Percutaneous Permeability Enhancement Of Essential Oil From Fructus Viticis And Volatile Oil From Cinnamomi

Objective:To determine the enhancing effect of essential oil from Ramulus Cinnamomi?RCEO?a drugs of pungent in flavor,and essential oil from Vitex trifolia?VTEO?a drugs of pungent in cool,on the transdermal absorption of diclofenac sodium?DS,Water-soluble drugs?and indomethacin?Ind,lipophilic drugs?in vitro and vivo.To investigate the action mode of RCEO and VTEO on activity of epidermal skin and to investigate action mode of RCEO and VTEO as penetration enhancer.The cumulative skin stimulation effect was determined by visual scoring in guinea pigs.HaCaT cells were cultured in vitro.Using CCK-8,examined the effect of the oil?different concentrations?and the results was compared with those of Azone,a classic permeation enhancer.Methods:1.The extraction of essential oil from Ramulus Cinnamomi and Vitex trifolia.The method of steam distillation was carried out to extract the essential oil from Ramulus Cinnamomi and Vitex trifolia.The extraction process was by orthogonal design.2.Effect of RCEO and VTEO on the transdermal absorption Inddand DS in vitro.The method to determine of Ind and DS was established by HPLC.Permeation studies was investigated by performing in vitro rat dorsal skin permeation studies using Franz-diffusion cells.Various parameters viz.steady state flux,cumulative penetration?Q?,and enhancement ratios?ER?were calculated from the permeation data.3.Effect of RCEO and VTEO on the transdermal absorption DS in vivo.Established a method for determination of DS in vivo microdialysis fluid of rat abdominal skin by HPLC.The samples were collected for 8hours,and the drug concentration was detected by HPLC.4.To investigate the skin stimulation effect of RCEO and VTEO.The cumulative skin stimulation effect was determined by visual scoring in guinea pigs.HaCaT cells were cultured in vitro.Using CCK-8,examined the effect of the oil?different rates?and the results were compared with those of Azone,a classic permeation enhancer.5.Effect of RCEO and VTEO on the microstructure of skin.The hair of rat dorsal skin was removed,one day after RCEO,VTEO and azone were administered for 8h in vivo,and the skin collected for HE staining and compared in SEM,to observe the structural change with LM and SEM.6.The secondary structure of stratum corneum with RCEO,VTEO and azone were studied by ATR-FTIR.The RCEO,VTEO and azone were administered in human skin in vivo for 8h.The administered skin was cleared and then determinated by ATR-FTIR.According to IR spectra of lipid substances.It was observed that the changes of CH₂ vibration peak position and position of keratinamide Amide Infrared Bands peak.7.The effect of RCEO,VTEO and azone on the Ca²⁺-ATP enzyme activity in HaCaT.The HaCaT cells were cultured in serum-free medium,and after more than 90%of cells adherented,different concentrations of drug-containing medium were added,cultured for 30min in 37?,centrifuged and discarded supernatant fluid,and then added a certain amount of normal saline,by sonication of cells,the Ca²⁺-ATP enzyme activity were determinated with the ultramicro-Ca²⁺-ATP enzyme kit,and the protein content in cells was determinated to calculate the Ca²⁺-ATP enzyme activity.Result:1.The optimal extraction process of RCEO is distillated for 6 h after volatile medical materials were macerated for 1h with 10 times amount of water.The optimum technical conditions of VTEO as follows:adding water 8 times as much as the medicine and extracting 6 h,the RCEO oyield was 0.6%,and The VTEO oyield was 0.2%.2.The results apparent permeability coefficient of Ind in vitro,as follows:RCEO?2%,1%,0.5%?:12.3,14.9,17.9,VTEO?2%,1%,0.5%?:7.59,15.6,19.2,Azone:2.4.DS results as follows:RCEO?2%,1%,0.5%?:8.27,9.63 and 7.72,VTEO?2%,1%,0.5%?:1.14,8.74,6.88;Azone:1.67.3.The cumulative penetration of micro-dialysis of DS treated by RCEO and azone in vivo were 174.37±10.56?g·cm^-2 and 129.88±5.39?g·cm^-2,which was significantly different from the blank group.The drug could not be detected after the time of VTEO.4.Except 2%RCEO group,no obvious erythema or edema were observed.The IC_?50?value of RCEO,VTEO and Azone for HaCaT cells was 97.06?g·mL^-1,285.10?g·mL^-1,17.08?g·mL^-1.5.Observational results of the skin under LM:Compared with blank group,the stratum corneum of skin dealed with RCEO,VTEO and azone all become more disordered,the gap between layers become bigger.A noticeable thickening,disorder,middling dense flocculation lump structure appeared.The conjunction between the stratum corneum cells had not significant difference.SEM observations showed that after treatment with RCEO,VTEO and azone,in transverse section of the skin:wrinkles increased significantly,interhemispheric fissures widened,and keratinocytes were loose and detached more clearly;In longitudinal section:stratum corneum was loosened,the gap between the epidermis and the dermis was widened,and the most outer layer of stratum corneum shedded obviously.6.ATR-FTIR results showed that compared to solvent group and blank group,the CH₂ stretching vibration peak position of lipids in the human after treatment with the VTEO,RCEO and azone shifted to higher wavelengths.The peak of keratin Amide I Infrared Bands shifted to the higher peak wavelength after treatment with the VTEO and RCEO,there was no significant change in Azone group.Both peak at keratin Amide II Infrared Bands shifted to the lower wavelength,and VTEO group peak of keratin Amide II Infrared Bands splits into two peak.7.The Ca²⁺-ATP enzyme activity was inhibited by VTEO?the rates of high and medium?,RCEO?differentrates?and azone?differents?,the difference being significant as compared with the normal control group?P<0.01?.And decreased with increasing concentration of the transdermal enhancers.Conclusion:The RCEO and VTEO had the effect of promoting transdermal absorption of DS and Ind in vitro,and the permeation effect is stronger than Azone.They can further increase the enhancing effect on fat soluble compound(ER_DS>ER_Ind).The RCEO permeation effect of transdermal absorption in vivo was superior to Azone,which is consistent with the results in vitro.However,the VTEO transdermal absorption is inconsistent with the results in vitro.The cause of these differences needs further researchs.The two oil safety is higher,so can become excellent natural penetration enhancers.Both improve the permeability of the drug by extracting lipids,increasing the degree of freedom of keratin amides,and ordered structure of the stratum corneum.The penetration enhancement mechanism of RCEO and VTEO was associated with the concentrations of Ca²⁺in keratinocytes,which changes the membrane fluidity and membrane potential of HaCaT cell.