br Methods br Transfollicular penetration of

Methods
Transfollicular penetration of herbs and their active constituents used for hair loss treatment The transfollicular administration of pharmacologically active molecules is recognised as a significant element for therapeutic interest, mainly with regard to delivery to specific sites of the hair follicle and the reduction of hepatic metabolism and systemic toxicity [18]. Moreover, hair follicles are privileged pathways for topically applied formulations with specific compounds, which enter faster into these shunts than through the stratum corneum, a main skin barrier [19]. The hair follicle itself can be divided into at least four target areas for topical application: (1) the sebaceous gland, (2) the bulge region, (3) the hair matrix cells and (4) the hair follicle infundibulum [20]. The latter provides an interrupted barrier with increased permeability and is surrounded by a high density of immune cells and an extensive capillary network important for penetration and systemic ionomycin receptor of active agents [21]. Topically applied active compounds from herbal products must be able to penetrate epidermal barrier and reach the specific sites of the hair follicles to affect hair growth via different biological pathways. Teichmann et al. [22] and Lademann et al. [23] demonstrated that a 2minute contact between the shampoo with caffeine and the skin surface was sufficient for its penetration deeply into the hair follicles and remains there for up to 48h, even after hair washing. This demonstrates the long-term reservoir function of the hair follicles for topically applied substances such as caffeine. Moreover, caffeine inhibits the activity of the 5α-reductase and leads to a significant stimulation of human hair follicle growth in vitro [24]. Despite caffeine, a large number of active components produced by plants is not able to pass through the skin barrier. Lipid solubility and molecular size are the major limiting factors for phytomolecules to pass the biological membrane after topical application. Therefore, the use of novel drug delivery systems containing herbal formulation has enhanced the therapeutic effects of plant extracts and facilitates their penetration through the skin [25]. Mixture of Aloe vera gel (1%) and Apium graveolens ethanolic extract (2%) in the form of O/W microemulsion was effective in penetrating into the pilosebaceous gland and showed the highest hair growth promoting activity [26]. Lipophilic curcumin (hair growing agent) incorporated in O/W microemulsion and in an amphiphilic cream was detected in human follicular infundibula and follicular orifices, respectively [27]. Monoolein cubosomal suspension (1%) containing 3% herbal extracts mixture (Poria cocos, Thuja orientalis, Espinosilla, Lycium chinense Mill, Coix lacryma-jobi, Polygonum multiflorum Thunberg) significantly enhances the skin permeation of the herbal extracts as potent as minoxidil solution (2.4%) [28]. Among them T. orientalis, P. multiflorum and Espinosilla extract showed hair growth promoting efficacy. Hair growing ingredients - hinokitiol loaded in monoolein cubosome revealed much higher skin permeation than hinokitiol dissolved in water, even the concentration of hinokitiol in cubosome suspension was lower than the concentration in the aqueous solution [29]. Ding et al. [30] showed that penetration of the flavonoid-rich Allium cepa leaf extract encapsulated in β-cyclodextrin proceeds across skin via both follicular and transcellular routes. Tsujimoto et al. [31] found that the dispersion liquids containing hair growing ingredient (hinokitiol, glycyrrhetinic acid) encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanospheres exerted human scalp-pore permeability 2.0- to 2.5-fold higher than samples containing only the hair growing ingredients. Moreover, the hair growing effect of the encapsulated PLGA nanospheres was improved in the in vivo tests, which showed that hair growth cycle was transformed from the resting phase to the growing phase.