Study On Separation Of Hepatoprotective Components In Rape Bee Pollen And Its Mechanisms Of Action

The bee pollen was one of the most nutritional food resources in the world. Rapebee pollen which is above300ton every year is the most productive bee pollen inChina. It has much nutrional components, including protein, polysaccharides, aminoacids, flavonoids compounds, and lots of bioactivity such as anti-tumor activity andhepatoprotective effects which also could treat prostatitis and improve immunity. Thefunctional components of rape bee pollen have became one of research emphasis.As the research object, rape bee pollen was was separated and purifiedsystematically. After cytological screening, mechanism of hepatoprotectivecomponents’ effect was explored which provided reference for the exploitation ofhepatoprotective components in rape bee pollen.1. The separation and purification of hepatoprotective components in rape beepollen. Solvent extraction, reversed phase column chromatography and HPLC wereused as the separation method. Finally four flavonoides and a mixture whichcontained component P1and component P2were isolated from rape bee pollen. Afterelucidated by MS and NMR, the four flavonoids （P2^P5） were identified asquercetin-3-O-β-D-glucosyl-（2→l）-β-glucoside（QMP）,kaemferol-3,4’-di-O-β-D-glucoside（KMG）,sexangularetin-3-O-ylβ-D-sophoroside（SMP）,kaemferol-3-O-β-D-glucosyl-（2→l）-β-D-glucoside（KMP）.SMP is isolated from rape bee pollen for the first time.2. Cytological screening of hepatoprotective components in rape bee pollen. Avitro model of L-02hepatocytes injured by CCl₄was established. Cell survival ratemeasured by MTT assay was considered as the screening criterion in this experiment.The result showed that the appropriate concentration of CCl₄that could inducehepatocytes injury was determined as10mM. N-butanol extract group showed thehighest hepatoprotective effect among all the extracts. Component A2separated fromN-butanol extract showed the highest hepatoprotective effect that the cell survival rateof100μg/mL group and200μg/mL group was2.41times and2.03times as much as control group. The result of qualitative analysis of A2suggested that A2may beflavonoides. After a further purification of component A2and cytological screening,hepatoprotective mixture C1and hepatoprotective component QMP were isolatedfrom A2. The hepatoprotective activity of QMP and C1were positively correlatedwith their dosage. The cell survival rate of high-dose group（200μg/mL） of QMP andC1was2.64times and2.66times respectively as well as control group. ComponentKMP and KMG showed negative hepatoprotective activity in vitro, and componentSMP’s hepatoprotective activity will be tested.3. Structure-activity relationship between flavonoids and flavonoids glycosides.To investigate the hepatoprotective ability of QMP, kaempferol and quercetin, IC₅₀values were measured by intervening CCl₄-induced L-02with QMP, kaempferol andquercetin in various concentrations. Quercetin showed the highest hepatoprotectiveeffect whose IC₅₀value was70.28μM, and the IC₅₀values of kaempferol and QMPwas102.26μM and132.64μM, respectively. Hydroxy glycosylation caused that thehepatoprotective effect of kaempferol and quercetin was much higher than QMP,KMP and KMG. Quercetin showed the higher hepatoprotective ability thankaempferol, because the strong ionization ability of C3’-OH of quercetin.4. Mechanism of hepatoprotective components’ hepatoprotective effect. Themechanism of hepatoprotective effect of QMP, kaempferol and quercetin wasinvestigated. The result showed that both of the three components could reduce thelipid peroxide level caused by CCl₄. Compared with control group, the level of MDAand LDH leakage rate decreased, meanwhile the level of SOD improved, whichmeans that QMP, kaempferol and quercetin showed a strong hepatoprotective effecton CCl₄-induced liver injury.