In Vitro Antioxidant And Antitumor Activities Of The Different Extracts From Shell Of Camellia Oleifera Abel

Camellia oleifera Abel., which belongs to Theaceae, is the special species of the edible oil trees in China. Camellia oleifera Abel, olive oil, palm and coconut were regarded as the four kinds of major woody oil plants in the world. Camellia oleifera seed is used as a common traditional Chinese medicine for the long time in China. Camellia Oleifera shells, as the by-products of the pressed oil from Camellia oleifera seed, are always discarded or used as fertilizer. Recently, many studies have also revealed their chemical properties and biological activities, such as antitumor, immunostimulation, and antioxidation.In this study, the 70% ethanol extract of Camellia Oleifera shells were separated into ethyl acetate extract, n-butanol extract, chloroforms extract and aqueous extract by systematic solvent extraction, We investigated that the effect of the different solvent extracts from the shells of Camellia Oleifera Abel on the proliferation of the hepatocellular carcinoma cell line(HepG2), Moreover,we clarified the mechanism of apoptosis induced by the ethyl acetate extract from the shells of Camellia Oleifera Abel, which will provide the experimental basis for the clinical application of the extracts from the shells of Camellia Oleifera.Total phenolics and total flavonoids were measured by the methods of Folin-Ciocalteu and aluminium chloride, respectively. And, the antioxidant activity was tested by DPPH, and hydroxyl assays in vitro. Besides, the proliferative activity of the different human cancer cell lines was detected by CCK-8 assay. Apoptosis of HepG2 cells induced by ethyl acetate extract at the different concentration was detected by FCM using apoptosis kit (the double staining of PI and Annexin V-FITC) Cell cycle of HepG2 cells was detected by FCM using PI staining The mKNA levels of Survivn, Bcl-2, Box and caspase-3 of HepG2 were tested by real-time PCR.Total phenolic content of extracts(Ethyl Acetate, N-butanol, Chloroforms, Aqueous) were (79.25±4.552)mg/g, (51.81±3.651)mg/g, (2.762±0.523)mg/g, (19.73±1.191)mg/g respectively, and total flavonoid content were (26.73±0.613)mg/g, (15.63±1.126)mg/g, (13.16±0.672)mg/g, (5.734±0.761mg/g, respectively. The antioxidant activities of these extracts in a range of concentrations from 15.625 to 250μg/mL increased in dose-dependent manner., and the antiproliferative activities of these extract in a range of concentrations from 62.5 to 250μg/mL increased in dose-dependent manner. Besides, the ethyl acetate extract induced the percentage of the early apoptosis cells (right lower quadrant), and the late apoptosis and necrosis cells (upper right quadrant) in the dose-dependent manner. The cell cycle was tested by PI staining, and the result showed the ethyl acetate extract could interfere with the cell cycle process of HepG2 cell, increase the number of cells in G0/G1 phase, and prevent cancer cells to enter into S phase. Moreover, the mRNA levels of the molecules related the apotosis were tested by the real-time PCR, and the results showed that the ethyl acetate extract could inhibit the expression of Survivin and increase the expression of Caspase-3, decrease the expression of Bcl-2, and increase the expression of Box. In addition, the correlation analysis showed that the expression of Survivin and Caspase-3 was negative correlation significantly (P< 0.01), the expression of Caspase-3 and Bax/Bcl-2 was positively correlated significantly (P<0.001) and there was no statistical difference in expression of Survivin and Bax/Bcl-2.In summary,the extract from the shell of Camellia oleifera Abel had antioxidant and anticancer activity, and ethyl acetate extraction has significant extraction efficiency. It is worth developing antioxidant and anticancer products by adding the extract from the shell of Camellia oleifera Abel in the future.