Comparative Study On Hypolipidemic Effect, Pharmacokinetics And Tissue Distribution Of Cis-and Trans-2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside In Mice

2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) is one of thewater soluble bioactive components in Polygoni Multiflori Radix. Manystudies have documented that it has many pharmacologic activities, such asantioxidation, antihyperlipidemic, against osteoporosis, preventing vascularendothelial dysfunction, neuroprotective, myocardial preservation, improvingblood flow, ameliorating vascular senescence, and so on. For the structure,THSG has cis-and trans-isomers. Research has shown that PolygoniMultiflori Radix contains trans-THSG and cis-THSG. Since trans-THSG ismore stable than cis-THSG, the main form of THSG in Polygoni MultifloriRadix is trans-THSG in natural conditions. The main object in the past studyis trans-THSG, while the study on cis-THSG is less.In this study, we first prepared the pure product of cis-THSG which wasidentified by four kinds of spectra to confirm its purity. The relative correctionfactor of cis-THSG to trans-THSG was determined, which could be used forthe determination of cis-THSG. Based on the above research, the comparativestudy on antihyperlipidemic effect of cis-THSG and trans-THSG wasinvestigated. An HPLC method for simultaneous determination of cis-THSGand trans-THSG in mice plasma and tissues was established and then thepharmacokinetics and tissue distribution of cis-THSG and trans-THSG usingthis method were studied. All that could contribute to the development andutilization of THSG.Pat one Preparation and identification of cis-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucosideObjective: To establish a method for preparing cis-THSG fromtrans-THSG, and confirm the structure of cis-THSG by four spectra analyses. Methods: Kinds of light (daylight,254nm and365nm ultraviolet UV),solvent (water and methanol), initial concentration (0.1,0.2,0.4,0.8,1.0,2.0,4.0,6.0,8.0and10.0mg/mL) and exposure time (5,10,15,20,25and30min)were investigated to choose the optimum conditions for convertingtrans-THSG into cis-THSG. The cis-THSG was separate from the supernatantof the converted solution by the HPLC system. Separation was performedusing a Diamonsil C18column (250mm×10.0mm,5μm), which was elutedwith methanol and water. The flow rate of mobile phase was maintained at1.0mL/min. The detection wavelength was set at285nm and the columntemperature was set to30℃. Cis-THSG fractions were collected and thesolvent was volatiled at50℃under vacuum condition. The residue wasdissolved with water and then the aqueous solution was lyophilized to obtainwhite solid powder. The MS, NMR, UV and IR were used to identify itsstructure.Results: The optimum conversion conditions are shown as follows:trans-THSG is dissolved in water with a concentration of6.0mg/mL andirradiated by365nm UV light for20min. MS scan showed the white powderhad the same molecular ion m/z405.1[M-H]-with trans-THSG, whichdemonstrated that they had the same molecular weight. Then the molecularions m/z405.1[M-H]-were conducted the second stage MS scan. Both ofthem produced the same fragment ion m/z243.1[M-C-6H11O5](neutral loss ofglycosyl) and other fragment ions, such as m/z225.1,173.0,145.2,137.1and109.0. The results showed that cis-THSG and trans-THSG had the samecleavage rule. The proton signal of cis double bond was found in1H-NMR ofthe product. UV full scan spectrum showed that the maximum absorptionwavelength was moved from320nm of trans-THSG to285nm of cis-THSG.IR spectra showed that it had absorption peaks in3500-3200(νO-H),2924and2885(νC-H),1609and1514(νC-C),1456(δC-H),690(γ=CH) cm-1, among which690cm-1was the outer surface vibration signal of the cis olefin. The whitepowder was confirmed to be cis-THSG.Conclusion: The preparation method is simple, fast and efficient. It is suitable for the preparation of cis-THSG from trans-THSG.Part two Determination of the relative correction factor of cis-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside to trans-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside based on liquidchromatographyObjective: To measure the relative correction factor of cis-THSG totrans-THSGMethods: Separation was performed using a Diamonsil C18columnwhich was eluted with acetonitrile-methanol-0.1%acetic acid (11:7:82) at aflow rate of1.0mL/min. The detection wavelength was set at285nm forcis-THSG and320nm for trans-THSG. The column temperature was set to30℃, and the injection volume was20μL.The standard solutions of cis-THSGand trans-THSG with different concentration were prepared and then injectedinto the liquid chromatograph. The calibration curves were obtained with peakarea as vertical axis and concentration as abscissa. The relative correctionfactor was obtained with the slope of the calibration curve of cis-THSGdivided by trans-THSG. The trans-THSG peak area was corrected by therelative correction factor, and the corrected calibration curve obtained with theconcentrations of trans-THSG and the corrected peak aera was set as thecalibration curve of cis-THSG. The standard material and the test smples wereused for the evaluation of the relative correction factor. The peak area of eachstandard solution of cis-THSG was substituted into the calibration curve ofcis-THSG to obtain the concentration, and the recovery was expressed by theratio of the obtained concentration and the true concentration. The evaluationof the test sample was expressed by relative recovery, which was obtainedwith the concentration of the cis-THSG calculated by relative correction factorand external standard method.Results: The calibration curves of cis-THSG and trans-THSG wereY=33.54X+1.190and Y=43.48X-0.5007. Calibration curves were generatedover a concentration range of0.112～112μg/mL (R2=0.9997) for cis-THSGand0.110～110μg/mL (R2=0.9991) for trans-THSG, respectively. The relative correction factor was0.771. Method validation results showed that theinstrument precision (RSD) of cis-THSG and trans-THSG were0.92%and1.07%, intra-day precision RSD were1.22%and3.81%, inter-day RSD were3.40%and2.63%. Recoveries were in the range of96.2～101.3%with RSDvalues between1.19%and4.89%. Recoveries evaluated by the standardmaterial were in the range of95.4～102.6%. Relative recoveries evaluated bythe test sample were within the range of100.1～102.7%.Conclusion: The relative correction factor of cis-THSG could be usedfor the determination of cis-THSG without standard of cis-THSG.Part three Comparative study on hypolipidemic effect of cis-and trans-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucosideObjective: To make the comparative study on hypolipidemic effect ofcis-THSG and trans-THSG.Methods: The concentration of total cholesterol (TC), triglyceride (TG),high density lipoprotein cholesterol (HDL-C) and low density lipoproteincholesterol (LDL-C) in mice serum were used to evaluate the blood lipid level.The effects on blood lipids of both high and low doses of cis-THSG andtrans-THSG were studied in normal mice. The investigation was employed inthe hyperlipidemic mice which induced by feeding with high-lipid dietaccompanied by giving cis-THSG and trans-THSG for15consecutive days,and observed the effect of high and low doses of drugs on mouse blood lipid.Acute hyperlipidemia mice were induced by intraperitoneal injection of Tritonreagent and the effect of high and low doses of cis-THSG and trans-THSG onmodel lipids was investigated.Results: In normal mice, cis-THSG and trans-THSG had no significanteffect on serum TC and TG, but could significantly decrease the level ofLDL-C. In addition, high doses of THSG could significantly increase the levelof HDL-C. In the chronic hyperlipidemic mice, cis-THSG could significantlyreduce the level of TC and TG. However, low dose of trans-THSG could onlysignificantly reduce the level of TG. While high dose of trans-THSG had nosignificant effect on TC, TG and HDL-C. In the acute hyperlipidemic mice, except the level of HDL-C at low doses of cis-THSG, the levels of TC, TG,HDL-C and LDL-C could significantly be decreased.Conclusion: Cis-THSG and trans-THSG have the same obviouslipid-regulating effect in normal mice and acute hyperlipidemic mice, butcis-THSG has stronger effect on lowering TC and TG in chronichyperlipidemic than trans-THSG. The results show that the hypolipidemiceffect of cis-THSG is better than trans-THSG.Part four Comparative study on pharmacokinetics of cis-and trans-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside in miceObjective: To establish an HPLC method for simultaneous determinationof cis-THSG and trans-THSG in mouse plasma, and then to compare thepharmacokinetic characteristics of cis-THSG and trans-THSG.Methods: Mixed solution contained cis-THSG and trans-THSG wasadministered to mice by intravenous injection and oral administration (iv20mg/kg, ig50mg/kg). Blood samples were collected by picking eyeball afteradministration drug at the prearranged time (5,10,20,30,40,50,60,90,120,150and180min). Plasma was obtained by centrifugation at approximately4743×g for5min. Analyses was performed using a Diamonsil C18（250mm×4.6mm,5μm）column with acetonitrile-methanol-0.1%acetic acid (11:7:82)as mobile phase at a flow rate of1.0mL/min. The detection wavelength ofcis-THSG was set at285nm, while trans-THSG and polydatin was320nm.Column temperature was30℃and the injection volume was20μL. Polydatinwas appointed as the internal standard.20μL of internal standard solutionwere added to a200μL aliquot of plasma, and then600μL of methanol wasadded for precipitating the plasma protein. Sample was centrifugated atapproximately16600×g for10min after vortex for10s.700μL supernatantwas dried in nitrogen stream at40℃. The obtained residue was reconstitutedin200μL of mobile phase, vortexed and centrifugated at16600×g for10min.A20μL of the supernatant was injected into the HPLC system for analysis.The elimination rate constants (k) were determined with the last four datapoints of the drug-time curve through linear regression analysis of the logarithmic transformation. AUC0-tand AUC0-∞was calculated with thetrapezoidal rule through a non-compartment model.Results: The calibration curve (Y=0.0261X-0.0060, R2=0.9986) ofcis-THSG in plasma was linear over the range of0.228～182μg/mL, and thecalibration curve (Y=0.0338X-0.0073, R2=0.9968) of trans-THSG in plasmawas linear over the range of0.245～196μg/mL. The accuracy and precisionwere evaluated by determining the QC samples at low, medium, highconcentrations and the lower limit of quantification of QC samples. The intra-and inter-day precision RSD values were less than8.30%, and the accuraciesof RE were from-9.69%to4.08%. The stabilities of cis-THSG andtrans-THSG in plasma under different storage conditions such as threefreeze-thaw cycles, short-term stability at room temperature (25℃) for4h,long-term stability at-20C for15days and stability after sample preparationwere evaluated. RE values were in the range of-6.59%～1.33%. The AUC0-tof cis-THSG and trans-THSG were2188.80and756.43μg·min/mL. TheAUC0-∞of cis-THSG and trans-THSG were2244.29and785.51μg·min/mL,and the half-life of cis-THSG and trans-THSG were163.61and82.13min byintravenous injection, respectively. The AUC0-tof cis-THSG and trans-THSGwere3759.49and823.48μg·min/mL. The AUC0-∞of cis-THSG andtrans-THSG were3807.42and846.71μg·min/mL, and the half-life ofcis-THSG and trans-THSG were140.84and65.58min. The Cmaxof cis-THSGand trans-THSG were115.15and53.92μg/mL. The Tmaxof cis-THSG andtrans-THSG were30and20min by oral administration, respectively.Conclusion: The developed method was specific, sensitive, simple, rapid,and suitable for simultaneous determination of cis-THSG and trans-THSG inmouse plasma. The result indicated that the pharmacokinetic parameters ofcis-THSG and trans-THSG in mice were significantly different. The half-lifeof cis-THSG is longer than that of trans-THSG, and the bioavailability ofcis-THSG is higher than that of trans-THSG. Part five Comparative study on tissue distribution of cis-and trans-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside in miceObjective: To establish a method for simultaneous determination ofcis-THSG and trans-THSG in mice, and to study the properties of tissuedistribution of cis-THSG and trans-THSG.Methods: Mixed solution contained cis-THSG and trans-THSG wasadministered to mice by intravenous injection and oral administration (iv20mg/kg, ig50mg/kg). Heart, liver, spleen, lung, kidney, stomach, smallintestine, brain and bladder samples were obtained at5,20, and50min,respectively. After washed with distilled water, dryed with paper and cut intopieces, tissue samples were homogenized in saline solution (1:2w/v).Polydatin was taked as the internal standard.200μL tissue samples weredetermined with the same way as the fourth part sample preparation andchromatographic conditions. The liver tissue samples were used for methodvalidationResults: The calibration curve (Y=0.0263X-0.0173, R2=0.9963) ofcis-THSG in tissue sample was linear over the range of0.228～182μg/mL,and the calibration curve (Y=0.0334X-0.0034, R2=0.9994) of trans-THSG intissue sample was linear over the range of0.245～196μg/mL. The accuracyand precision were evaluated by determining the QC samples at low, medium,high concentrations and the lower limit of quantification of QC samples. Theintra-and inter-day precision RSD values were less than9.47%, and theaccuracies of RE were ranged from-6.81%to4.18%. The stabilities ofcis-THSG and trans-THSG in tissue under different storage conditions such asthree freeze-thaw cycles, short-term stability at room temperature (25℃) for4h, long-term stability at-20C for15days and stability after samplepreparation were evaluated. RE values were in the range of-9.49%～-0.88%.The results showed that both cis-THSG and trans-THSG were widelydistributed in the various tissues, but each of them has its own distributioncharacteristics. At the same dosage, the concentrations of trans-THSG intissues were greater than that of cis-THSG.Conclusion: The developed method was specific, sensitive, simple, rapid,and suitable for the study on tissue distribution of cis-THSG and trans-THSG in mice. The results suggested that there are differences between cis-andtrans-isomers of THSG in tissue distribution, which suggested that the twoisomers may have different pharmacological effects and side effects.