Biological Activities Of Broccoli Waste And Its Active Ingredients

Broccoli(Brassica oleracea var.italica),a popular floral green vegetable,was widely cultivated in China due to its flavor,taste and high nutritional value.However,the leaves and stalks of broccoli,occupying more than the 70%of the broccoli plant,generate a vast amount of bio-waste after harvest.According to an statistical analysis,significant amounts of broccoli wastes were disposed as rubbish in China.Therefore,the reutilization of this vegetable by-products and its exploitation as potential functional food ingredients are important.The results obtained from this study will provide a scientific foundation for developing broccoli waste-based functional foods and its applications.Firstly,the study was designed to examine total phenolics,flavonoid,crude polysaccharides,lipids,Vc,crude protein,minerals contents and antioxidant properties of four varieties of broccoli bio-waste from Zhejiang Province.Based on the results,it showed that there was a wide variation of bioactive compounds contents.Notably,the'NaiHanYouXiu' variety had the higher content of crude polysaccharides,and the'YanXiu' variety had the higher content of total phenolics.Therefore,the two varietys were chosen for further study.The 70%EtOH extract of 'YanXiu' variety of vegetable by-products was fractionated sequentially by petroleum ether,chloroform,ethyl acetate and n-butyl alcohol.The antioxidant,?-glycosidase and tyrosinase inhibitory activities of ethanolic crude extract(EF)and its five different solvent sub-fractions(namely,petroleum ether fraction(PF),chloroform fraction(CF),ethyl acetate fraction(AF),n-butyl alcohol fraction(BF)and the water fraction(WF))from ethanolic crude extract were investigated.Moerover,the total phenolics and flavonoid contents of all fractions were evaluated.Our results revealed that all the fractions and EF have some certain extent antioxidant activities.The AF demonstrated the highest total phenolic content and total flavonoids content,accompanied with the highest antioxidant,a-glycosidase and tyrosinase inhibitory activities compared to other fractions.Phytochemical investigation of the AF led to isolation of one known compound(cholesterol).The a-glycosidase inhibitory activity of the cholesterol was determined.It was observed that cholesterol exhibited good inhibitory effect on a-glycosidase.The vegetable by-products of 'NaiHanYouXiu' variety were used as the experimental materials.Three independent variables,including extraction temperature,ratio of water to raw material,and extraction time were screened as the significant factor affected the extraction yield of polysaccharides from broccoli byproducts(PBB)by Plackett-Burman design.Response surface methodology based on a Box-Behnken design was applied to optimize the extraction of PBB.The optimal extraction conditions with a yield of 16.64%for extraction of PBB were determined as extraction temperature 82 ?,extraction time 212 min and ratio of water to raw material 26 mL/g.Under the determined conditions,Five verification experiments were performed and the PBB yield was(16.59±0.04)%,which was not significant from predicted result.Therefore,the results suggest that the model was accurate and adequate for the extraction of PBB.And,the curde PBB was prepared using the optimal extraction conditions,and the curde PBB was separated by deproteinization and decolorization to obtain the PBB-1.The PBB-lwas purified using a DEAE Fast Flow column,eluting with a stepwise gradient of NaCl(0,0.1 M,0.3 M,0.5 M)to afford fractions PBB-1a,PBB-lb,PBB-lc,PBB-1d,respectively.As the yields of PBB-la and PBB-lc were high,they were chosen for further study.The carbohydrate contents,protein contents,uronic acid contents,sulfate radical contents,total phenolics contents,FT-IR spectrum of the PBB-1,PBB-la and PBB-lc were also investigated.Notably,the PBB-lc obtained from 0.3 M NaCl eluent had the higher content of carbohydrate,protein,uronic acid,as well as sulfate radical.Evaluation of the antioxidant and hypoglycemic activities in vitro suggested that PBB-lc exhibited better bioactivities than PBB-land PBB-1a.Furthmore,the PBB-1c was further subjected to a Sephadex G-100 chromatography to generate only one single peak(PBB-1c-1).The anti-cancer cell proliferation activity of PBB-1c-1 were determined by MTT assay.One human hepatoma cell line was chosen,that was HepG2.PBB-1c-1 exhibited significant anti-cancer cell proliferation activity,and a dose-dependent behaviour was observed for proliferation inhibition of the line.At the same time,nucleus shrinkage or broken were found in cells incubated with PBB-1c-1 through fluorescent microscope.These results showed that PBB-1c-1 could induce apoptosis in human hepatoma cell HepG2.