全 文 :微波法提取全叶马兰总黄酮成分工艺优化
闫 红,王伟娥,彭惜媛,吴 岚,杜 娟
(佳木斯大学药学院,黑龙江 佳木斯154007)
摘要:目的:考察影响微波法辅助提取全叶马兰总黄酮的主要因素,用正交实验确定了最优提取工艺。方法:采用微波辅
助法进行黄酮类化合物的提取,分析比较料液比例、乙醇浓度提取时间和微波功率对总黄酮提取量的影响,用紫外分光光度
法测定黄酮含量。结果:优选了测定波长及显色条件等分析条件,测定方法的回归方程y=0.0213x+0.0051相关系数
R2=0.9933。单因素实验考察了乙醇浓度、微波功率、提取时间、料液比四个因素对全叶马兰黄酮类化合物提取效率的影响。
通过正交试验确定了最佳的提取工艺条件为物料比为1:10、提取时间40s、微波功率406W、乙醇浓度60%。结论:该方法与
传统的提取方法相比,提取率高,省溶剂,大大提高了提取效率。
关键词:全叶马兰;总黄酮;微波提取;工艺优化
中图分类号:R284;R931 文献标识码:A 文章编号:1008-0104(2014)04-0043-02
全叶马兰(Kalimeris integrtifolia Turcz.Ex DC.)为菊
科(Asteraceae)马兰属(Kalimeris)多年生草本植物,别名全
缘叶马兰、野白菊(湖北)、黄花三草、扫帚花、扫帚鸡肠、全叶
紫菀、全叶鸡儿肠、路边菊等,主要生于山坡、林缘、灌丛、路
旁。广泛分布于我国东北部、西部和中部,国外在俄罗斯的
东部西伯利亚、蒙古和朝鲜也有分布。《中华本草》[1]中记
载,全叶马兰味苦、性寒,具有清热解毒、止咳、散瘀止血的功
效。全叶马兰以其嫩茎叶供蔬食,也可做优良的牧草。黄酮
类化合物具有抑菌、消炎、抗氧化、抗衰老、抗癌防癌、抗病毒
等诸多功效,有着广阔的市场前景全叶马兰含有丰富的黄酮
类物质[2],本文应用微波法对黄酮成分进行提取工艺研究,
旨在确立最佳的提取条件,为全叶马兰的开发利用提供依
据[3]。
1 仪器与材料
1.1 实验仪器
S54型紫外可见分光光度计(上海棱光技术有限公司);
电子分析天平(上海恒平科学仪器有限公司);格兰仕微波炉
(佛山市格兰仕微波炉电器有限公司);架盘药物天平(北京
医用天平厂);玻璃仪器气流烘干器(英峪仪器厂);乙醇(天
津市凯通化学试剂有限公司);芸香叶苷 (国药集团化学试
剂有限公司);亚硝酸钠 (哈尔滨新达化工厂),氢氧化钠 (天
津市博迪化工有限公司);硝酸铝 (北京化工厂);试剂均为
分析纯。
1.2 实验材料
全叶马兰采摘于黑龙江省佳木斯市亮子河国家森林公
园,经佳木斯大学药学院刘娟教授鉴定为菊科植物全叶马兰
(Kalimeris integrtifolia Turcz.Ex DC.)。
2 实验方法
2.1 全叶马兰总黄酮的含量测定
2.1.1 药材的处理
将马兰药材低温烘干后备用。
2.1.2 标准溶液和储备液的配制
精密称取1.000g的芸香叶苷,用60%的乙醇溶解并定
容于100mL容量瓶中,配成1.000g/100mL的标准溶液。精
密称取亚硝酸钠5g,加入100mL容量瓶中,加入适量蒸馏
水,定容至100mL容量瓶中,于冰箱中备用。精密称取硝酸
铝10g,加入100mL容量瓶中,加入适量蒸馏水,定容至
100mL容量瓶中,于冰箱中备用。精密称取氢氧化钠4g,加
入100mL容量瓶中,加入适量蒸馏水,定容至100mL容量瓶
中,于冰箱中备用。
2.1.3 标准曲线的制备
精密移取0、0.5、1、2、3、4、5mL芦丁标准溶液,分别加
入到离心试管中,分别加入25mL的60%浓度的乙醇,用中
火微波加热30s后过滤,精密移取滤液1mL,首先加入0.
3mL亚硝酸钠储备液,静止放置6min,其次加入0.3mL硝酸
铝储备液,静止放置6min,再次加入4mL氢氧化钠储备液混
匀,最后加入2.4mL 60%的乙醇溶液,然后在室温下避光放
置15min,以0样为空白,在最佳波长处测定吸光值,以吸光
值为纵坐标,标准液浓度为横坐标,绘制标准曲线,计算回归
方程[4]。
2.1.4 样品溶液的配制
精密移取10mL提取液(微波优化条件后)置于50mL的
容量瓶中,照上述方法配制并测定吸光度。
2.2 微波工艺的优化实验
2.2.1 微波炉的主要技术参数
工作频率:2450MHz;最大的输出功率:700W;功率调节
共分为5档:小火、解冻、中火、中高火、高火;工作时间调节:
0~60min。
2.2.2 微波法实验条件的工艺优化
在上述单因素试验的基础上,以料液比、微波功率、提取
时间、溶剂浓度为因素,进行L9(34)正交试验,见表1,研究
微波提取马兰成分中黄酮的最佳工艺条件[5]。
表1 微波法提取全叶马兰总黄酮的正交试验
因素
A B C D
乙醇浓度(%) 料液比 提取时间(s) 微波功率(W)
1 50 1:10 10 解冻
2 60 1:15 15 中火
3 70 1:20 20 中高火
3 实验结果
3.1 标准曲线的绘制
通过紫外分光光度计测定,对芦丁溶液含量(X)和吸光
度(Y)进行回归处理得到回归方程为y=0.0213x+0.0051,
R2=0.9933,表明芦丁的含量和吸光度呈良好的线性关系,
见图1。
图1 芦丁标准曲线
3.2 全叶马兰总黄酮提取工艺正交试验
在上述单因素试验的基础上,以溶剂浓度、微波功率、提
取时间和物料比为因素,进行L9(34)正交试验,得出微波提
取全叶马兰总黄酮的最佳工艺条件,结果见表2。
由正交试验结果可以看出,四种因素对全叶马兰总黄酮
化合物的提取均有影响。通过R值的大小顺序C2>D3>A1
>B1,可以得出影响顺序依次为:提取时间>物料比>溶剂
浓度>微波功率,并且微波法提取全叶马兰黄酮的优化条件
是:提取时间40s、物料比1:10、乙醇浓度60%、微波功率
·34·HEILONGJIANG MEDICINE AND PHARMACY Aug.2014,Vol.37No.4
406W。
表2 微波法提取全叶马兰总黄酮的正交试验
实验号
A B C D
浓度 料液比 提取时间 功率
总黄酮含量(%)
1 1 1 1 1 2.868
2 2 1 2 2 2.600
3 3 1 3 3 3.532
4 1 2 2 3 3.369
5 2 2 3 1 2.915
6 3 2 1 2 2.269
7 1 3 3 2 3.172
8 2 3 1 3 2.032
9 3 3 2 1 2.804
K1 9.564 9.125 9.098 9.134
K2 9.719 9.111 10.145 10.111
K3 9.015 9.106 9.118 10.126
R 0.156 0.101 0.315 0.218
参考文献:
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上海科学技术出版社,1999,130 130
[2]张特立,于春月.马齿苋中总黄酮的提取工艺研究[J].黑龙江医
药科学,2013,36(4):37 38
[3]黄展,王晶.正交设计优化苦瓜多糖的超声提取工艺研究[J].黑
龙江医药科学,2013,36(3):22 23
[4]闫红,赵岩,张楠楠,等.微波法提取苍耳子中多酚的工艺研究
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(收稿日期:2014-03-20)
作者简介:闫红(1965~)女,黑龙江佳木斯人,本科,高级实验师。
通讯作者:杜娟(1974~)女,黑龙江绥化人,硕士,副教授。E-mail:jmsdujuan@126.com。
Optimization technique of flavonoids from Kalimeris integrtifolia
by microwave extraction method
YAN Hong,WANG Wei-e,PENG Xi-yuan,WU Lan,DU Juan
(Pharmacy Department of Jamusi University,Jiamusi 154007,China)
Abstract:Objective:To study the main factors those afect the extraction of total flavones in Kalimeris inte-
grtifolia by microware extraction method according to the orthogonal design in this paper.Methods:Flavonoids
compounds were extracted by microwave method.The efects of extraction microwave power,alcoholconcentra-
tion,extraction time and solid-liquid mass ratio on the extraction of flavonoids of Kalimeris integrtifolia were ana-
lyzed.The content of flavonoids was determinated by ultraviolet.Results:The determination conditions of UV-
Vis,the color reaction selection,color terms and wavelength detection were studied in this paper.The regression
equation is y=0.0213x+0.0051,r=0.9933.Four main factors including extracting solvent concentration,
extraction temperature,extraction time and ratio of solvent to sample were studied.By using single factor
analysis method and the orthogonal L9(34)analysis,the best extraction conditions were determined,that
is,40%ethanol concentration,microwave power was 406W;the extraction time was 40s;solid-liquid
mass ratio was 1:10.Conclusion:Compared with traditional methods of extraction,this method serves
high yield and provincial solvent,which greatly improves extraction efficiency.
Key words:Kalimeris integrtifolia;flavonoids;microwave extraction;optimization technique
(上接第42页)
Effect of Hawthorn leaves flavonoids(HLF)on diabetes melitus
rats'testis and the expression of EGFR in diabetes melitus rats
WANG Chong1,MA Xiao-ru2,SONG Ming-ming3,TIAN Guo zhong2
(1.Department of Pathology,Basic Medical Colege,Mudanjiang Medical Colege,Mudanjiang 157011,China;2.Department of Patho-
physiology,Basic Medical Colege of Jiamusi University,Jiamusi 154007,China;3.Department of Ophthalmology,the Central Hospital of Jia-
musi,Jiamusi 154003,China)
Abstract:Objective:To investigate the changes of EGFR expression in diabetes melitus rats'testis
and to explore the effect of HLF on diabetes melitus rats'procreation function.Methods:40male Wistar
rats were divided into 4group(control group,diabetes melitus model group,insulinize group and HLF
group)with ten in each group.Diabetic rat model was induced by a vena caudalis injection of streptozoto-
cin(30mg/kg).Rats in HLF group received HLF for 200mg/kg·d by gavage.One testis were taken and
fixed in formaldehyde to make immunofluorescence section and pathological section.Results:Type 2diabe-
tes melitus models were successfuly constructed.Rats in model group showed obviously pathology chan-
ges in testis.Convoluted seminiferous tubule seems chaos,and there was little sperma.After treatment by
HLF spermatogenic cels quantity and morphology recovered obviously.The EGFR expression in HLF
group was significant higher than that in model group(P<0.01),and there was no significant difference
compared with insulin group(P>0.05).Conclusions:HLF could decrease blood glucose concentration;
repair damage of testis;and promote the expression of EGFR.Therefore,HLF could protect testis tissue.
Key words:type 2diabetes melitus;Hawthorn leaves flavonoids;testicular pathological damage;EGFR
·44· 黑龙江医药科学 2014年8月第37卷第4期