Preliminary Study On The Mechanism Of Transdermal Penetration Of Traditional Chinese Medicine And Its Mechanism

"The medicated plaster must use these herbs like Zingiberis Rhizoma, Zanthoxyli Pericarpium and Ricini Senen as messenger drugs, which can dredge meridian and induce resuscitation", according to the statement on Li Yue Pian Wen (Published in 1870), an ancient classic literature on topical remedies in China. These statements reflect the combination characteristics of TCM(traditional Chinese medicine) topical preparations, these herbs which are rich in essential oil, can effectively promote the percutaneous absorption of the active components in a prescription, and resemble the role of the transdermal penetration enhancers in modern transdermal delivery system. In order to shed light on the transdermal promoting regularity of these essential oils on TCM effective compounds and understand their promoting mechanisms, Zanthoxyli Pericarpium and Menthae Haplocalycis Herba, which were commonly used in TCM topical preparations, were chosen to investigate the effect of their essential oil on the transdermal absorption of TCM compounds. This study would provide the theoretical guidance and data for the development of TCM transdermal penetration enhancers, and add weight to understand the rationality of combination characteristic of TCM topical preparations.Gas Chromatography-Mass Spectrometer (GC-MS) was employed to study the chemical compositions of essential oil from Zanthoxyli Pericarpium and Menthae Haplocalycis Herba. The apparatus like ultraviolet spectrophotometer and conductivity meter were used to measure the physico-chemical parameters of the essential oils. Gas chromatography (GC) was utilized to establish the fingerprint spectrum of the different batches of the oils. The cytotoxicity of the essential oils on HaCaT keratinocytes and CCC-ESF-1 fibroblasts was studied using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Scanning electron microscope(SEM) was used to observe the morphological change of rat skin. The major compounds of Z.bungeanum oil (essential oil from Zanthoxylum bungeanum Maxim.) were terpinen-4-ol(18.42%), 1,8-cineole(15.49%) and limonene(7.47%). The major compounds of M.haplocalyx oil (essential oil from Mentha haplocalyx Briq.) were menthol (51.00%), 2-isopropylidenecyclohexanone (11.49%) and menthone (6.11%). Compared with the well-established and standard penetration enhancer Azone, both of Z.bungeanum oil and M.haplocalyx oil showed lower toxicities in HaCaT keratinocytes and CCC-ESF-1 fibroblasts. Meanwhile, there were no obvious morphological alterations in the rat skin after treated with the essential oil, suggesting that both Z.bungeanum oil and M.haplocalyx oil possessed relatively low skin irritation potential.According to the study of quantitative structure-permeation relationships (QSPRs) models, a series of TCM active compounds with a wide range of lipophilicity (logP values), namely osthole (OT, logP=3.85), tetramethypyrazine (TMP, logP=2.34), ferulic acid (FA, logP=1.26), puerarin (PR, logP=-0.35) and geniposide (GP, log.P=-1.01), were chosen to replace the complex active compounds in TCM external preparations. The transdermal promoting effect of the essential oils on these model drugs were tested using in vitro permeation studies in which vertical Franz diffusion cells and rat abdominal skin were employed. It was found that both of Z.bungeanum oil and M.haplocalyx oil could effectively enhance the transdermal absorption of five model drugs in a concentration-dependence manner. After treatment with different concentrations of Z.bungeanum oil, the logarithm values of enhancement ratio(ER) tended to be a negative linear relationship to the logP values of model drugs:1%Z.bungeanum oil:logER=-0.0681og?+0.0641(r=0.3620); 3%Z.bunganum oil:logER=-0.34881ogP +2.4051(r=0.9567); 5% Z.bungeanum oil:logER=-0.068471ogP+3.4342 (r=0.9941), implying that Z.bungeanum oil exhibited the optimum permeation effect for the hydrophilic drugs. While the logarithm values of enhancement ratio(ER) appeared to be parabolic curve with the logP values of model drugs in the treatment with various concentrations of M.haplocalyx oil:3% M.haplocalyx oil: logER=-0.0557logP2+0.0751ogP+0.7292(r=0.5540); 5%M.haplocalyx oil: logER=-0.0166logP2-0.48061ogP+2.8279 (r=0.9318), suggesting that M.haplocalyx oil displayed a higher capacity for the transdermal absorption of moderate hydrophilic drugs (an estimated log? value of-0.50).Then, tetramethypyrazine(TMP) and puerarin (PR) were chosen as lipophilic and hydrophilic model drug respectively, and transdermal microdialysis was employed to investigate effect of essential oils on the the in vivo transdermal absorption of two model drugs. The effect of drug concentration and perfusion flow of the perfusate on the recovery of the linear probe was investigated in the in vitro microdialysis system. The consistency of the recovery and delivery of microdialysis probe was assessed by a concentration difference method, and then the retrodialysis method was used to measure the in vivo recovery of the microdialysis probe for TMP and PR, respectively. Similar to the drug administration in the in vitro permeation studies, the promoting effect of essential oil on the transdermal absorption of model drugs was assessed in the in vivo microdialysis system. According to the results of the study, the recovery of microdialysis probe for TMP and PR was independent of drug concentration, and was exponentially associated with the perfusion flow of the perfusate. The recovery of the probe was identical to its delivery, which indicated that drug molecules in the dialysis membrane had no interaction. The in vivo recoveries of microdialysis probe for TMP and PR were 59.17±1.22% and 19.85±1.60%, respectively. The results of in vivo transdermal microdialysis demonstrated that Z. bungeanum oil could promote the transdermal absorption of both of TMP and PR in a concentration-dependent manner. After treatment with different concentrations of Z.bungeanum oil, the in vivo-in vitro correlation coefficients of enhancement ratio(ER) for TMP and PR were 0.928 and 0.999, respectively. The low concentration of M.haplocalyx oil did not significantly enhance the transdermal absorption of TMP. While the oil concentration reached up to 5%, M.haplocalyx oil showed an obvious effect on TMP with an ER value of 12.50. Meanwhile, the in vivo-in vitro correlation coefficient of enhancement ratio for TMP was 1.000 in the treatment of M.haplocalyx oil. The PR content could not be effectively detected using HPLC method on account of the poor transdermal absorption of the hydrophilic drug. In addition, the low recovery of probe for PR and weak promoting effect of M.haplocalyx oil probably leaded to the difficulty in PR analysis.To compare the skin penetration enhancement effect of essential oil and its main compounds on five model drugs, terpinen-4-ol(18.42%), 1,8-cineole(15.49%) and limonene(7.47%) in Z.bungeanum oil and menthol(51.00%), menthone(6.11%) and pulegone(1.32%) in M.haploclayx oil were tested using in vitro permeation studies, respectively. The enhancement permeation capacities by Z.bungeanum oil and its main compounds were in the following increasing order:terpinen-4-ol= l,8-cineole<limonene<Z. bungeanum oil. While the compounds in M.haplocalyx oil exhibited better promoting effect than M.haplocalyx oil in the decreasing order: menthone>menthol>pulegone>M.haplocalyx oil.The saturation solubilities of five model drugs were measured to monitor the effect of essential oils on the drug thermodynamic activity. Stratum corneum(SC)/vehicle partition coefficients of model drugs with and without essential oils were also determined to investigate the effect of essential oil on the partition of model drugs in skin tissue. It was found that both of Z. bungeanum oil and M.haplocalyx oil could improve the saturation solubilities of lipophilic drugs, while had negligible impact on hydrophilic drugs, suggesting that the essential oils had certain effect on the thermodynamic activity of lipophilic drugs. Meanwhile, Z.bungeanum oil could promote the partition of lipophilic drug into stratum corneum, while M.haplocalyx oil tended to enhance the transfer of hydrophilic drug into stratum corneum.Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and molecular dynamic simulation were employed to investigate the effect of essential oils on lipophilic tails and polar head groups of intercellular lipids, respectively. Transmission electron microscopy (TEM) was used to observe the SC alteration after treated with essential oils. FTIR studies revealed that the SC treated with Z.bungeanum oil produced a higher shift in asymmetric and symmetric C-H vibration peak positions, and the higher the oil concentration was, the greater the shift in C-H stretching vibration was, which implied that Z.bungeanum oil reduced the SC barrier function by disrupting and extracting the SC lipid. M.haplocalyx oil changed the SC barrier function mainly by perturbing the asymmetric C-H vibration peak. In addition, the decrease in peak area of two amides and the shift in wavenumber were not observed after treated with essential oils, suggesting that both of Z.bungeanum oil and M.haplocalyx oil had negligible impact on SC keratin. TEM studies demonstrated that the rat SC gradually decreased with the oil concentration increasing, and the SC gap was gradually enlarged as well.To investigate the effect of essential oils on the inner epidermis, the cell functions associated with drug diffusion were measured using HaCaT keratinocytes, such as membrane fluidity and membrane potential. It was found that the essential oils studied could alter the fluidity of the inner epidermis by improving membrane fluidity and decreasing membrane potential. Meanwhile, the essential oils perhaps change the Ca2+ balance of the cell inside and outside by decreasing Ca2+-ATPase activity and increasing intracellular Ca2+ of HaCaT cells, which indicated that the alteration of cell functions was probably associated with the Ca2+ balance.