不显著,说明在试验过程中,试验的误差等偶然因素不会对实验结果带来显著影响。RAdj 2=0.8712(>0.800)有12.88%的变异不能由该模型解释,综合分析表明用此模型对柳芽总黄酮提取工艺结果进行很好的分析和预测。
图2a~2c展示了三维响应面曲线,它表明乙醇溶液浓度(X1)、超声提取时间(X2)、料液比(X3)对总类黄酮得率的影响。从图2a~2c可以看出,总黄酮的得率在一定范围内随三个因素各因素的增加而提高。
利用Design-Expert得到总黄酮的最终方程并成功应用于响应面优化。最优提取条件为:乙醇溶液的浓度为50%,超声提取时间为35min,液料比为70:1。
实施例3
柳芽总黄酮提取
垂柳吐出嫩芽约一厘米左右时为采摘对象,垂柳经沈阳工学院生命工程学院傅俊范教授鉴定,柳芽在洗净后自然晾干。将采摘柳芽100g烘干制成粉状,过16目筛,置于三角瓶中,用超声辅助提取柳芽总黄酮,乙醇溶液的浓度为50%,超声提取两次,每次超声提取时间为35min,液料比为70:1,过滤,合并两次滤液,浓缩至尽干,备用。
实施例4
柳芽总黄酮含量测定
本试验采用吕法测定柳芽总黄酮含量,首先建立芦丁标准曲线。以芦丁浓度为X轴、吸光度为Y轴,绘制标准曲线;标准曲线的回归方程为:Y=9.6607X-0.002,R2=0.9995。取柳芽提取的上清液定容到50mL容量瓶,取1ml提取液于25mL的比色管定容到10mL,按照上述方法,然后在510nm处测其吸光度,通过回归方程计算实施例3中提取液中的黄酮含量。其总黄酮的含量为7.55%(N=3)。
总黄酮的含量=m1/m2×100% m1=C×V1×V2
注:C为测得吸光度值,带入回归方程计算出的总黄酮浓度(mg/mL),V1为总黄酮提取液体积(mL),V2为测量总黄酮含量时的定容体积(mL),m2为柳芽样品重量(mg),m1为总黄酮提取量。
实施例5
柳芽总黄酮成分鉴定
制备柳芽总黄酮粉末。称量粉末样本约200mg,加入1mL甲醇:水(8:2,V:V),涡旋混匀。加入2~3颗二氧化锆研磨珠,研磨提取3min;4℃条件下离心10min,离心力20000xg;上清液用0.22um滤膜过滤,取滤液上机分析。
质谱条件离子源:电喷雾电离源(ESI);扫描方式:正负离子切换扫描,检测方式:Full mass/dd-MS2;分辨率:70000(full mass);17500(dd-MS2);扫描范围(scan range):150.0~2000.0m/z;电喷雾电压(Spary Voltage):3.8kV(Positive);毛细管温(CapillaryTemperature):300℃;碰撞气:高纯氩气(纯度≥99.999%);鞘气:氮气(纯度≥99.999%),40Arb;辅助气(Aux gus heatertemp):氮气(纯度≥99.999%),350℃数据采集时间:30.0min。
色谱条件色谱柱:RP-C18 150×2.1mm 1.8um,Welch,流速0.30mL/min,流动相:0.1%甲酸乙腈溶液(A)-0.1%甲酸水溶液(B),梯度洗脱(洗脱条件见表3),洗针液选用甲醇,柱温箱温度:35℃,自动进样器温度10.0℃,进样针高度:2.00mm,自动进样器清洗设置:Both,自动进样器洗针体积:200.00uL,自动进样器进针清洗时浸泡时间:3.00ms,自动进样器进样体积:5.00uL。
表3色谱梯度洗脱条件
如图3所示,柳芽总黄酮提取物的色谱记录均显示出丰富多样的色谱峰信号。有研究表明柳属植物普遍含有黄酮类化合物,其中主要是黄酮类、黄酮醇类、黄烷醇类,此外还有二氢黄酮类、二氢黄酮醇类、花色素类、查尔酮类等化合物。本研究经高分辨液质采集的数据通过CD2.1(Thermo Fisher)完成数据初步整理后与数据库(mzCloud,mzVault,ChemSpider)检索比对,最终鉴定出10个有效化合物,涉及到黄酮醇(槲皮素、异槲皮素、山奈酚、异鼠李素、芦丁、黄芪苷)、二氢黄酮(柚皮素)、黄烷醇(儿茶素),1个酚苷类化合物(水杨苷)和1种生物碱(葫芦巴碱)。本试验选取色相液谱峰面积≥1×108的化合物进行详细分析讨论,并且结合相关文献,将每个化合物的Rt(time)、[M-H]-、MS/MS[M-H]-、[M+H]-、MS/MS[M+H]、Calculated mass、Formula列在表4中。
表4柳芽总黄酮提取物化学成分的鉴定结果
注:a为黄酮类化合物,b为酚苷类化合物,c生物碱类化合物
实施例6
体外抗氧化试验
以维生素C(Vitamin C,VC)为对照,通过检测其总抗氧化能力,清除1,1-二苯基-2-三硝基苯肼自由基(1,1-Diphenyl-2-picrylhydrazyl,DPPH)、超氧阴离子自由基(·O2 -)和羟自由基(·OH-)能力,评价柳芽总黄酮体外抗氧化效果。
清除DPPH自由基试验
参考DPPH自由基清除能力试剂盒(南京建成)说明书,配制相关试剂:
试剂一:临用前甩几下使试剂落入底部,再加入40mL无水乙醇充分溶解后备用;用不完的试剂4℃避光保存。
分别取400μL不同浓度(62.5、125、250、500、1000mg/mL)的样本溶液放入测定管,按照上述方法加样,测定管吸光度值为A测定,对照管吸光度值为A对照,空白管吸光度值为A空白。Vc作为阳性对照组,计算样品各个浓度的清除率及IC50的值。DPPH自由基清除能力的计算公式:
抑制羟自由基试验
参考羟自由基测定试剂盒(南京建成)说明书,配制相关试剂,将配制好的应用液先在37℃水浴中预温3min,以下操作在37℃水浴中进行。
分别取0.2mL不同浓度(62.5、125、250、500、1000mg/mL)的样本溶液放入测定管,按照上述方法加样,测定管吸光度值为A测定,对照管吸光度值为A对照,空白管吸光度值为A空白,标准管吸光度值为A标准。Vc作为阳性对照组,计算样品各个浓度的清除率及IC50的值。抑制羟自由基的计算公式:
抑制超氧阴离子试验
参考抑制超氧阴离子自由基试剂盒(南京建成)说明书,配制相关试剂:
分别取0.05mL不同浓度(625、1250、2500、5000、10000mg/mL)的样本溶液放入测定管,按照上述方法加样,测定管吸光度值为A测定,对照管吸光度值为A对照,空白管吸光度值为A空白。Vc作为阳性对照组,计算样品各个浓度的清除率及IC50的值。抑制超氧阴离子自由基的计算公式:
总抗氧化能力试验
参考总抗氧化能力试剂盒(南京建成)说明书,配制相关试剂:
分别取0.1mL相同浓度(1mg/mL)的样本溶液放入测定管,按照上述方法加样,重复三次。测定管吸光度值为A测定,对照管吸光度值为A对照。Vc作为阳性对照组,计算该浓度样品的清除率及IC50的值。总抗氧化能力的计算公式:
结果如表5所示。
表5柳芽总黄酮提取物抗氧化结果
结果表明:柳芽总黄酮质量浓度为1mg/mL时,抑制超氧阴离子自由基56.58%,表现出理想的抑制作用。羟自由基可与活细胞中分子发生反应,引发组织病变和衰老等。当柳芽总黄酮质量浓度为1mg/mL时,抑制羟自由基达84.04%,表现出良好的抗氧化活性。
对柳芽总黄酮的·OH、O2 -·抑制率、DPPH自由基清除率与浓度进行线性拟合,其线性拟合方程分别为:Y(·OH)=-164.47X2+260.26X-11.737,R2=0.9946;Y(O2 -·)=-10.972X2+35.998X+31.476,R2=0.9969;Y(DPPH)=-79.409X2+136.02X+21.966,R2=0.9948,均呈现出良好的线性量效关系;由此可计算出柳芽总黄酮的·OH、O2 -·、DPPH自由基的IC50分别为217.3、619.2、206.3μg/m L。
研究表明柳芽总黄酮具清除DPPH自由基能力且对超氧阴离子自由基(O2 -·)和羟基自由基(·OH)均有不同程度的抑制作用。本试验研究结果表明,柳芽总黄酮对羟基自由基(·OH)的抑制作用尤为突出,与VC(IC50189.25μg/mL)相当。此外,总抗氧化能力是体现物质清除不同自由基的有效和,柳芽总黄酮总抗氧化能力测定结果表明:柳芽总黄酮总抗氧化能力检测值为65.19U/mg。
综上所述,柳芽总黄酮体外表现出一定程度的抗氧化活性。
实施例7
柳芽总黄酮对二甲苯诱导小鼠耳肿胀的影响
选取小鼠30只,随机分为5组,每组6只。分别设模型对照组、地塞米松对照组(5mg/(kg·bw))、柳芽总黄酮低、中、高剂量组(125mg/(kg·bw)、250mg/(kg·bw)、500mg/(kg·bw))。采取灌胃给药,每日定时一次,持续喂药5d。最后一次喂药40min后,使用50μL二甲苯均匀涂抹在小鼠右耳前后两面,左耳作为对照,涂抹同等体积的蒸馏水,40min后脱颈处死小鼠。将双耳顺着小鼠耳廓基线剪下,用直径8mm的打孔器分别在左右两耳相同位置切下圆耳片,用分析天平快速称重。用左、右耳质量的差值表达小鼠耳廓肿胀度。耳肿胀抑制率通过式(1)计算。结果见表6。
表6柳芽总黄酮对小鼠耳廓肿胀的影响
注:与模型对照组相比,*代表差异显著(P<0.05);**代表差异极显著(P<0.01)。
由表6可知,不同剂量柳芽总黄酮均可以抑制小鼠耳廓肿胀,且呈剂量依赖性,与模型组相比,不同剂量柳芽总黄酮能显著抑制小鼠耳廓肿胀(P<0.01)。此外,高剂量柳芽总黄酮抑制效果近似于地塞米松,由此可见剂量增大,抑制效果更明显。
实施例8
柳芽总黄酮提取物的抑菌试验
体外抑菌试验方法
样品的制备用二甲基亚砜(DMSO)作为溶剂,称取柳芽总黄酮提取物400mg,加入5mL 1%DMSO,超声辅助溶解,经0.22μm微孔滤膜过滤,配成质量浓度80mg/mL药液。用MHB液体培养基依次倍比稀释成质量浓度40、20、10、5、2.5、1.25、0.625、0.3125、0.1562、0.078mg/mL。
菌悬液制备将供试细菌Escherichia coli(ATCC25922)、Streptococcus(ATCC49619)、Staphyloccocus aureus(ATCC29213)、Salmonella(ATCC 51812)分别接种于LB固体琼脂培养基上,37摄氏度静置培养24-36h。培养后挑取3—5个菌落于LB液体培养基中。在37摄氏度,以150rpm振摇,至细菌生长达到对数生长中期。约为1x108CFU,将1x108的菌液用MHB液体培养基稀释1000倍,即1x105CFU/MI。
对照药配制以水作为溶剂,分别称取左氧氟沙星与多西环素0.0128g,加入10mL水中溶解,超声辅助溶解,过0.22um无菌滤膜,配成1280ug/mL储备药液。用液体MHB培养基依次稀释成质量浓度为64、32、16、8、4、2、1、0.5、0.25、0.125ug/mL。
MIC测定采用双倍稀释法,取5mL LB液体培养基、500μL不同浓度样品和500μL菌液加入到试管中,充分混匀。试管置于37℃,180rpm摇床培养24h。以肉眼不见浑浊的最低浓度即为MIC。以多西环素、左氧氟沙星作为阳性对照,1%DMSO溶液和MHB培养基为阴性对照。对照药物左氧氟沙星和多西环素MIC值均符合CLSI2018标准。结果见表7所示。
表7柳芽总黄酮提取物抑菌活性的研究
从表7可以看出,柳芽总黄酮对四种细菌均有抑制效果,其中对金黄色抑菌效果最好MIC为2.5mg/mL,对沙门氏菌的抑菌效果相对较差,MIC为10mg/mL。
本发明柳芽总黄酮提取物含有大量的黄酮类化合物,并且展现出良好的抑菌效果,试验结果证明,其对革兰氏阳性菌和革兰氏阴性菌均有抑菌效果,有望在食品加工、美容保健以及替代抗生素中发挥积极作用。
实施例9
柳芽总黄酮对冰醋酸致小鼠扭体的影响
小鼠分组及喂药方法同实施例7。其中,用等量的阿司匹林替代地塞米松对照组中地塞米松。末次给药后1h,向小鼠腹腔注射0.1mL/10g的0.6%冰醋酸溶液,诱发小鼠扭体,疼痛反应指标以30min后采用摄影机记录小鼠20min内扭体频率为准。疼痛抑制率通过式(2)计算。
表8柳芽总黄酮对冰醋酸致小鼠扭体的抑制作用
由表8可知,低、中、高剂量柳芽总黄酮提取物均可以抑制小鼠扭体,且呈剂量依赖性。不同剂量柳芽总黄酮组显著抑制小鼠扭体(P<0.01),阿司匹林对照组极显著抑制小鼠扭体(P<0.01)。高剂量柳芽总黄酮抑制效果(60.53%)近似于阳性药物阿司匹林(68.54%)。因此,不同剂量的柳芽总黄酮均有镇痛效果,尤其是高剂量柳芽总黄酮效果更加明显。
可见,本发明的柳芽总黄酮提取物具有明显的抗氧化、抗炎、抑菌、镇痛的作用。
实施例10小鼠急性毒性实验
1.小鼠急性毒性LD50预实验
取昆明小鼠40只,每天按0.2mL/10g小鼠体质量灌胃给药1次,连续观察7d,进行LD50的测定。
连续7天按最大给药体积和TFW提取液最大溶解浓度给药未发现小鼠死亡,LD50>10g·Kg-1,未得出半数致死量。
2.急性毒性MTD法试验
取昆明小鼠40只,随机分为2组,为柳芽总黄酮TFW组和对照组,每组为20只。小鼠试验前一日晚上禁食不禁水,次日上午TFW组小鼠灌服提取液,灌胃给药最大容积0.2mL/10g,给药剂量为0.5g/mL;对照组灌服同量的生理盐水;每天灌胃1次,连续灌胃7天。灌胃后当天密切观察6h内各组小鼠灌胃后的症状,灌胃第二天后每天观察1次,记录动物的行为活动,精神状态,被毛,摄食,饮水,呼吸,排泄物情况和鼻,眼,死亡及体质量变化等情况。小鼠急性毒性症状见图4。同时称量灌胃前,灌胃后第1,3,5,7天的体质量,记录小鼠体质量变化。第8天脱颈处死各组小鼠,对其主要脏器进行肉眼观察,与对照组内脏进行比较,并对病变脏器及肝、肾进行病理组织学检查,在此之前计算脏器指数:
脏器指数=脏器湿重(mg)/体质量(g)
2.1小鼠急性毒性MTD症状观察
灌胃后15min内,个别小鼠行动迟缓,但无不良反应;15-30min,小鼠逐步恢复正常,对外界刺激灵敏,饮食、饮水正常,有爬笼现象;观察期内无小鼠死亡。7天后将全部存活小鼠处死,经剖检未发现TFW组与对照组小鼠内脏器官存在异常(图4)。
2.2小鼠急性毒性MTD值测定
根据小鼠灌胃剂量计算MTD值,结果TFW的MTD值为10g·Kg-1·d-1(注:最大耐受量指每日每千克小鼠最大给药克数)。
2.3小鼠急性毒性MTD体质量变化
小鼠灌胃后连续观察7d,称取TFW组和对照组小鼠的体质量。结果表明:TFW组和对照组的体质量变化无显著性差异(表9,图5)。
表9TFW对小鼠急性毒性MTD试验体质量变化的影响
2.4小鼠脏器指数
与对照组比较,TFW组的小鼠各脏器外观、质地均无差异;各脏器指数均无显著性差异(表10)。
表10小鼠脏器指数
2.5小鼠肾脏、肝脏组织病理学变化检测结果
小鼠肝和肾组织病理染色结果见图6。可见,肝细胞形态正常,排列紧密,肝小叶结构正常。小鼠肾小管排列结构正常。肝脏和肾脏均无纤维化、明显炎性细胞浸润等病理性变化。小鼠肝脏和肾脏未发现显著病理变化。
在该试验条件下,TFW的小鼠灌胃给药最大耐受剂量为10g·Kg-1·d-1,小鼠未见相关毒性反应及死亡现象,说明临床剂量下该药安全可靠。
本发明的柳芽含有多种有效成分,从柳芽中提取的柳芽总黄酮具有良好的抗氧化、抑菌、抗炎和镇痛效果。
Claims (6)
1.柳芽总黄酮提取物在制备抑制细菌药物中的应用,所述的细菌为大肠杆菌、链球菌、金黄色葡萄球菌或沙门氏菌,其特征在于,所述的柳芽总黄酮提取物通过如下方法制备:柳芽用乙醇溶液超声辅助提取,乙醇溶液的浓度为30-70%,液料比为40-70:1,超声提取2-3次,超声提取时间每次15-35 min,过滤,合并滤液,浓缩至尽干,即得。
2.权利要求1所述的应用,其特征在于,乙醇溶液的浓度为30-50%,料液比为50-70:1。
3.权利要求1所述的应用,其特征在于,乙醇溶液的浓度为50%,液料比为70:1,超声提取时间为35 min。
4.权利要求1所述的应用,其特征在于,所述的柳芽总黄酮提取物包含黄酮醇、二氢黄酮、黄烷醇、酚苷类化合物、葫芦巴碱。
5.权利要求1所述的应用,其特征在于,所述的柳芽总黄酮提取物包含槲皮素、异槲皮素、山奈酚、异鼠李素、芦丁、黄芪苷、柚皮素、儿茶素、水杨苷和葫芦巴碱。
6.如权利要求1-5任何一项所述的应用,其特征在于,所述的柳芽总黄酮提取物和药学上可接受的载体或赋形剂组成药物组合物。
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吴艳芳.柳芽中总黄酮含量的测定.《光谱实验室》.2011,第28卷(第6期),第2871-2873页,尤其是第2871页第2.3项和第2873页第4项. * |
柳叶黄酮提取纯化及其生物活性研究进展;赵二劳等;《北方园艺》(第23期);第206-210页,尤其是第206页左栏第1段 * |
柳芽中总黄酮含量的测定;吴艳芳;《光谱实验室》;第28卷(第6期);第2871-2873页,尤其是第2871页第2.3项和第2873页第4项 * |
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