Biological Evaluation Of Glucosinolate And Proanthocyanidin From Broccoli Leaves

Five experiments were conducted to define the optimum drying temperature anddrying duration of broccoli leaves and evaluate the biological value of the glucosinolate andprocyanidins extracted from broccoli leaves using both in vitro and in vivo techniques.Experiment1was conducted to assess the effect of variation in drying temperatureand duration on the content of crude protein, moisture content, procyanidins, andglucosinolates of broccoli leaves. The experiment is single-factor completely randomdesign, with the drying temperature of50℃,65℃,80℃,105℃and the drying time of1h,2h,4h,8h,12h,16h,24h and36h. Each treatment had6repeats, and in each repeat1kg of fresh broccoli leaves were oven-dried, and afterwards all leaves were vacuumfreeze-dried. In the control group, fresh leaves were directly vacum freeze-dried. Crudeprotein, moisture content, procyanidins, and glucosinolates were measured. The resultsshowed that:(1) to achieve less than17%of the moisture content of the end product, ittook36h to dry the leaves at50℃,16h at65℃, at least8h at80℃, and at least4h at105℃.(2) The drying temperature and duration have no effect on the crude proteincontent in broccoli leaf.(3)The content of procyanidins in broccoli leaf was maximum at50℃,65℃(8h,12h), and80℃(4h,8h).(4) With the increase of drying temperature(50℃,65℃and65℃) and duration, the content of glucosinolates remained stable.However, the glucosinolates content in the treatment at105℃obviously increased, and asignificant difference was shown compared with the control group (P <0.05). It isconclused that the drying temperature at80℃for8hours can be the best condition formaximum retention of crude protein, procyanidins and glucosinolates in the end-product.Experiment2was to test the sensitivity and minimum bacteriostatic concentration(MIC) of the glucosinolate acid hydrolyzate (ITCS) in Broccoli leaf on Salmonella, E.coli,S.aureus and Bacillus subtilis using both the agar plate punching and tube double dilutionmethods. Antibacterial effects were also observed in mice infected with Staphylococcusaureus and Salmonella in vivo. The results showed:(1) with dilution of ITCS, antibacterialeffects reduced gradually. The rank for antibacterial effects at the same concentration was Bacillus subtilis> Staphylococcus aureus> Salmonella typhi> Escherichia coli.(2) TheMIC of ITCS for E. coli, Salmonella, Staphylococcus aureus and Bacillus subtilis was1/40(0.234umol/mL),1/40(0.234umol/mL),1/80(0.117umol/ml) and1/80(0.117umol/mL),respectively.(3) The ITCS had certain effects on protecting mice infected withStaphylococcus aureus and Salmonella, and better protection obtained for resistingStaphylococcus aureus.Experiment3was conducted to study the in vitro inhibitory effects of ITCS oncancer of MKN–45and SW620. MTT assay and flow cytometry were carried out toassess the apoptosis effect of different concentrations of ITCS on cancer cells of MKN–45and SW620. The results showed:(1) after72h of incubation at different concentrations ofITCS in SW620MKN–45cells, the proliferation of cancer cells was subdued, and showeda significant dose-dependent effect;(2) Cancer cells took changes in their form andquantity, featuring the typical apoptosis morphology.(3)With the ITCS concentrationincreasing, cancer cell apoptosis rate also increased gradually. Cancer cells apoptosis ratein each test group was higher than that in normal control group and positive control groupfor cisplatin. It was concluded that ITCS has a good ability to induce the cell apoptosis ofMKN-45and SW620.Experiment4was to investigate the protective effect of ITCS on the mice against thepathogenic bacteria infection. Different concentrations of ITCS were perfused into thestomach of the model mice. Three days later, the mice were injected in their abdominalcavity0.5mL of MLD of staphylococcus aureus fluid and salmonella typhi bacteria fluid togenerate infection. Then, the drugs were continuously given to the7thd, and observed themortality rate of each group within the7d. The curative effect of drugs on animals wasevaluated based on its mortality rate. The results showed that the ITCS has certainprotective effect on mice infected with staphylococcus aureus and salmonella typhi. Theantibacterial effect against staphylococcus aureus was better than salmonella typhi in mice.The conclusion is drawn as follows: the ITCS has certain antibacterial effect in in vivo test.Experiment5was to study the effect of procyanidins extraction from broccoli leafon antioxidant activity, viscera index and growth performance of mice. Fourty healthymale Kunming mice were selected and randomly divided into5groups,8in each. Every day, mice in each group were continuously gavaged0.5mL procyanidins crude extractwith three different doses (100mg/kg,200mg/kg,400mg/kg), the VC control solution(100mg/kg) and distilled water as the normal control. After4weeks, SOD, MDA,GSH-PX, CAT of blood plasma and liver homogenate, organ indexes and growthperformance (body weight, feed intake) were measured. It found that (1) procyanidins frombroccoli leaf powder could significantly improve the activity of SOD, MDA, GSH-PX andCAT, and reduce the content of MDA.(2) There was no significant difference in organindexes of mice among treatment groups,(3) There was no significant change in growthperformance (body weight, feed intake) of mice among treatment groups.Thusprocyanidins from broccoli leaf has a significant antioxidant activity in mice, and not toxicto mice.