Studies On The Anticancer Efficacy Material Base And Pharmacokinetics Of Ginkgo Biloba Leaf Falvonoids

Ginkgo biloba has been the oldest living tree on earth, dating back to the Paleozoic period, over 225 million years ago. Many of the commercial preparations of Ginkgo biloba extract (EGB 761) were formulated to contain 6% terpene trilactones (i.e., bilobalide and ginkgolides A, B, C, and J) and 24% flavonoid glycosides. In EGB 761 many different flavonoid glycosides occur most of them being derivatives of Quercetin, Kaempferol and Isorhamnetin. The aglycones themselves occur only in relatively low concentrations.EGB 761 has been used as a therapeutic agent for same cardiovascular and neurolgical disorders, expecially dementia. Many reports suggested general effects of EGB 761 on the vascular system, memory, cognition, anxiety, and gene regulation. Flavonoid compounds have been demonstrated the action could be affect by the number of OH group, methylation and glycosylation. The biological actions of aglycones of flavonoids are higher than glycosides of flavonoids. The bioavailability of flavonoid aglycones is higher than flavonoid glycosides. The Criteria of EGB 761 containing 5%-7% terpene trilactones, along with 22%-24% of flavonoids. But it isn't defined the concentration of flavonoid aglycones. In this study, we apply EGB 761 prepare three different ingredients samples: EGB 761 devoid of terpene trilactones (EGB 761-DT, mainly containing the flavonoid glycosides) sample; EGB 761 hydrolysate (EGB 761-H, mainly containing the aglycones of Quercetin, Kaempferol, Isorhamnetin and terpene trilactones) sample; EGB 761 devoid of terpene trilactones hydrolysate (EGB 761-DT-H, mainly containing the aglycones of Quercetin, Kaempferol and Isorhamnetin). We investigate the difference of flavonoid aglycones and flavonoid glycosides and the affect of terpene trilactones on the anticancer effect of EGB 761. At the same time, compare the pharmacokinetics of flavonoids and aglycone in EGB 761 and evaluate the effects of terpene trilactones on the pharmacokinetics of flavonoids.Part one: Separation and preparation of falvonoids in Ginkgo biloba extractsObjective: In this study, we apply EGB 761 prepare three different ingredients samples: EGB 761 devoid of terpene trilactones (EGB 761-DT, mainly containing the flavonoid glycosides) sample; EGB 761 hydrolysate (EGB 761-H, mainly containing the aglycones of quercetin, kaempferol and isorhamnetin and terpene trilactones) sample; EGB 761 devoid of terpene trilactones hydrolysate (EGB 761-DT-H, mainly containing the aglycones of quercetin, kaempferol and isorhamnetin), and prepare quercetin, kaempferol and isorhamnetin.Methods: Using liquid-liquid extraction method devoid the terpene trilactones in EGB 761 sample and hydrolyzed total flavonoid glycosides in MeOH-Hydrochloric acid (25%) (4:1, v/v). Using polyamide Collumn isolate the three aglycones of quercetin, kaempferol and isorhamnetin. High purity aglycones of quercetin, kaempferol and isorhamnetin were prepared by preparation HPLC method.Results: The prepared sample of EGB 761-DT, EGB 761-H, and EGB 761-DT-H were satisfied the requirement. The purity of aglycones of quercetin, kaempferol and isorhamnetin were greater than 98%.Conclusion: The methods used in the experiment are simple and the costs are low. It could devoid the terpene trilactones in EGB 761 sample and the concentration of aglcones are higher. The sample could be used in the pharmacology and pharmacokinetics study.Part two: The pharmacokinetics of Ginkgo biloba extracts falconoid in the ratObjective: Developed an HPLC method for determination of quercetin, kaempferol and isorhamnetin simultaneously in rat plasma for studying the pharmacokinetics of different ingredients of EGB 761 after a single oral or i.v. administration. Comparing the pharmacokinetics of flavonoids and aglycone in EGB 761 and evaluate the effects of terpene trilactones on the pharmacokinetics of flavonoids in EGB 761.Methods: After rats were orally or i.v. administrated different ingredients of EGB 761 sample, blood samples were obtained from fossa orbitalis vein according to the specific schedule, and collected in heparinized centrifuge tube (containing Vc), respectively. Sample was pretreated by a single-step protein precipitation with MeOH-Hydrochloric acid (25%) (4:1, v/v). The Alltech brava BDS C18 (250×4.6mm, 5μm) was used as the stationary phase with the mobile phase consisting of methanol-phosphoric acid (0.5%, v/v) (55:45, v/v). The column temperature was maintained at 30℃and the UV detector was set at 370nm. The corresponding pharmacokinetic parameters were calculate with the computer program 3p97 (the Chinese Society of Mathematical Pharmacology) using the non-compartment analysis.Results: 1. The calibration curve in plasma was linear over the range of 10~2000 ng·mL-1, the RSD values of intra-day and inter-day were less than 15% and the recovery in rat plasma was 92.3%~94.9% for quercetin; the calibration curve in plasma was linear over the range of 10~2000 ng·mL-1, the RSD values of intra-day and inter-dary were less than 15% and the recovery in rat plasma was 91.8%~94.4% for kaempferol; the calibration curve in plasma was linear over the range of 10~2000 ng·mL-1, the RSD values of intra-day and inter-day were less than 15% and the recovery in rat plasma was 98.4%~100.1% for isorhamnetin. 2. After single oral administration of EGB 761, EGB 761-DT, EGB 761-H, EGB 761-DT-H, quercetin, kaempferol, and isorhamnetin to rats, the main pharmacokinetic parameters were as follows: quercetin, kaempferol and isorhamnetin in EGB 761 sample: Cmax were 0.264±0.056, 2.504±0.839 and 0.714±0.184μg·mL-1, Tmax were 12.00h, AUC0-t were 3.956±1.066, 33.220±12.386 and 9.575±2.875μg·h·mL-1, MRT were 16.85±0.83, 16.37±0.96 and 16.45±0.67h; in EGB 761-DT sample: Cmax were 0.400±0.073, 0.991±0.347 and 0.374±0.150μg·mL-1, Tmax were 3.20±1.43, 7.20±3.25 and 8.40±1.96h, AUC0-t were 4.803±0.561, 15.536±5.570 and 4.727±1.750μg·h·mL-1μg·h·mL-1, MRT were 10.1±0.38, 12.16±0.72 and 13.48±1.85h; in EGB 761-H sample Cmax were 2.566±0.431, 10.114±1.559 and 1.799±0.247μg·mL-1, Tmax were 3.14±0.43, 2.29±0.25 and 2.59±0.22h, MRT were 9.35±0.32, 7.12±0.10 and 10.42±0.21h, AUC0-t were 28.165±7.414, 94.513±30.532 and 25.491±6.934μg·h·mL-1 ; in EGB 761-DT-H sample: Cmaxwere 3.456±0.287, 18.628±1.134 and 3.753±0.248μg·mL-1, Tmax were 0.69±0.16, 1.09±0.25 and 0.94±0.22h, MRT were 7.36±0.18, 8.14±0.09 and 8.43±0.14h, AUC0-t were 24.215±2.866, 187.280±27.916 and 37.043±7.178μg·h·mL-1;Quercetin, kaemoferol and isorhamnetin aglycones: Cmax were 1.088±0.337, 14.644±4.081 and 0.180±0.037μg·mL-1, Tmaxwere 0.44±0.21, 0.54±0.08 and 2.70±2.05h, MRT were 9.16±0.15, 8.25±0.36 and 9.74±2.01h, AUC0-t were 8.995±2.216, 141.298±28.188 and 2.087±0.247μg·h·mL-1. The relative bioavailability in EGB 761-H and EGB 761-DT-H sample is higher than in EGB 761 and EGB 761-DT samples. 3. After single i.v. administration of EGB 761, EGB 761-DT, EGB 761-H, EGB 761-DT-H, quercetin, kaempferol, and isorhamnetin to rats, the main pharmacokinetic parameters were as follows: quercetin, kaempferol and isorhamnetin in EGB 761 sample: AUC0-t were 0.822±0.125, 3.441±0.797 and 0.861±0.292μg·h·mL-1, MRT were 8.39±0.16, 8.68±0.32 and 7.59±0.27; EGB 761-DT sample: MRT were 7.620±0.77, 8.31±1.57 and 7.32±0.35h, AUC0-t were 0.835±0.231, 5.250±1.083 and 1.227±0.054μg·h·mL-1;EGB 761-H sample: MRT were 4.78±0.27, 8.22±0.14 and 7.78±0.20h, AUC0-t were 10.657±3.057, 110.832±16.869 and 37.740±3.670μg·h·mL-1;EGB 761-DT-H sample: MRT were 4.119±0.14, 7.82±0.20 and 7.57±0.20h, AUC0-t were 11.834±2.667, 112.790±23.520 and 37.712±5.860μg·h·mL-1 ; Quercetin, kaemoferol and isorhamnetin aglycones: MRT were 6.36±0.18, 7.88±0.18 and 8.91±0.14h, AUC0-t were 11.073±1.581, 127.596±16.734 and 7.223±0.796μg·h·mL-1.Conclusion: 1. The aglycone form flavonoid (EGB 761-H and EGB 761-DT-H sample) presented a strong improved absorption and relative bioavailability after oral administration. 2. Flavonoids glycosides were hardly transformed to aglycones in rats after i.v. administration, relative bioavailability of aglycones are higher than glycosides flavonoids. 3. Terpene trilactones increased the absorption and decreased the excretion of flavonoid glycosides, but decreased the absorption and increased the exertion of flavonoid aglycones after oral administration, and had no effects on the pharmacokinetics of flavones after i.v. administration.Part three: In Vitro anticancer activities of Ginkgo biloba extract falconoidsObjective: By comparing the anticancer effect of the four samples (EGB 761, EGB 761-DT, EGB 761-H, EGB 761-DT-H) on SGC-7901, HepG2, L1210 cancer cells, we investigate the difference of flavonoid aglycones and flavonoid glycosides and the affect of terpene trilactones on the anticancer effect of EGB 761.Methods: SGC-7901, HepG2, and L1210 cancer cells were sub-cultured in DMEM with 10% fetal ovine serum (FBS) at 37℃with 5% CO2. The cytotoxicity assay was performed by MTT method. The inhibitory rates were calculated according to the formula: IR= [1-(the mean of treated group)/(the mean of control group)]×100%. The cytotoxicity was expressed as IC50 (concentration of 50% cytotoxicity, which was extrapolated from linear regression analysis of experimental data).Results: The cytotoxicity of EGB 761, EGB 761-DT, EGB 761-H and EGB 761-DT-H on cells was dose-dependent and each cell had a different sensitivity to the inhibitive effect. The EGB 761 and EGB 761-DT sample had similar inhibitory effects on two cell lines. EGB 761-H sample had a more efficient than EGB 761-DT-H sample on HepG2 cell lines. EGB 761-H and EGB 761-DT-H samples (mainly containing the aglycones) had a more efficient than EGB 761 and EGB 761-DT samples (mainly containing the flavonoid glycosides). These date suggested that the concentration of aglycones in the EGB 761 sample could affect the anticancer activity. The sample mainly containing aglycones had a high efficient. Terpene trilactones didn't affect the anticancer effect of flavonoid glycosides, but increased the anticancer effect of aglycones on HepG2 cell lines. The cytotoxicity of EGB 761, EGB 761-H and EGB 761-DT-H on L1210 was The IC50 values on cytotoxicity were 46.36±2.43, 10.27±0.88 and 14.93±0.73μM for EGB 761, EGB 761-H and EGB 761-DT-H samples, respectively. EGB 761-H sample had a more efficient than EGB 761-DT-H samples. EGB 761-H and EGB 761-DT-H samples (mainly containing the aglycones) had a more efficient than EGB 761 samples (mainly containing the flavonoid glycosides).Conclusion: 1. the anticancer effect in different ingredients of EGB 761 isn't equal. EGB 761-H and EGB 761-DT-H samples (mainly containing the aglycones) has higher efficient than EGB 761 and EGB 761-DT samples (mainly containing the flavonoid glycosides). 2. Terpene trilactones can increase the anticancer effect of aglycones on HepG2 and L1210 cell lines. Increased the concentration of aglycone form flavonoids in the EGB 761 sample could improve the anticancer activity.Part four: Therapeutic mechanisms of Ginkgo biloba leaf falvonoids on gastric cancerObjective: Although several molecules and pathways had been proposed as targets of EGB 761, the precise mechanism by which these compounds exert their cancer-protective effects were poorly understood. In the present work the objective was to elucidate the mechanism of different ingredients of EGB 761 sample.Methods: SGC-7901 cells from exponentially growing cultures were seeded within 24-well culture plates and were allowed to attach for 48 h before treatment. The cells were treated with 25μM EGB 761, EGB 761-H, EGB 761-DT-H and without (vehicle control, 0.1% DMSO) for 48 h. To observe the induction of apoptosis cells were stained with Hoechst 33258 and Giemsa. Telomerase activity was quantitated by PCR-TRAP-ELISA method. The expression of hTERT mRNA was determined by RT-PCR.Results: Staining with Hoechst 33258 and Giemsa showed that 25μM EGB 761, EGB 761-H, EGB 761-DT-H increased the number of apoptotic cells. The telomerase activity was significantly decreased after treat with EGB 761, EGB 761-H, and EGB 761-DT-H. EGB 761-H and EGB 761-DT-H were more efficient inhibitor of telomerase activity than EGB 761. The EGB 761, EGB 761-H and EGB 761-DT-H were able to down regulate hTERT expression. The effect of EGB 761-H and EGB 761-DT-H sample was stronger than EGB 761 sample.Conclusion: The inhibitory effects of different ingredients of EGB 761 samples on cell proliferation may be attributed to its inhibition on telomerase enzymatic activity by down regulate hTERT mRNA expression and induced apoptotic. The effect of EGB 761-H and EGB 761-DT-H sample was stronger than EGB 761 sample.Part five: Comparative anticancer and antioxidant activities of Ginkgo biloba leaf falvonoidsObjective: By compare the anticancer effect of the three samples on S180-bearing mice and EAC-bearing mice, we investigate the difference of flavonoid aglycones and flavonoid glycosides and the affect of terpene trilactones on the anticancer effect of EGB 761 and assesse the immunomodulatory effects of the different ingredients of EGB 761 samples. At the same time, it is in our interests to examine whether EGB 761 might suppress S180 growth through the antioxidant system in vivo.Methods: Seven-day-old S180 ascites (0.2mL, 2×107 cells·mL-1) were transplanted subcutaneously into the right axilla of each mouse of the rest groups. The mice were treated as follows: Normal control group (8%Tween 80, v/v); Model control group (8%Tween 80, v/v); 5-Fu group (10mg·kg-1); EGB 761 group; EGB 761-H group; EGB 761-DT-H group. All the groups were administered by p.o. every day for 10 days, starting 24h after tumor implantation. Twenty-four hours after the last drug administration, mice were anaesthetized with ether and sacrificed by cervical dislocation. The organs and solid tumors were excised and weighted. The anticancer activity in vivo was expressed as an inhibitory rate calculated as [(A-B)/A]×100%, where A and B were the average tumor weight of the model control and experimental groups, respectively. Blood was immediately transferred to a glass tube and allowed to clot, then centrifuged at 3000 rpm for 10 min, followed by the removal of plasma for measured of CAT, GST, MDA and SOD, according to the manufacture's protocol.Results: Treatment with three different EGB 761 samples caused marked suppression of the tumor weight. Compared with the model control, the inhibitory rates of EGB 761, EGB 761-DT-H and EGB 761-H on the tumor weight in S180-bearing mice were 41.74%, 60.72% and 63.76%, respectively. However, the anticancer effect was more pronounced in the group treated with EGB 761-H and EGB 761-DT-H as compared with treatment with EGB 761 samples. Spleen index and thymus index of different EGB 761 samples group did not have significantly decrease compared to model control group. But 5-Fu group had significantly difference compared to model control group (p<0.05). Treatment with three diffetent EGB 761 samples showed significantly decreased the MDA levels as compared to the Model control group. Treatment with EGB 761-H and EGB 761-DT-H returned MDA levels close to normal control values and lowered the MDA levels (P<0.05) as compared with the EGB 761 group. Treatments with different EGB 761 samples revealed no significantly change in blood GST level when compared with normal control values and significantly increase GST level when compared with model control values. Administration of different EGB 761 samples revealed significantly increased SOD and CAT activities when compared to the model control group and significantly lowered the SOD activities when compared to the normal control group. Treatment with EGB 761-H and EGB 761-DT-H returned CAT activities close to normal control values and significantly increased the CAT activities (p<0.05) as compared with the EGB 761 group.Conclusion: Different EGB 761 samples could inhibit tumor weight in S180-bearing mice and prolong the life time of EAC-bearing mice in vivo and without any visible serious toxic effect on the immune systems, the anticancer activity of EGB 761-H and EGB 761-T-H sample (mainly containing aglycones) were higher than EGB 761 samples, increase the concentration of aglycone form flavonoids in the EGB 761 sample could improve the anticancer activity. The terpene trilactones didn't affect the anticancer effect of flavonoid. Elevation the levels free radical scavenging GST, SOD and CAT content might be one of the mechanisms that lead to cancer prevention.