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Extraction method and application of anti-tumor active component from tobacco

An anti-tumor activity and extraction method technology, which is applied in the field of extraction of tobacco anti-tumor active components, can solve the problems of volatile, flammable, toxic and the like, and achieve the effects of improving the extraction content, simple preparation method and broad application prospect.

Inactive Publication Date: 2019-07-12
GUIZHOU TOBACCO SCI RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, low-polarity solvents such as petroleum ether, n-hexane, medium-polarity solvents acetone, and methylene chloride are mostly volatile, flammable, and toxic solvents, which have high requirements for industrial production equipment and personnel, and the obtained tobacco activity The content of components is small, which greatly limits the further research and application of tobacco active components

Method used

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  • Extraction method and application of anti-tumor active component from tobacco
  • Extraction method and application of anti-tumor active component from tobacco
  • Extraction method and application of anti-tumor active component from tobacco

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] 本发明提供了一种烟草活性组分的提取、分离方法,包括以下步骤:

[0059] (1)预处理。

[0060] 称取30Kg烟草原料,放置在烘箱中进行烘干,烘干温度50℃,烘制时间4h,烘至烟草含水量小于5%取出;取烘干处理后的烟草原料,使用50目筛网的粉碎机粉碎后,并放入200目筛网的粉碎机中进行超微粉碎处理,得到烟草超微粉。

[0061] (2)常温水提。

[0062] 称取30Kg烟草超微粉加入到200L提取罐中,加入100Kg纯化水,常温搅拌提取0.5h;搅拌提取结束后转移至离心瓶中,8℃离心0.5h,转速为3000rpm / min;收集离心处理后的沉淀物。

[0063] (3)高温水提。

[0064] 称取步骤(2)中的沉淀物25Kg,加入到100L浓缩提取罐中;加入60Kg纯化水,打开浓缩提取罐加热开关;加热到90℃后,在90℃的温度下浓缩提取0.5h;向浓缩提取罐夹套中通入循环水,以对浓缩提取液进行降温处理,使其降温至室温;将浓缩提取液转移至离心瓶中进行离心处理,8℃离心0.5h,转速为3000rpm / min;并收集离心处理后的沉淀物。

[0065] (4)甲醇提取。

[0066] 将步骤(3)中得到的沉淀物加入到不锈钢桶中,每桶10Kg;每桶中加入15Kg的甲醇,甲醇的质量分数为92%,在0℃的提取温度下浸渍提取操作;其中,提取时间为12h,每隔0.5h对桶内物质搅拌一次;将浸渍提取物置于离心瓶中3℃离心1.5h,转速为3000rpm / min;收集离心处理后的上清,将收集的上清转移至50L旋蒸中60℃浓缩处理,在浓缩完成后转移至烘盘中,60℃烘制成浸膏,并收集离心处理后的沉淀物。

[0067] (5)丙酮提取。

[0068] 将步骤(4)中得到的沉淀物加入到不锈钢桶中,每桶10Kg;每桶中加入15Kg的丙酮,丙酮的质量分数为92%,在0℃的提取温度下浸渍提取操作;其中,提取时间为12h,每隔0.5h对桶内物质搅拌一次;将浸渍提取物置于离心瓶中,3℃离心1.5h,转速为3000rpm / min;收集离心处理后的上清,将收集的上清转移至50L旋蒸中60℃浓缩处理,在浓缩完成后转移至烘盘中,60℃烘制成浸膏,得到烟草丙酮提取物(TA),即烟草活性成分。

[0069] (6)烟草...

Embodiment 2

[0075] 实施例2:抑制人肺肿瘤A549细胞生长的试验

[0076] 细胞培养:在5%CO 2 ,37℃,饱和湿度下,用RPMI 1640(10%FBS+1%PS)培养基培养人肺肿瘤A549细胞,选取对数期生长的细胞作为实验细胞。细胞计数后用培养基稀释为一定浓度的细胞悬液。

[0077] 细胞生长情况监测:将细胞实时监测仪放入37℃、5%CO 2 ,饱和湿度培养箱中。取8孔板,每孔加入150mL RPMI 1640培养基,放入细胞实时监测仪中走基线,走完基线后取出八孔板,每孔加入稀好的A549细胞悬液345mL,至每孔细胞数约2×10 4 个,静置30min,在倒置显微镜下观察细胞是否均匀。每孔加入5mL稀释好的药物(实施例1得到的烟草活性组分TA-150-12-7)至终浓度为50μg / mL,含1%DMSO的培养基作为空白对照组,放入细胞实时监测仪中检测,检测完毕时拍照记录。

[0078] 空白对照孔:不加药物,加1%DMSO;

[0079] 实验孔:加药物

[0080] Experimental results such as Figure 4 所示:上方线为空白对照孔、下方线为实验孔。

[0081] 根据A549细胞的增殖生长曲线,说明本发明烟草活性组分TA-150-12-7对A549细胞生长抑制作用。

Embodiment 3

[0082] 实施例3:抑制人肝癌Hep-G2细胞生长的试验

[0083] 细胞培养:在5%CO2,37℃,饱和湿度下,用DMEM(10%FBS、1%PS)培养基培养人肺癌Hep-G2细胞,选取生长状态良好的细胞作为实验细胞。细胞计数后用培养基稀释为一定浓度的细胞悬液。

[0084] 细胞生长情况监测:将细胞实时监测仪放入5%CO 2 ,37℃饱和湿度培养箱中。取八孔板,每孔加入150mLDMEM培养基,放入细胞实时监测仪中走基线,走完基线后取出八孔板,每孔加入稀释好的Hep-G2细胞悬液345mL,至每孔细胞数约4×10 4 个,静置30min,在倒置显微镜下观察细胞是否均匀。每孔加入5mL稀释好的药物(实施例1得到的烟草活性组分TA-150-12-7)至终浓度为50μg / mL,含1%DMSO的培养基作为空白对照组,放入细胞实时监测仪中检测,检测完毕时拍照记录。

[0085] 空白对照孔:不加药物,加入1%DMSO;

[0086] 实验孔:加药物

[0087] Experimental results such as Figure 5 所示:上方线为空白对照孔、下方线为实验孔。

[0088] 根据Hep-G2细胞的增殖生长曲线,说明本发明烟草活性组分TA-150-12-7对Hep-G2细胞生长抑制作用。

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Abstract

The invention provides an extraction method and application of an anti-tumor active component from tobacco. The method includes the following steps that tobacco raw materials are subjected to ultrafine crushing firstly and then subjected to normal-temperature extraction, high-temperature extraction, methanol extraction and acetone extraction in sequence, wherein a high polar solvent, purified water, methanol and acetone are used as extracting solvents in sequence; a concentrated extractum of tobacco acetone extract is taken and subjected to chromatographic separation through a preparative liquid phase, obtained components are screened by a cell activity test to obtain components higher in activity, the obtained components higher in activity are separated and screened by a cell activity test again so as to obtain the component highest in activity, and the component highest in activity refers to the anti-tumor active component. The method has good repeatability and the obtained anti-tumor active component can be used for preparing anti-tumor drugs.

Description

technical field [0001] The invention relates to the technical field of tobacco product processing and extraction, in particular to an extraction method and application of tobacco antitumor active components. Background technique [0002] Tobacco, also known as fairy grass, is a traditional Chinese medicinal material. Its inorganic components include water and mineral elements; organic components mainly include sugars, alkaloids, heterocyclics, pigments, phenols, terpenes, organic acids, lipids Substances, alcohols, aldehydes and ketones, etc. Among them, the organic components in tobacco have various functions such as antibacterial, antiviral, antitumor, antioxidant activity, and scavenging free radicals in the body. [0003] At present, in order to realize the extraction of active components of tobacco, supercritical extraction, Soxhlet extraction, and ultrasonic extraction are generally used. Absolute ethanol, n-hexane, acetone, ethyl acetate, petroleum ether, and dichlo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K36/81A61P35/00
CPCA61K36/81A61P35/00
Inventor 苏贤坤赵瑞娟耿召良曹军陈梅张时山
Owner GUIZHOU TOBACCO SCI RES INST
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