Studies On Characteristic Flavonoids In Bamboo Leaves

This study at first investigated in vivo the metabolic behavior of four flavone C-glycosides, characteristic flavonoids in bamboo leaves, when experimental rats took up the antioxidant of bamboo leaves (AOB). Based on an effective HPLC inspective method, the research of metabolic kinetics of AOB was conducted. Some important information including absorption form and site, metabolic pathway and concentration change of objective compounds in different time phases were obtained, in order to supply much more scientific evidence on AOB for its certification of GRAS, generally regard as safe. Then, two kinds of characteristic flavonoids, i.e. four flavone C-glycosides (orientin, homoorientin, vitexin and isovitexin) and a multiple-methoxyl flavone (tricin) were isolated and obtained from AOB mainly using by the preparative HPLC method. Subsequently, the antioxidant activities of homoorientin and tricin were evaluated using by Rancimat, DPPH, FRAP and chemiluminescence methods, taking luteolin-7-O-glucoside, luteolin and apigenin as comparative compounds. The relationship between antioxidant capacity and chemical structure, such as aglycones and glycosides, C-glycosides and O-glycosides, methoxylation and non-methoxylation, were studied for elucidating the dose-dependent and/or structure-dependent relationships of flavonoids. Furthermore, the effect of bamboo-leaf-flavonoids (BLF) on the inhibition of potential dependent Ca²⁺channel (PDC) and receptor operated Ca²⁺channel (ROC) was studied via the hyperpotassium-induced and phenylephrine (PE)-induced pre-constriction pathways in a perfusion model of isolated thoracic aortic rings from rabbits. This study also investigated the effects of BLF on cumulative concentration-response curves with KC1 and PE-induced vasoconstriction from intracellular Ca²⁺ release and extracellular Ca²⁺influx; effects on the pre-treatment of the hyperpotassium and guanylate cyclase (sGC) inhibitor, i.e. methylene blue (MB); effects on the pre-treatment of the cyclooxygenase inhibitor, i.e. indomethacin; and effects on the pre-treatment of the nitric oxide synthase (NOS) inhibitor, i.e. L-NAME. The present study further observed the synergistic effects of BLF on the NO production with and without the presence of extracellular Ca²⁺using the Ca²⁺ antagonist of nifedipine as a positive control, and demonstrated their vasodilatation effects on the thoracic aortic rings isolated from rabbits and relative mechanism. Results were summarized as follows:a) Based on our established HPLC method to effectively determine the contents of flavone C-glycosides in various biological samples, the absorption form and site, metabolic pathway and level change of objective compounds were obtained via the inspection of organs and excreta extracts after the oral administration of AOB in rats. It has been primary elucidated that four flavone C-glycosides were absorbed and metabolized via the gastrointestinal pathway, instead of the blood and urine pathway, which was significant different from the routine metabolism pathway of flavone O-glycosides.b) Totally 6.5 g of flavone C-glycoside rich fraction and 174 g of tricin-rich fraction was obtained from 5 L of AOB concentrated solution via the AB-8 macroporous adsorption resin based column chromatography. Then, flavone C-glycosides and tricin were isolated from the above fractions by the preparative HPLC method and further purified by the drowning-out crystallization and membrane dialysis methods. Totally 49 mg of orientin,142 mg of homoorientin,15 mg of vitexin,62 mg of isovitexin and 3.09 g of tricin were finally obtained all with a purity of more than 90%.c) The antioxidant activities of selected flavonoid samples evaluated by Rancimat method with the addition level range of 0.02-0.05%were ordered as follows:luteolin> tricin> luteolin-7-O-glucoside> apigenin> homoorientin. The DPPH'scavenging ability was ordered as follows:luteolin≈homoorientin> luteolin-7-O-glucoside >> tricin >> apigenin. Relative IC₅0 value range is 0.089-105.328 mmol/L. The ferric reducing ability with the addition level range of 0.02-0.1 mmol/L was ordered as follows:homoorientin> luteolin≈luteolin-7-O-glucoside> tricin> apigenin. The O₂ scavenging ability was ordered as follows:tricin> luteolin-7-O-glucoside> homoorientin> luteolin >> apigenin. Relative IC₅0 value range is 0.05-10.59 mmol/L. The'OH scavenging ability was ordered as follows:luteolin> luteolin-7-O-glucoside > tricin> homoorientin >> apigenin. Relative IC₅0 value range is 0.24-1691.03 mmol/L. The H₂O₂ scavenging ability was ordered as follows:tricin> luteolin-7-O-glucoside> luteolin> homoorientin >> apigenin. Relative IC₅0 value range is 0.37-130.58 mmol/L.d) The effect of BLF, which is a standard preparation with total flavonoid content above 50%, on the vascular rings was observed in a perfusion model of isolated thoracic aortic rings from rabbits. Results showed that there is a concentration-dependent relationship of the vasodilatation effect of BLF on PE-and hyperpotassium-induced pre-constricted vascular rings, which indicated that BLF may inhibit intracellular Ca²⁺release or extracellular Ca²⁺influx induced by hyperpotassium and PE, respectively. And so it presented a vasodilatation effect via reducing the intracellular Ca²⁺levels. Such effect was presented as the non-endothelium related form, which indicated that BLF exerted the effect directly via vascular smooth muscle cells. Comparison study of NO production with and without the presence-of extracellular Ca²⁺in the human umbilical vein endothelium cell line demonstrated that extracellular Ca²⁺only enhanced its NO production. The vasodilatation effect of BLF was significantly reduced after the addition of L-NAME and MB, which indicated their mechanism of action via the NO-sGC-cGMP pathway. Furthermore, BLF showed a synergistic effect with the presence of nifedipine, a positive control. After the pretreatment of indomethacin at 10μmol/L, a significant vasodilatation effect on PE pretreated isolated vascular rings was observed when the addition level of BLF was above 3000μg/mL (p<0.05,p<0.01), and that indicated the vasodilatation effect of BLF in a high level may be related to cyclooxygenase via the production of prostaglandin.