An in vitro Permeation of Phyllanthus amarus Extract Through Human and Shed Snake Skins

An in vitro permeation of a hydroalcoholic extract of Phyllanthus amarus (PaE) was investigated using excised human epidermis and shed king cobra skin as the barrier membranes. Donor and receptor compartments of diffusion cells were pH−controlled to simulate the permeation environment of the human skin. The PaE was analyzed by using normal−phase densitometric TLC detected at λ 280 nm and toluene:ethyl acetate (17:3) as the mobile phase. There were four major components observed in the saturated solution of the donor at pH 5.5. Over 24 h, only one component, possibly phyllanthin, was found in the receptor solution after permeation across the human epidermis, while two components, possibly phyllanthin and an another unknown permeated, permeated through shed snake skin. When compared to the saturated donor concentration, phyllanthin gave permeation fluxes of 0.04±0.01 and 0.12±0.02 %⋅cm −2 ⋅h −1 through the human and shed snake skins, respectively. It seems that only certain component(s) of the P. amarus extract could permeate through the skins, and by comparison, at a slower rate across the human skin than shed snake skin.


INTRODUCTION
Dermatological applications of herbal extracts for pharmaceutical and cosmetic purposes may have delivered some components which were not well documented. In vitro permeation studies by using various designs of diffusion cells have been one of the methods used to simulate percutaneous absorption of drugs or other compounds [1] . In vitro permeation studies of herbal extracts could be a supportive part for development of rationale use of phytotherapy. Phyllanthus amarus Schum & Thonn. (P. amarus), family Euphorbiaceae, was selected for study due to its traditional claims for treatment of skin diseases and recent reports on its activities as an antiinflammatory [2,3] , antibacterial [4] , antifungus particularly dermatophytic fungi [5] and potent antioxidant properties [6] . Active components of P. amarus such as phyllanthin and hypophyllanthin have been isolated and identified [7,8] . Different species of Phyllanthus could be similar in morphology but different in pharmacological activities to P.amarus [2,8] , it is necessary to confirm the plants being used.
The skin plays a vital role in controlling the passage of substances.
The permeation of any compounds through the skin involves physicochemical characteristics of the compounds, the vehicle and the barrier membrane [9,10] . Passage of a dissolved compound across the human skin generally occurs from an outer skin pH of about 5 through various layers, and if possible, to a sink condition with a physiological pH of 7.4. Human skin is not as readily available as the waste material like shed snake skin, and both have been shown to give comparable in vitro permeation [9,10,11,12] . This includes king cobras (Ophiophagus hannah) [12] which shed large pieces of skin readily usable for several experiments.
This study employed the human epidermis and shed snake skin as the barrier membranes and controlled the pH, which is likely to be one of the vital factors influencing the permeation of a hydroalcoholic extract of P.amarus (PaE). The permeation profile studied by this type of herbal extract could provide a vital information for further product development. Preparation of the human epidermis and shed snake skin: Fresh, surgically excised samples of human skin were obtained directly after breast plastic surgery with informed consent following ethical approval. Shed king cobras skin were kindly gifted and collected freshly from the Khok Sanga King Cobra Club, Khon Kaen, Thailand. These were kept at −21°C until use. The human epidermis was separated as previously described 15 while the dorsal scales of the shed snake skin were used as received. DNA identification of P. amarus by sequence characterized amplified region (SCAR): Fresh leaves of P. amarus were used for genomic DNA extraction following a previously described method 16 . The PCR amplification and reactions were performed in a thermocycler (PCR Sprint, U.S.A.) and the products were electrophoretically separated in 1.2% (w/v) agarose gel and staining-visualized with ethidium bromide. The RAPD fingerprint pattern resulting from amplification was compared with the SCAR primers of P. amarus collection at the Department of Biology, Khon Kaen University.  PCR amplification of DNA of both collections (P1 and P2) using P. amarus−specific primers gave rise to a specific band of approximately 500 bp. The extraction process gave reproducible yields of a fine, hygroscopic and light-brown powder. PaE was hydrophilic, i.e. soluble in water, ethanol and insoluble in chloroform and n−hexane, and hygroscopic.
The TLC chromatograms of PaE saturated in the donor solution showed 2−3 resolution color bands on silica plate at R f value about 0.1−0.5. The extract showed a good resolution of peak no. 1 and 2 at average R f of 0.21 and 0.28, as shown in Figure 2, which resembles the peaks of phyllanthin [8] and an unknown. The receptor solutions using shed snake skin as the barrier at 6, 12 and 24 h could separate 3−4 peaks (as shown in Figure 3).
At sink conditions, the permeation of phyllanthin linearly increased with time from donor solution with the skin pH of 5.5 to receptor solution at pH 7.4. An attempt to estimate the permeation rates of peak no. 1 (at R f 0.21) during 6−24 h, analyzed by linear regression, were 0.04±0.01 and 0.12±0.02 %⋅cm −2 ⋅h −1 through the human epidermis and shed snake skin, respectively, with correlation coefficient of more than 0.999. Another unknown substance as shown by a presence of peak no.2 at R f 0.28 permeated when using shed snake skin at a slower rate of 0.006 %⋅cm −2 ⋅h −1 .
It is concluded that phyllanthin, one of the major components of P.amarus, could permeate through the barrier structure of both human and shed snake skins. Further study is required to qualitatively identify each component and investigate the competitive permeation profile of the mixtures which are usually found when using any herbal extracts. CONCLUSION Some components of the extract of P.amarus were shown to permeate through the scales of the shed skin of king cobras and the human epidermis. The permeation could be a passive transport mechanism influenced by the physicochemical properties of the compounds involved. The human skin gave a slower rate of permeation than the scales of shed snake skin which could be the result of differences in lipid compositions. This, however, suggests a potential development of a topical use of PaE, provided that the activities of the substance(s) were proved.