摘要
黄荆是马鞭草科牡荆属(Vitex L.)植物,主产于长江以南。其果实、根、茎及叶等部位均可入药,具有镇痛、抗炎、抗菌、抗肿瘤、抑制淋巴细胞及激素调节、抗氧化、蜕皮、驱虫等功效。前期试验对该属植物中的黄荆子抗肿瘤有效部位进行了筛选,得到黄荆子抗肿瘤有效部位,并对其进行了系统化学成分研究,得到VBE-1、VBE-2等多个芳基二氢萘类木脂素类化合物,体内外试验表明该木脂素类化合物对人乳腺癌、人卵巢癌、人肝癌和人结肠癌细胞的生长均有较强的抑制活性。且初步的作用机制研究证实:该类化合物是抗肿瘤药物作用的重要靶点的Akt-mTOR通路的抑制剂。
为了控制黄荆子的质量,本课题对黄荆子进行了VBE-1、VBE-2的含量测定方法研究,首次建立了其高效液相含量测定方法。测定条件为:Kromasil ODS-1柱(250 mm×4.6 mm,5μm)为色谱柱;流动相为乙腈-0.1%冰醋酸水溶液梯度洗脱(0 min,15∶85;10 min,15∶85;40 min,28∶72);检测波长为255 nm;体积流量为1.0mL/min,柱温为25℃。自制对照品VBE-1与VBE-2在0.430～6.45ug与0.239～3.59ug范围内,峰面积与其含量同样呈良好的线性关系(R~2=1)。
为了更好地开发利用该属植物,寻找资源丰富、疗效显著的抗癌药物,本课题对牡荆属植物黄荆、牡荆的药材资源进行了初步调查,结果发现:与牡荆相比较,黄荆子中VBE-1、VBE-2含量较高;在黄荆药材的各个部位中,以其果实含量最高,其根、茎、叶中含量明显低于果实;不同产地黄荆子的VBE-1、VBE-2含量相差较大,以贵州、湖南双牌产的药材含量较高。
由于黄荆子木脂素类化学成分有明显的抗肿瘤活性,本课题对该类木脂素成分进行了富集纯化,考察了其提取条件,并利用聚酰胺进行了纯化,确立其最佳提取纯化工艺为:40%的乙醇为提取溶剂,提取两次,溶剂的体积分别为8、7倍药材量,提取时间分别为1.5h、1.0h,合并提取液,滤过;滤液回收乙醇,取0.4倍药材量体积30-60目聚酰胺拌样至干,上样到0.6倍药材量体积30-60目聚酰胺柱上,2个BV水洗,4个BV0.5%氨水洗,所得氨水洗脱液再用0.4倍药材量体积30-60目聚酰胺拌样至干;上样到0.6倍药材量体积30-60目聚酰胺柱上,2个BV水洗,4个BV40%乙醇洗,所得乙醇洗脱液60℃减压浓缩至1倍药材量体积;经同体积乙酸乙酯萃取两次,合并乙酸乙酯层,回收乙酸乙酯并蒸干,60℃真空干燥,获得了VBE-1与VBE-2总含量达50%的有效部位。为该部位进行抗肿瘤新药开发打下坚实基础。
Vitex negundo L.grows widely throughout the south of Yangtze River.The fruits,roots,stems,leaves,and other parts of this plant can be used as a medicine with analgesical,anti-inflammatory,anti-bacterial, antioxidant and anti-tumor activities,meanwhile it can regulate the hormone secretion.In the early investigations,the studies of the chemical constituents from the effective anti-tumor parts of Vitex negundo L..led to the isolations of a series of phenyldihydronaphthalene -type lignans,particularily VBE-1 and VBE-2.Both of them showed strong inhibitory in vitro and in vivo activities against the growth of human breast cancer,ovarian cancer,human hepatocellular carcinoma and human colon cancer cells.
A HPLC method to determine the content was established by the mean of study for the VBE-1 and the VBE-2 at the first time.The experimental conditions as follows:Kromasil ODS-1 column(250mm×4.6mm,5μm) was used as the chromatogram column,the mobile phase was acetonitrile -0.1%acetic acid aqueous solution gradient elution(0 min,15:85;10 min,15:85;40 min,28:72);detection wavelength was at 255 nm,the flow rate was 1.0 mL/min,and the column temperature was at 25℃.A good linear correlation was obtained when concentrations of VBE-1 and VBE-2 ranged from 0.430～6.45ug and 0.239～3.59ug separately.
The investigation about the resource of this plant was studied for the purpose of finding sufficient and highly efficient anti-cancer drugs.The results showed that the content of VBE-1 and VBE-2 from Vitex negundo L.were higher than those in Vitexin;And in all parts of Viticis negundo L.,the content of the fruits was much higher than those of the roots, stems and leaves.Besides,the different environments of different areas also influenced the content of VBE-1 and VBE-2,according to the research the content was higher relatively in Guizhou province and the Shuangpai city from Hunan province.
The subject aimed at gathering the phenyldihydronaphthalene-type lignans from fruits of Vitex Negundo which have obvious anti-tumor activity.The process of this experiment focused on extraction and purification by polyamide column chromatography.Finally the process was optimized as follows:The fructus of Vitex Negundo was extracted with 40%ethanol twice(1.5h and 1.0h,respectively) and the volume of solvent was respectively 8,7 times of the medicine amount.The extract was concentrated to remove the ethanol and absorbed in polyamide (30-60 mesh) with 0.4 times of the medicine volume,which was subjected to column chromatography over polyamide(30-60 mesh) with 0.6 times of the medicine volume,using water(2BV) and 0.5%ammonia water(4BV) as gradient elution.The eluent of 0.5%ammonia water was absorbed in polyamide(30-60 mesh) with 0.4 times of the medicine volume,which was subjected to column chromatography over polyamide (30-60 mesh) with 0.6 times of the medicine volume,using H2O-EtOH as gradient elution(H2O:2BV,40%EtOH:4BV).The ethanol of 40% EtOH eluent was removed under pressure and extracted twice with ethyl acetate(v:v 1:1).The ethyl acetate layer was evaporated in vacuum at 60℃to afford the effective portion with VBE-1 and VBE-2 accounting for 50%.
引文
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