| Cinnamomum longepaniculatum (Gamble) N. Chao is an endemic Lauraceae (Lauraceae) Cinnamomum (Cinnamomum) plant species in China, has been listed as national key protected wild plants Ⅱ level, which is mainly distributed in Sichuan Yibin, in Hubei,Hunan, Shaanxi,Yunnan, Jiangxi, Guangxi, Guangdong and other places. Cinnamomum longepaniculatum leaves contains a lot of essential oil (3.8～4.5%),which is an important raw material of perfume, medicine, industrial consumer goods and Products of the chemical industry. In order to take full advantage of cinnamomum longepaniculatum in resources, we studied the anti-hepatoma cell activity of the extraction from leaves of cinnamomum longepaniculatum in vitro, and discussed the mechanism of anti-hepatocellular carcinoma of the effective component. The main result are as follows:1Preparation of extracts from leaves of cinnamomum longepaniculatum:Systematic solvent methode was used to extract crude extracts from Cinnamomum longepaniculatum leaves. The essential oil was extracted by steam distillation. The residue were extracted by ethanol and fermentation extraction by petroleum,ethyl acetate and N-butanol. The active part of the present invention was different polar parts obtained by extracting and separating Cinnamomum longepaniculatum leaves, for example, the ethanol extract, petroleum extract, ethyl acetate extract, N-butanol extract and water extract. Five monomeric substances was separated by rectificating from the essential oil of cinnamomum longepaniculatum, such as1,8-cineole, safrole, γ-terpilenol,α-terpinene, terpinene-4-alcohol.2Anticancer activity of the extraction from Cinnamomum longepaniculatum leaves against human BEL-7402cell in vitro:By MTT fast staining method to study the growth inhibition rate of the extraction from Cinnamomum longepaniculatum leaves on human BEL-7402cells. In result, when the ethanol extract, petroleum extract, ethyl acetate extract, N-butanol extract, the essential oil,1,8-cineole,safrole, y-terpilenol, a-terpinene and terpinene-4-alcohol affected on BEL-7402cell, the IC50is>1280μg/mL,>1280μg/mL,1122.4μg/mL,>1280μg/mL,1047.6μg/mL,823.4μg/mL,313.7μg/mL,405.3μg/mL,773.6μg/mL, and964.5μg/mL. By soft agar cloning formation assay to study the effect of the extraction from Cinnamomum longepaniculatum leaves against human BEL-7402cells. In result, when the ethanol extract, petroleum extract, ethyl acetate extract, N-butanol extract, the essential oil,1,8-cineole,safrole, γ-terpilenol,-terpinene and terpinene-4-alcohol affected on BEL-7402cell, the IC50is>1280μg/mL,>1280μg/mL,1123.4μg/mL,>1280μg/mL,1047.6μg/mL,823.4μg/mL,313.7μg/mL,405.3μg/mL,773.6μg/mLand964.5μg/mL. The research results show that:among the extractions from Cinnamomum longepaniculatum leaves, the effect of safrole and γ-terpilenol against BEL-7402cell is the best; the second is the essential oil and the ethyl acetate extract; the effect of the ethanol extract, petroleum extract,1,8-cineole, α-terpinene and terpinene-4-alcohol against BEL-7402cell is poor; the N-butanol extract almost has no inhibition on BEL-7402cell.3The mechanism of safrole angainst human hepatoma BEL-7402cells in vitro:By light microscopy assay and transmission electron microscopy assay to study the morphological changes of safrole on human hepatoma BEL-7402cells, flow cytometric analysis was used to observed the distribution of cell cycles and apoptosis precentage, and by DNA fragmentation assay to study the fragmentation of DNA. The results show that:After treated with safrole, BEL-7402cell showed typical apoptosis morphological changes, such as cell shrinkage, nuclear condensation. Whit treated time prolong, the nuclear fragment which have been condensation. Observed by transmission electron microscopy, the nucleus electron density is reduced, and the nucleolus appeared vacuole. At the same time, the organelle have been changed, such as swelling of mitochondria, deformation and vacuolization of mitochondria, and apoptotic bodies appeared. Sfrole can arrest cell cycle at the G0/G1phase and S phase, the number of G2was reduced. In this way, safrole can block the normal cell proliferation.In another way, safrole can induce human hepatoma BEL-7402cells to occur apoptosis.Compared with the control group, the apoptpsis rate of the experiment group was significantly increaed, and with the treated time prolong, the cell apopotosis rate gradually increases. After treated with safrole, DNA of cells would occur fragmentation.By gel electrophoresis, there are some striped tail and typical "DNA-ladder". The results show that: safrole can change the normal physiological activity of the human hepatoma cell by various ways.4The mechanism of γ-terpilenol angainst human hepatoma BEL-7402cells in vitro:By light microscopy assay and transmission electron microscopy assay to study the morphological changes of γ-terpilenol on human hepatoma BEL-7402cells, flow cytometric analysis was used to observed the distribution of cell cycles and apoptosis precentage, and by DNA fragmentation assay to study the fragmentation of DNA. The results show that:After treated with γ-terpilenol, BEL-7402cell showed typical apoptosis morphological changes, such as cell shrinkage, nuclear condensation. Whit treated time prolong, the nuclear fragment which have been condensation. Observed by transmission electron microscopy, not only find the morphological changes of nuclear, but also the organelle have been changed. Such as swelling of mitochondria, deformation and vacuolization of mitochondria, and apoptotic bodies appeared0. γ-terpilenol can arrest cell cycle at the G0/G1phase and S phase, the number of G2was reduced. In this way, γ-terpilenol can block the normal cell proliferation. In another way, γ-terpilenol can induce human hepatoma BEL-7402cells to occur apoptosis.Compared with the control group, the apoptpsis rate of the experiment group was significantly increaed, and with the treated time prolong, the cell apopotosis rate gradually increases. After treated with γ-terpilenol, DNA of cells would occur fragmentation. DNA fragmentation caused by γ-terpilenol have time-dependent manner. Before24h, DNA almost not occur dissociation; with treated time prolong, there are some striped tail and typical "DNA-ladder". By gel electrophoresis, there are some striped tail and different bands appeared. The results show that: safrole can change the normal physiological activity of the human hepatoma cell by various ways. |
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