| Osteoarthritis(OA)is a disease of the degeneration and destruction of the whole joint,and severely diminishes the patients’ life quality and mainly causes joint pain on knees,hips,ankles and spines attributed to the fibrosis,rhagadia,ulcer,attrition of articular cartilages induced by multiple factors.This chronic disease imposes a substantial economic burden on both of the sufferers’ families and the society and is reported to be one of the leading causes of the disability around the world.“Huangjingzi” is the dry fruits of the Verbenaceae plant Vitex negundo L.,which conventionally used as a remedy for rheumatoid arthritis(RA)in folk medicine and functions as “dispelling wind and relieving pain,relaxing sinew and activating collaterals” in TCM.Our group previously elucidated that a series of benzene-naphthalene types of lignans were the chemical basis of its anti-rheumatoid arthritis activity.Purified by the first generation of preparation process,at a scale of 10 kg,a TOV(total lignans of Vitex negundo)sample with a purity of 50% was obtained in laboratory.The most potent lignans of it includes 6-Hydroxy-4-(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde(VNL),vitedoin A,vitexdoin A and vitexdoamine A.Nonetheless,the method is immature and failed to meet the demands of purity and transfer rate of lage-scale industrial production process.In view of that many similarities between RA and OA with regard to the pathogenic manifestation,this study was conducted to systematically evaluate the anti-osteoarthritis activity and demonstrate the pharmacokinetic characteristics of TOV on the basis of its optimized preparation process.Hence,this study focuses mainly on the contents as following:1.Optimization of the Preparation of TOV and Its Pilot Scale TestsOn the basis of the former preparation process of TOV,the procedure with polyamide resins was removed,and the ethyl acetate extraction was adjusted prior to the macroporous resin purification.Several novel and effective types of macroporous resins were re-screened and a single-factor experiment was employed to acquire the optimum parameters of each step of the purification process.Then pilot scale tests were magnified at the scale of 100kg and210kg,each for a three-time repetition.As a result,the Vitex negundo fruits were properly powdered and sifted(10 mesh)then extracted twice with 70%ethanol(10×)under reflux for1h each time to obtain the crude extract,which was later evaporated to the density of 1g/L.This extract was partitioned with 4 times of ethyl acetate than water at a pertinent p H value 6,twice in total.The macroporous resin LX-3020 was adopted to purify the ethyl acetate portion at a concentration 1.5mg/m L of TOV of the suspension,which was 14 times of the column’s bed volume(BV).Subsequently the resin column was washed with 20%ethanol for 4-time BV to remove the pre-impurities and 40%ethanol for 10-time BV successively to afford the targeted fraction.The 40%ethanol eluent was condensed at 50℃and desiccated at 45℃both under vacuum to give the raw material of TOV.This coarse product was smashed into powder screened through the 80-mesh sieve to gain TOV finally.The process of pilot scale tests attained TOV with a purity of 64.4%-72.8%,the transfer rate of 58.0%-66.6%and yield up to5.5-6.0‰.Therefore,this preparation process is stable,brief and reliable to meet the requirement of industrial productions.2.Anti-osteoarthritis Activity of TOVIn view of our preliminary investigation on the anti-RA activity of TOV,we conducted a research on the anti-osteoarthritis activity of TOV acquired from the new preparation process.This research employed SD rats with OA induced by the sodium iodoacetate(MIA)and randomly disposed into 6 groups(n=8)including control,model,positive drug(celecoxib,17.1mg/kg),low,medium and high dose-treated groups of TOV(20,40,80mg/kg).One week after modeled,rats were administrated drugs intragastrically for a week and then were anesthetized to collect the blood samples and the hind limbs.Electric Von Frey dolorimeter was employed to measure the values of pain threshold,H&E and Safranin O-Fast Green staining to observe the pathological state of the right knee joints,ELISA kits to evaluate the level of TNF-α,IL-1β,IL-6,PGE2 in serum.The results showed that after TOV-treatment for a week,rats’ pain thresholds were significantly increased vs.model group(p<0.001)dose-dependently,and the celecoxib group displayed no remarkable differences compared with the control group(p>0.05).Observing the H&E and Safranin O-Fast Green stained slices,90mg/kg TOV significantly improved knee articular cartilages surfaces(p<0.01),and decreased the loss of chondrocytes(p<0.001)and the total joint scores(p<0.001).ELISA assays exhibited that the level of TNF-α,IL-1β,PGE2 in serum decreased notably in the TOV groups vs.model group(p<0.001),except IL-6.Accordingly,TOV has a therapeutical effect on the osteoarthritis model induced by MIA and has the potential for the treatment of OA.3.Effects of TOV on the Acute Blood Stasis in RatsSPF SD rats were divided into the seven groups,including a model group(n=15),two positive drug groups(50mg/kg aspirin and 0.5g/kg “Naoxintong” capsules,n=11),a control group(n=10),three TOV groups(10mg/kg,30mg/kg,90mg/kg,n=10)stochastically.After taking drugs for one week constantly,all of the groups were modeled with the injections of the adrenalin hydrochloride and ice-bath,except the control group.Afterwards rats were anesthetized instantly to draw the blood samples with both of heparin-anticoagulant and procoagulant pipe for each.Observation index:1.The weight differences between all groups.2.The viscosity of plasma and whole blood,rate of erythrocyte aggregation,platelet aggregation induced by ADP.3.Cytokines:The level of TXB2,6-Keto-PGF1α,and TXB2/6-Keto-PGF1αrate in serum.As a result,the control group,model group and drug-treated group were no prominent differences in feeding,drinking,coat color,movement and weight(p>0.05).“Naoxintong”capsules and TOV-treated groups were more durable to the adrenalin hydrochloride and with high livability vs.model group(p<0.05);aspirin,“Naoxintong”capsules and TOV group significantly resisted against the increment of the viscosity of plasma and whole blood(p<0.01);aspirin(p<0.001)and“Naoxintong”capsules(p<0.01)significantly decreased the TXB2;all of drug-treated groups diminished6-Keto-PGF1α(p<0.001);the exhibited inclination of down-regulating the rate of TXB2/6-Keto-PGF1αin serum vs.model group(p<0.001).4.Pharmacokinetics of TOVA UPLC-MS analytical method was established to delineate the pharmacokinetics of the two main constituents VNL and vitedoin A of TOV.30 SD rats were divided randomly into 4intragastric groups(n=6)and 1 intravenous group(n=6),and blood samples were collected after administration at 14 point-in-time,centrifugated and extracted to obtain the plasma for UPLC-MS analysis,which determines the final drug concentration.Data were processed by non-compartment analysis using the software winnonlin 6.2 to afford the main pharmacokinetics parameters.The results showed that the standard curves are of superior linearity and the method is stable,precise,reproducible enough,thus,applicable for the quantificaition of the two compounds in plasma.Consequently,the main pharmacokinetics parameters(Cmax,AUC 0-24 and MRT 0-inf＿obs)of VNL and vitedoin A exhibited favorable dose-dependent relationship with the four oral dosages(25、50、100、200mg/kg)and showed similar fate in body as rapid absorbtion and metabolism process with a F=15.34%～21.89%,16.29%～22.11%;t1/2=2.8h,1.3h respectively;Tmax≈10.0min similarly. |
