| Cinnamomum camphora is widely distributed in the south of the Yangtze River in China,such as Hunan,jiangxi and Fujian.As known that Cinnamomum Camphora belongs to a kind of evergreen Lauraceae,which is also a representative species of tropical and subtropical evergreen broad-leaved forest.The main chemical constituents of Cinnamomum Camphora were volatile oil,alkaloids,flavonoids,lignans,organic acids and Zi alcohol.Lignans have been widely studied as one of the hotpots because of its multiple biological activities,such as antioxidant,anti-tumor,regulate plasma cholesterol,protect the liver,anti-inflammatory,antiviral,antibacterial and other active functions.In order to make full development and utilization of Cinnamomum Camphora resources,the following work is focused on the the extraction,separation,purification process and antitumor effect in vitro of lignans extracted from Camphor leaves and the main findings are as follows.1.The content of lignans was determined by the method of chromotropic acid as chromogenic with camphora leaves as the raw materials,which uses schisantherin A as the reference substance.In order to optimize the hot refluxing extraction process of lignans,the method of response surface analysis with four factors and three levels was carried out by using the extraction rate of lignans as response value and the ethanol concentration,liquid-to-solid ratio,reflux times and temperature as response factor.The optimum extraction conditions of the lignans from camphora leaves as follows:concentration of ethanol was 87%,ratio of liquid-to-solid was 11:1 mL/g,reflux times were 3,the extraction temperature was 70℃ and the extraction rate of lignans was 43.39 mg/g.(The mass of the powder is 5.0 g.)2.The purification pocess of lignans alcohol-extraction from camphora leaves by macroporous resin as below:The adsorption rate and desorption rate of lignans in camphor leaves regard as the indexes,according to adsorption and desorption property of macroporous resins to lignans,D101,AB-8,X-5,NKA-9,HPD722 and HPD600 resins were compared to purifying lignans from camphora leaves and selecting AB-8 macroporous resin as purified material.The effect of concentration,flow rate and volume of sample on adsorption performance of macroporous resin and the effect of concentration,flow rate and dosage of eluent on desorption performance of AB-8 macroporous resin are investigated.The optimal purification process of lignans with AB-8 macroporous resins is studied by the orthogonal experiment.The purification parameters optimized by adsorption and desorption were determined as follows:The sample amount was 7 BV(bed volume)at 1.0 mL/min flow rate,the sample concentration was 2.12 mg/mL.After saturation adsorption,desorbing with 80%ethanol 8 BV at 2BV/h flow rate.The yield of lignans was 66.68%after concentrating and drying the elution,which was significantly better than before(22.34%).Lignans were further separated and purified and it was identified by ultraviolet spectrum,high performance liquid chromatography-mass spectrometry and high performance liquid chromatography and the purity of sesamin was 90.13%.3.Human hepatic cancer HepG2 cells were cultured in vitro,then those cells were co-cultured with different concentrations of sesamin(40,60,80,100,120 μg/mL)extracting from camphor leaves and the effects of sesamin on the viability,morphology,migration,morphological changes of apoptosis and the apoptosis rate of human hepatoma HepG2 cells were determined.The results indicated that sesamin in the range of 40～120 μg/mL could inhibit the proliferation of HepG2 cells in a dose and time dependent manner(p<0.01),displaying IC50 values at 100 μg/mL.The observation of inverted microscope found that cell morphology was crinkled and round,the intercellular connectivity was loose and the capacity of adherence was declined.Wound healing test showed that the rate of migration were 34.26±4.57%and 25.25±2.13%when 40 μg/mL and 80 μg/mL sesamin were cultivated with HepG2 cells for 48 h respectively,the migration capacity of cells was significantly decreased(p<0.001)when compared with the control group(71.35±4.92%).Hoechst 33258 staining appeared bright blue apoptotic bodies.Flow cytometry detection showed that the rate of apoptosis of HepG2 cells was increased with the increasing of concentration of sesamin from 40 to 120 μg/mL.The apoptosis rate are 27.1%,36.1%,41.6%,48.4%,60.2%,respectively.4.The glassy carbon electrode was modified by chitosan and carboxylated multi-walled carbon nanotubes and the effect of sesamin on the activity of human hepatoma HepG2 cells was detected by the changes of voltammetric behavior of the cells.The results showed that the response of modified electrode with 4 μL CS-MWCNTs was better and there was no obvious toxicity effect on cells.The growth of human hepatoma HepG2 cells treated with different concentrations of sesamin(40,60,80,100,120 μg/mL)were measured by the change of current signal.The inhibitory rate of sesamin on cells were 24.06%,35.28%,43.38%,51.67%,67.50%,which was consistent with the results of traditional methods that inhibited the cells in a concentration dependent manner.This method offers a quick and simple way to detect the effect of sesamin on the apoptosis of human hepatoma HepG2 cells. |
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