Study On The Penetration-enhancing Effects Of Menthol And Its Mechanisms

Objective:To determine thepenetration effect of menthol on the transdermal absorption of 5-Fu. in combination with azone;and to explore the percutaneous penetration mechanism of menthol.Methods:1 To evaluate penetrating function of menthol and azoneUsing two-room diffusion cell device in vitro and selecting the rats back skin as transdermal barrier and 5-Fu as a model drug, the steady-state flow and permeability coefficient of 5-Fu were calculated, the penetrating effects of different concentrations of menthol and azone were explored with the time-quantity method.2 The effect of menthol on the skin micro-structure2.1 The structures of skin dealed with menthol were observated by light microscopyThe hair of rat skin was removed,and one day after menthol was administered for 8h in vivo,then the rats were killed and the skin taking medication were peeled off and fixed in 10% formaldehyde solution for 24h,routine paraffin section and hematoxylin - eosin (HE) staine,and then the structures of skin observated by light microscopy.2.2 The structures of skin dealed with menthol were observated by scanning electron microscopyThe hair of rat skin was removed, and one day after menthol was administered for 8h in vivo,then the rats were killed and the skin taking medication were peeled off and fixed in 2.5% glutaraldehyde for 24h. The skins were cleared in 4℃phosphate buffer (pH7.4), dehydrated in series of alcohol and alcohol was replaceed with acetone.Then the skin were immersed in the isoamyl acetate for 30min and dried in CO2 critical point, finally sticked, film was plated with ion sputtering method,and then the structures of skin observated by scanning electron microscopy.2.3 The structures of skin dealed with menthol were observated by transmission electron microscopyThe hair of rat skin was removed, and one day after menthol was administered for 8h in vivo,then the rats were killed and the skin taking medication were peeled off and fixed in 2.5% glutaraldehyde for 24h. The skins were cleared in 4℃phosphate buffer (pH7.4), postfixed in 1% osmium tetroxide and dehydrated in series of acetone. Finally the skins were penetrated in ethoxyline resin and embedded orientation,sliced for ultrathin sections,and the ultrathin sections stained in uranyl acetate-lead citrate,and then the structures of skin observated by transmission electron microscopy.3 The structures of skin lipids dealed with menthol were studied by ATR-FTIRThe hair of rat skin was removed, and one day after the rats were killed and the skin were peeled off and the stratum corneum were stripped. Then the stratum corneum were immersed in the menthol solution for 24h in 37℃. Finally the stratum corneum were dried completely and the infrared spectrum of stratum corneum were determinated by ATR-FTIR. The menthol was administered in human skin in vivo for 8h.The administered skin were cleared and then determinated by ATR-FTIR.According to IR spectra of lipid substances,the changes of CH vibration peak position were observated,and the changes of two types of skin lipid structure and conformation were explored in order to determine the effect of menthol on the skin stratum corneum structure.4 The effect of menthol on the Ca²⁺ and Ca²⁺-ATP enzyme activity in keratinocytes4.1 The effect of menthol on the Ca²⁺ in keratinocytesThe human keratinocytes were cultured in serum-free medium,and after more than 90% of cells adherented,different concentrations of menthol was added in the medium.By using flow cytometer(FCM) bound with Fluo-3/AM staining technology,the effects of menthol on the free intracellular calcium concentration of kerationocytes were semi-quantitated.4.2 The effect of menthol on the Ca²⁺-ATP enzyme activity in keratinocytesThe human keratinocytes 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,cells were broken with freeze-thaw method rapidly, the Ca²⁺-ATP enzyme activity were determinated with the ultramicro-Ca²⁺-ATP enzyme kit.Results:1 To evaluate penetrating function of menthol and azoneThe concentration of menthol in 250mg/mL with azone in the 250μl/mL have some synergy, but the concentration of menthol greater than 500mg/mL with azone greater than 500μl/mL did not show synergy.2 The effect of menthol on the skin micro-structure2.1 Light microscope observationsCompared with solvent group and blank group,the stratum corneum of menthol group become rarefaction and more stratum corneum shedded,what's more,the outer stratum corneum of menthol group in some regions become girdle-shaped dissociation. The cell spaces become bigger. The structure of stratum corneum were changed by menthol,which made the stratum corneum become rarefaction and the cell space become bigger,so it cut down the barrier effect of the skin.2.2 Scanning electron microscopy observationsCompared with solvent group and blank group,the wrinkle of skin dealed with menthol become more obviously,stratum corneum shedded obviously,become rarefaction and have hole-like structure,and the speace of epidermis widen.Longitudinal section observation: the structure of stratum corneum loose obviously; the most outer layer of stratum corneum shedded obviously, at all levels a clear gap could observate at all levels, and the epidermis separated from the dermis. Menthol affected the structure of epidermis,made them become rarefaction and the order-pykno-structure were broken.So they reduced the barrier effect of the skin and increase the permeability of the skin. 2.3 Transmission electron microscopy observationsCompared with solvent group and blank group,the stratum corneum of skin dealed with menthol become more disordered,the gap between layers become bigger. The normal lamellar-membrane structure of lipids all or mostly disappeared,and a noticeable thickening,disorder,middling dense flocculation lump structure appeared. The conjunction between the stratum corneum cells had not significant difference. Menthol disturbed the ordered arrangement of stratum corneum,made the space of stratum corneum widen and the structure become rarefaction,and so it increased the permeability of the skin.3 The structures of skin lipids dealed with menthol were studied by ATR-FTIRCompared to solvent group and blank group, the CH2 stretching vibration peak position of lipids in the human and rats stratum corneum treated with menthol shifted to the peak for 3-4 wavelength, which showed the conformation of lipids had changed, and the randomness of stratum corneum lipids increased.4 The effect of menthol on the Ca²⁺ and Ca²⁺-ATP enzyme activity in keratinocytes.4.1 The effect of menthol on the Ca²⁺ in keratinocytesCompared wiht blank group, the fluorescence intensity of Ca²⁺ in the KC treated with different concentrations of menthol shifted to right and increased; Compared with solvent group,menthol whose concentration excel to 25.00μmol/L could significantly increase fluorescence intensity of Ca²⁺ in the KC(p<0.01), the Ca²⁺ fluorescence intensity of 25.00μmol/L of menthol and 50.00μmol/L menthol group had no significant difference( p> 0.05); 100μmol/L of menthol compared with the other groups had significant difference (p<0.01); 200μmol/L, 400μmol/L of menthol group had no significant difference(p>0.05); the change of Ca²⁺ fluorescence intensity does not exist a concentration-dependent manner.4.2 The effect of menthol on the Ca²⁺-ATP enzyme activity in keratinocytesCompared wiht blank group,the solvent group and different concentration of menthol all reduced the Ca²⁺-ATP enzyme activity and had significant difference(P<0.01).which showd that 0.1%DMSO and different concentration of menthol had an different effect on the Ca²⁺-ATP enzyme activity. Compared wiht solvent group,the effect of menthol group whose concentration was from 25.00μmol/L to 400μmol/L on the Ca²⁺-ATP enzyme activity had significant difference(P<0.01,P<0.05).Conclusions:1 The concentration of menthol in 250mg/mL with azone in the 250ul/mL have some synergy, but the concentration of menthol greater than 500mg/mL with azone greater than 500ul/mL did not show synergy.2 Menthol had an impact on the skin micro-structure,which made the wrinkle of skin increased obviously,the arrangement of stratum corneum disordered, the cranny between epidermis widen,the gap between layers bigger and the stratum corneum become rarefaction and shedded from epidermis.The results showed that menthol changed the order dense structure,made the epidermis become rarefaction and the permeability of skin increased.3 Menthol changed the conformation of lipids in the stratum corneum of human and rat epidermis,so which made the order dense structure of stratum corneum changed..4 Different concentrations menthol increased the Ca²⁺ concentration and reduced the Ca²⁺-ATP enzyme activity in the keratinocytes.