| The group obtained wikstroelide E(WE),wikstroelide R,6α,7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate,pimelotide A,pimelea factor S6,genkwadane C,simplexin,laurifolioside A and other diterpenoids from the bud of Wikstroemia chamaedaphne in the early stage,all of which showed good activation of latent HIV,among which WE had the best activation effect,and its activation effect was500 times higher than that of the positive control prostratin.The activation effect was 500times higher than that of the positive control prostratin.Since WE and other compounds are ryanodine diterpenes with a 5/7/6 tricyclic backbone,they are highly oxidized and complex in structure,and are difficult to synthesize artificially,which highly restricts their subsequent pharmacogenetic studies.Therefore,based on the strong activation effect of WE and other diterpenes in the bud of W.chamaedaphne on latent HIV,it is possible to optimize the enrichment preparation process to prepare WE-rich diterpene extracts from the bud of W.chamaedaphne and to carry out activation studies on latent HIV.This study will provide a basis for the study of the efficacy of diterpene extracts from the bud of W.chamaedaphne in activating latent HIV.In this study,seven major diterpenoids were isolated from the bud of W.chamaedaphne and identified by HPLC analysis.Thus,a method was developed for the simultaneous determination of four diterpenes in the enriched diterpene extracts.By comparing the adsorption and desorption of diterpene components by seven macroporous resins with different physical properties,the D101 macroporous resin was further screened by the adsorption and desorption amounts in static tests to obtain the most suitable resin for the enrichment of diterpene components.In the study of the adsorption kinetics of diterpene components by D101 resin,a comparison of the rate control in the adsorption process using the proposed primary kinetic model and the proposed secondary kinetic model was conducted,and it was found that the adsorption process of D101 resin was more in accordance with the proposed secondary kinetic model.In order to better explain the adsorption effect of D101 resin on the diterpene components in the bud of W.chamaedaphne crude extract at certain temperature,the adsorption effect on these diterpene components at 25°C,30°C and 35°C was investigated by using Langmuir,Freundlich and Temkin isothermal adsorption equation model,and it was found that the adsorption of diterpene components in crude extracts on D101 resin was a heat absorption process,and the equilibrium adsorption capacity was positively correlated with the increase of the adsorption equilibrium concentration(C_e)at the same temperature.Finally,the best process parameters for the separation and purification of diterpenoids by D101resin were determined by dynamic adsorption test:the diameter to height ratio of the wet loading column was 1:5,the mass concentration of the loading solution was 16.67 mg/m L,the loading volume was 5 BV,the loading flow rate was 0.5 m L/min and the dynamic adsorption was maintained for 5 h.When desorption,10 BV was eluted with 50%ethanol to remove the larger polar impurities.And then eluted with 80%ethanol for 40 BV and collected at a desorption flow rate of 3.0 m L/min.The purification process of silica gel column chromatography is as follows:take 1.5 times of sample 60～80 mesh silica gel for mixing,dry sample,take 10 times of sample 100～200 mesh silica gel for wet column loading,elution procedure is gradient elution,that is,the first gradient with petroleum ether:acetone(12:1)for 42 BV to remove the less polar impurities;the second gradient with petroleum ether:acetone(6:1)for elution 53 BV and collected,the solvent was recovered under reduced pressure and dried to obtain the target extract.The final results of content determination showed that after enrichment and purification,the contents of the four major diterpene components were 26.46±0.23%,34.91±0.53%,11.04±0.19%,and8.14±0.21%,respectively,and the total diterpene extract content was 80.55±0.81%,and the transfer rate during purification was 93.32%,which was 8.97,9.09,5.38,and 16.28times higher compared to the bud of W.chamaedaphne crude extract its content increased by 8.97,9.09,5.38,16.28 times,respectively,and overall by 8.61 times.It indicates that the purity of the four major diterpene components in the bud of W.chamaedaphne was significantly improved after enrichment with D101 macroporous resin and purification by silica gel column chromatography,and further indicates that the diterpene extract with high content can be obtained after simple enrichment and purification,which indicates that the enrichment and purification process has the advantages of time and labor saving,high efficiency and rapid,and cost saving,and has achieved the goal of enrichment and purification was achieved.In this experiment,the cytotoxicity and activation of latent HIV activity of the isolated and purified seven diterpene compounds and the enriched and purified diterpene extracts were determined.It was found that the seven diterpene compounds isolated and purified were non-toxic to NH2 cells at 10μM and all showed some activation effect in terms of activity,i.e.,activation ploidy in the range of 10-19-fold.It was also found that the bud of W.chamaedaphne crude extract,80%ethanolic part and diterpene extract were non-toxic to NH2 cells at two concentrations of 0.1μg/m L and 1.0μg/m L,but exhibited different activating activities.That is,the activation ploidy of the diterpene extract at two concentrations of 0.1μg/m L and 1.0μg/m L was 21 and 19 times,respectively,which was close to 22 times that of the positive drug prostratin(10μM),the activation ploidy of the80%ethanolic site was 13 and 16 times,respectively,and the activation ploidy of the bud of W.chamaedaphne crude extract was 10 and 12 times,respectively.The activation potential of the bud of W.chamaedaphne showed a certain dependence with the increase of diterpene content.Finally,it can be seen from the activation of latent HIV activity that the activity of the diterpene extract is better than that of the diterpene monomeric compounds,which can further indicate that a variety of active diterpene compounds including WE were enriched,indicating that the enrichment and purification process of the diterpene extract is stable and reliable. |
PDF Full Text Request