The Study On The Promoting Penetration Mechanism Of Borneol And Menthol Involving The P-glycoprotein

ObjectiveThis study was to explore the influence of P-gp on the effect of borneol and menthol atbehavioral and cellular levels by means of investigating the relationship between thetranslation function of P-gp and the effect of transdermal drug, and discover its potentialvalue to the transdermal drug delivery system. It provided a theoretical foundation for thedesign and application of the penetration enhancer by better understanding of themechanism of the promoting penetration of borneol and menthol.Methods1. Immunohistochemical experimentThe distribution of the staining of the mice back skins were observed by microscopeusing the immunohistochemical SABC method.2. In vitro skin permeability experimentA modified Franz diffusion cell device were used, the mice back skins were selected astransdermal barrier. The promoting penetration effects were evaluated by the differentcumulative permeation amount between each group.3. Drug concentration screening experiment--MTTMTT method was used for toxicity experiment based on cell model, OD values weredetermined by Microplate reader and calculated the cell survival rate. 4. Immuno-fluorescence experimentImmuno-fluorescence experiment were conducted to determine the expression index ofP-gp in HDMEC cell, which were incubated with various concentrations of menthol,borneol, DMSO control or blank control.5. RT-PCR experimentHDMEC cells were cultured in medium supplemented with various concentrations ofmenthol, borneol or control. The expression levels of MDR1mRNA gene were determinedby RT-PCR method.6. Western-blot experimentHDMEC cells were cultured in medium supplemented with various concentrations ofmenthol, borneol or control. The expression level of P-gp protein were determined byWestern-blot.7. Acculation and efflux experimentHDMEC cells were cultured in medium supplemented with various concentrations ofmenthol, borneol, verapamil or control. The fluorescence level were semiquantitatived byimmunofluorescence method.8. Transdermal permeation experiment in vitroA modified Franz diffusion cell device were used, the mice back skins were selected astransdermal barrier. Various concentrations of menthol, borneol or control were added toRh123or sodium fluorescein solution, set verapamil as positive control. The promotingpenetration effects were evaluated by the different cumulative permeation amount betweeneach group.9. Transdermal permeation experiment in vivoThe minces were treated with various concentrations of menthol, borneol or control.Tissue extracts samples were collected, the permeation amount of Rh123were determinedby HPLC. Results1. Distribution and Expression of P-gp in skinDAB brown staining results indicated that P-gp were distributed in epidermis, dermisand subcutaneous tissue area.2. Function of the P-gp in skinThe results from in vitro skin permeability experiment indicated that the cumulativepermeation amount of Rh123were more than sodium fluorescein(P<0.01), The cumulativepermeation amount of Rh123were increased with verapamil(P<0.01), but the cumulativepermeation amount of sodium fluorescein had no significant difference（P>0.05）.3. Drug concentration determinationThe results of MTT test indicated that if the viability of HDMEC cell keep more than80%, the concentration of borneol should lower than500μM, the concentration of mentholshould lower than600μM and the concentration of verapamil should lower than30μM.4. The influence of borneol and menthol on expression of P-gp in HDMEC cellThe immuno-fluorescence results showed that there were green fluorescences inHDMEC cells. Compare to blank control, the DMSO group had no significantdifference(P>0.05) on fluorescence level. The fluorescence level decreased when theconcentration of borneol increase (P<0.05). The fluorescence level increased when theconcentration of menthol increase (P<0.05).5. The influence of borneol and menthol on expression of MDR1RNAThe HDMEC cells were incubated with borneol and methol for48hours, theexpression level of MDR1mRNA were decreased when the concentration of borneolincreased, and increased when the concentration of menthol increased. All had a significantdifference compare to blank control（p<0.01）.6. The influence of borneol and menthol on the expression of P-gp proteinThe HDMEC cells were incubated with borneol and methol for48hours, theexpression level of P-gp protein were decreased when the concentration of borneolincreased, and increased when the concentration of menthol increased. All had a significantdifference compare to blank control（p<0.01). 7. The influence of borneol and menthol on the function of P-gpThe cumulative permeation amount of Rh123in HDMEC increased when theconcentration of borneol increased and decreased when the concentration of mentholincreased（p<0.01）. The efflux amount of Rh123decreased when the concentration ofborneol increased（p<0.01）. But the efflux amount of Rh123didn’t increased when theconcentration of menthol increased, but they all higher than the blank（p<0.01）.8. The influence of borneol and menthol on promoting penetration involving the P-gpIn vitro test indicated the permeation amount of Rh123in menthol groups were morethan the sodium fluorescein groups (p<0.01). The permeation amount of Rh123inborneol groups were less than the sodium fluorescein groups (p<0.01).The permeationamount of Rh123in menthol groups were more than borneol groups (p<0.01).9. Transdermal permeation experiment in vivoThe results from HPLC were all lower than LOQ.Conclusion1. P-gp were distributed and expressed in skin.2. The P-gp in skin can improve the permeability of its substrate but not work fornon-substrate.3. Borneol can restrain the expression of MDR1mRNA and P-gp, but methnol can inducethe expression of MDR1mRNA and P-gp.4. Borneol can improve the permeation amount of the substance and decrease the effluxamount of the substance. Menthol can decrease the permeation amount of the substance andimprove the efflux amount of the substance.5. P-gp can decrease the permeation enhancing effect of borneol for Rh123, and increasethe permeation enhancing effect of menthol. But it didn’t influent the permeability ofsodium fluorescein.