Study On The Extration Of The Isothiocyanate From Broccolini Seed And Its Anticancer Activity

Cabbage mustard and broccoli are both acknowledged as owning cancer fighting properties, the broccolini is a hybrid of a broccoli and cabbage mustard and it contains a large percentage of cancer fighting substance. Isothiocyanates are the product of glucosinolates and have been shown to play an improtant role in the anticancer activity of brassicaceous vegetable. In this pape, the hydrolysis condition of Glucosinolates, the extraction and identification of isothiocyanates from broccolini seed, especially the anticancer activity and its mechanism of isothiocyanates were studied. The main contents and results were as follows:1. Separation and qualitative analysis of Glucosinolates in broccolini seed, cabbage mustard seed and broccoli seedWe abtained the different content and components of glucosinolates in these three seeds by using the palladium chloride analytical method, the results are shown as follows: broccolixi seed 39.959μmol/g , broccoli seed 37.771μmol/g, cabbage mustard seed 37.246μmol/g.Analysing by HPLC-MS–MS method, the kinds of glucosinolates in these three different seeds were six, eight and four.2. Extraction, identifation of isothiocyanates and the optimized hydrolysis condition of Glucosinolates in broccolini seedThe effects of defatting treatments, freeze drying on the extracting of isothiocyanates were studied. In order to have maximum ITCs content, the optimized extraction processes were as follows: the broccoli seed were smashed. Then using water to wet the seed meal and left to autohydrolysis at room temperature for 8 hours. Following autohydrolysis, the meal was extracted with dichloromethane for 3 times. The crude dichloromethane extract was filter and concentrated in vacuo on a rotatry cvaporator (30℃). The concentrate was washed with a ration of normal hexane and centrifugated for the quantitative analysis. The effects of time, pH, and temperature on the hydrolysis of glucoraphanin were determinated. The optimum hydrolytic conditions of Glucosinolates were: 8 hours; pH=7.0; 25℃. Five categories of isothiocyanates were identified through GC-MS analysis.3. The anticancer effect of isothiocyanates was studiedThe MTT assay results showed that the IC50 for SW-480, A549, OVCAR-3 were 77.72, 81.94, 78.6μg/ml, respectively. In vitro treatment of the SW-480 cells as a representative cell line with ITCs (10~120μg/ml), typical signs for apoptosis in a dose-dependent manner were observed by microscope. Compared with the control group, cell morphology further changed into rather irregular, crumb-like structures, nuclear fragmentation occurred, and apoptotic bodies appeared by FM, and the apoptosis rate in SW-480 cells was found to be 1.94%,42.60%,49.08%,52.06% and 76.43% using flow cytometry (FCM). Together, these results suggested that ITCs could induce SW-480 cells apoptosis and inducing necrotic cell death.4. The anticancer action mechanism of isothiocyanates was studiedIn order to determine the different protein levels in SW-480 cells following recombinant ITCS treatment, a proteomic analyses (2-DE, image analysis, and mass spectrometry with protein database interrogation strategies) was introduced. Compared with the proteomic profiles of control, 107 protein spots (75 upregulated and 32 downregulated proteins) whose contents were changed in response to ITCs administration, fifteen of protein spots were unambiguously identified.