Study On The Anticancer Compound 24-OH-PD From Red Ginseng

Red ginseng is a steamed Panax ginseng.In this study we began with the chemical compound extraction from red ginseng and got the new compound 20（S）,25-epoxydammarane-3β,12β,24α-triol which was also known as 24-OH-PD.Then we conducted the research on the anticancer activity in vitro,the docking with the cancer associated protein and the pharmacokinetic（PK）characteristics of 24-OH-PD in vivo.1.Obtained two new dammarane-type saponins from red ginseng.By various methods of isolation,the physicochemical properties and spectroscopic methods（¹HNMR,13 CNMR,2D-NMR,MS）,13 compounds were identified as:20（R）-ginsenoside Rh₁（1）,20（S）,25（R）-epoxydammarane-3β,12β,24β,26-tetraol（2）,20（S）-Protopanaxadiol（3）,20（S）,25-epoxydammarane-3β,12β,24α-triol（4）、(20（S）,25-epoxydammarane-3β,12β,24β-triol（5）,20（R）-ginsenoside Rg3（6）,20（S）,24（R）-dammar-20,24-epoxy-3α,12β,25-triol（7）,20（R）-ginsenoside Rf（8）,20（S）-ginsenoside Rg3（9）,20（R）-ginsenoside Rg₂（10）,ginsenoside Rg₁（11）,ginsenoside Rb₁（12）,ginsenoside Re（13）.Among them,compound 2 and compound4 were identified as new compounds,and compound 5 and compound 7 were obtained from the red ginseng for the first time.2.A first research on anticancer activity of 24-OH-PD in vitro.The anticancer activity of 24-OH-PD was evaluated in M14、MD-MBA-231、CCRF-CEM and Jeko-1cancer cell lines.The effects of 24-OH-PD on inhibiting proliferation and inducing apoptosis were evaluated.And the anticancer mechanism was studied by docking analysis with cancer related proteins.It was found that the addition of 24-OH-PD to the culture medium significantly inhibited cell proliferation and induced apoptosis in all four cancer cell lines.24-OH-PD docked successfully with 18 out of 50 proteins.The data of docking result demonstrated the promising targets of 24-OH-PD.3.First establishment of an UPLC-MS/MS assay for 24-OH-PD in biosamples.The 24-OH-PD and internal standard of panaxatriol（PT）were extracted from the sample by methanol precipitation technique,and the mass spectrometer was operated in the positive ion mode using multiple ion pairs were m/z 477.1→143.0 for26-OH-PD and 477.4→127.1 for PT.These methods were fully validated according to documents from the State Food and Drug Administration.4.First research on the pharmacokinetic of 24-OH-PD in rat.Using the established UPLC-MS/MS method,we investigated the rat plasma at different time after intravenous challenging of 24-OH-PD at the doses of 2.5¹0mg/kg.The major PK parameters were as follows: the mean maximum plasma concentration Cmax was 3316.86±517.39 8577.11±2792.83 μg/L.The elimination half-time T_1/2 was 4.82±1.99 5.45±1.83 h,and the CL and V was 0.44±0.05 ⁰.76±0.61 L/h/kg and 3.09 ±0.89 6.59±7.66 L/kg,respectively.5.The research on plasma protein binding.The binding rate（BR）of 24-OH-PD to rat plasma protein was investigated using equilibrium dialysis.The results showed that the BR were 88.10 ± 9.10% 92.30 ±2.40% at 200²000ng/m L,and showed no concentration-dependent effect.This indicated the high plasma-protein binding rates of 24-OH-PD in combination mode.6.The research on the tissue distribution of 4-OH-PD in rat.According to the established UPLC-MS/MS method,the concentration of 24-OH-PD at 15 min,1h,3h,and 5h were detected in heart,liver,spleen,lung,kidney,stomach,intestine,uterus,testicle,muscle and fat after intravenous challenging of 24-OH-PD at a single dose of 5.0mg/kg.After the intravenous injection of 24-OH-PD,the drug extensively and quickly distributed in the examined tissues,the highest level was observed in lung.And showed no long-term accumulation in all the tissues.7.The research on the excretion of 24-OH-PD in rat.To study the excretion of 24-OH-PD,we collected rat feces and urine at different time after intravenous challenging of 24-OH-PD at a single 5.0 mg/kg dose in rat.By the UPLC-MS/MS method,the results were as following: The maximum excretion rate of 26-OH-Panaxadiol in both feces and urine were shown between 8 h and 12 h.The percentages of cumulative amount of 24-OH-PD over a 96 h period in feces and urine were 1.3844 ± 0.3878% and 0.0018 ± 0.0013%,respectively.8.The first study on the metabolites of 24-OH-PD in rat.An analysis method based on ultra-performance liquid chromatography-quadrupole time-of flight mass spectrometry（UPLC-Q-TOF-MS）and Waters UNIFI Scientific Information System were employed for the detection of 24-OH-PD metabolites in rat plasma,urine and feces samples.The results showed both phaseⅠand phase Ⅱmetabolisms in the metabolic processes of 24-OH-PD in rat.Besides the metabolic pathways of 24-OH-PD included dehydroxylation,dehydration,demethylation,methylation,hydroxylation,sulfation,phosphorylation,acetylation,glucuronidaation.