INTRODUCTION

The melanophores form a very responsive organiza- tion and changes in their environment may contribute to either dispersion or aggregation of melanin granules with a corresponding darkening or blanching of the

ARTICLE INFORMATION:

*Corresponding Author: Received 20th November, 2015

Accepted after revision 10th December, 2015 BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 NAAS Journal Score : 3.48

©A Society of Science and Nature Publication, 2015. All rights reserved.

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skin. Various hormonal and pharmacological agents have been found to induce melanophores aggregation or dispersion in amphibian species via the involvement of alpha or beta adrenergic receptors (Bagnara & Hadley., 1973, Novales & Davis., 1969). Melanophores, the black pigment cells of the amphibian provide an ideal and

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unique model system and have been pharmacologically used since the past many years and have been yielded fruitful in"uences which are being used for the thera- peutic applications of pigmentary disorders, (Aspergren., 2009, Ali et al.,2015 and Ali and Naaz 2015).

Various agents such as kojic acid, hydroquinone and phenolic compounds though are reported to be tyrosi- nase inhibitors causing skin depigmentation, but have adverse coetaneous toxicity; including tumors hence there has been an increasing impetus to develop alter- native hypopigmenting agents (Ishikawa et al., 2007, kim et al., 2007). Recently it has been also that some secondary metabolites in various plants, affect the for- mation of melanin pigment with inhibiting tyrosinase activity. Resveratrol a natural occurring stilbene isolated from the fruit skin of the A. hypogaea has been reported to show several biological properties like inhibition of platelet aggregation, antioxidant, anti-in"ammatory and free radical scavenging (Wang et al., 2002; Ovesna et al., 2006; Udenigwe et al., 2008; Lee et al., 2010).

The present study was carried out to determine the effects of seed skin extract of A. hypogaea along with active ingredient pure resveratrol on the isolated dorsal skin melanophores of the toad Bufo melanostictus which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents of plant origin leading to skin paling via stimulation of adrenoceptors.

MATERIAL AND METHODS

Ethanolic extract was prepared according to the method of Nepote et al. (2004), where seed skins were obtained from the peanuts by boiling. To obtain the extract the seed skins were defatted by n-hexane (5 ml each 10g) during 12 h at room temperature. De-fatted seed skins (20g) were extracted with 300ml ethanol during 24 hrs by maceration in darkness at room temperature. The extract was !ltered and the residue was extracted again under the same conditions. The combined !ltrate was evaporated to dryness in evaporator in 35°C. Sticky brown extract weighing 2.95g was obtained. The extract was re-dissolved in distilled water for in vitro studies using different concentration. The extract was subjected to the analysis of its chemical constituent by using High performance thin layer chromatography (HPTLC). The standard Resveratrol, CAT No- R5010, obtained from Sigma-Aldrich, USA was used in the present study. A stock solution of standard Resveratrol was prepared in 0.2% dimethylsulphoxide (DMSO) and used in further dilutions

Adult Bufo melanostictus during the rainy season and kept in large aquaria. Prior to the experiments, they were

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allowed to acclimatize to laboratory conditions for two days. Diseased, injured or lethargic toads were removed and only active, uniformly sized toads were used. For in vitro studies the dorsal skin was peeled away, a series of nearly 4mm diameter skin pieces were cut out with a sharpened steel scissor and the pieces were kept in 10 ml of Amphibian Ringer Saline (ARS), containing 111 mm of sodium chloride, potassium chloride 2 mM, calcium chloride 1 mM and sodium hydroxide 2 mM, in 100 ml of double distilled water at pH 7.4, in small petri dishes and they were equilibrated in saline medium for 15-20 min with frequent stirring. dorsal skin pieces of the toad, containing about 50-100 melanophores, in the control ARS had the tendency of dispersion within a period of 30-60 minutes, hence they were brought to the intermedi- ate state of neither aggregation nor dispersion by using a very low concentration of adrenaline (2×10-8 g/ml) as per the modi!ed standard method of Ali et al. (1998).

This facilitated the accurate measurement of the aggregation responses of the melanophores induced by the seed skin extract of A. hypogaea and its active ingre- dient resveratrol. The responses of control as well as of those melanophores which were incubated in 10 ml ARS saline containing various concentrations of extract of A. hypogaea (1×10-6 to 6.4×10-5 g/ml) and pure resvera- trol (1×10-6 to 6.4×10-5 g/ml) along with their speci!c antagonists were measured accordingly to the method of Bhattacharya et al. (1976) and the values recorded were staged as a mean melanophores size index (MMSI), which is a modi!ed method of Hogben and Slome (1931) where the actual diameter (length X breadth) of ten ran- domly selected melanophores was recorded using Leitz Ocular Micrometer, calibrated previously with 10x10 magni!cation. The value thus obtained was then mul- tiplied by the unit of micrometer which was 15µ. There- after the arithmetical mean was calculated. This was the mean melanophores size index (MMSI).

Randomized selection of melanophores was the experimental design used in this study. Each experi- ment consisted of different dose concentration and each experiment was repeated seven times. Standard Devia- tions (S. D.) and Standard Errors of Means (S. E.) were calculated in each experiment. All data were analyzed using standard methods of Lewis et al., (1971) and recal- culated using GraphPad Prism software (UK). P<0. 05 indicate statistically signi!cant difference.

RESULTS AND DISCUSSION

The effect of seed skin extract of A. hypogaea was stud- ied on the isolated skin melanophores of B. melanostic- tus. It was found that the extract of A. hypogaea per se gradually aggregated all the dorsal skin melanophores

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of B. melanostictus in a dose range 1 × 10-6 g/ml to 6.4

×10-5 g/ml. The effective concentration of A. hypogaea as low as 1 × 10-6 g/ml could decrease the MMSI from a control value of 4.01 ± 0.07 to 3.68 ± 0.05 ( Fig. 1).

The increasing concentrations of 2 × 10−6 , 4 × 10−6 and 8 × 10−6 g/ml of seed skin extract of A. hypogaea decreased the MMSI values to 3.18 ± 0.17, 2.9 ± 0.11 and 1.99 ± 0.07 respectively from the control value of (4.01 ± 0.07) . This trend of melanophore aggregation by the seed skin extract of A. hypogaea continued and the concentration of 6.4 ×10−5 g/ml decreased the MMSI from a control of 4.01 ± 0.07 to 1.2 ± 0.07 (P < 0.0001)

. At this stage all the melanophores had become highly aggregated and showed retardation of branches in a highly punctuate state making the skin appear lighter as compared to the control.

It was found that when the pretreated melanophores by seed skin extract A. hypogaea at a maximal dose i.e. 6.4 × 10-5 g/ml were washed repeatedly with amphibian ringer saline (ARS) and re-immersed in it, the melano-

phore aggregating effect got completely disappeared. At this stage the MMSI of the dorsal skin melanophores had become 3.72 ± 0.07 which is near the control value of 4.01 ± 0.07 (Fig. 1).

It was found that the powerful per se melanophore aggregation effects of extracts of A. hypogaea were completely blocked by 4×10-5 g/ml of propranolol (Ε blocker) and partially blocked by prazosin ( blocker). In response to the highest concentration of 6.4×10-5 g/ ml of extract of A. hypogaea, in presence of propranolol and prazosin the MMSI was found to be 3.84 ± 0.5 and

1.18± 0.03 whereas the same without pretreatment with was 1.2±0.07 (Fig. 1). Skin pieces containing melano- phores of B. melanostictus were subjected to reserpine treatment with a constant dose of (4×10-5), after which they were further treated with increasing concentrations of extract of A. hypogaea (1×10-6 to 6.4×10-5 g/ml). It was found that the per se melanin aggregating effects of A. hypogaea extracts got highly potentiated by 4×10- 5g/ml of reserpine. The MMSI had become 0.79 ± 0.04

in comparison to per se (1.2 ± 0.07) treatment of the skin melanophores with extract of A. hypogaea (Fig. 1). It was also found that the potentiated melanophore aggregation effects of A. hypogaea by reserpine were also blocked by 4×10-5 g/ml of propranolol and prazosin.

In order to further validate the exact role of A. hypogaea extract and its active ingredient in inducing melanin aggregation leading to lightening of melano- phores of B. melanostictus, the skin melanophores were incubated with resveratrol in concentrations rang- ing from 1×10-6 to 6.4×10-5 g/ ml. It was observed that resveratrol induced a physiologically signi!cant and reversible melanophore aggregation in all concentra- tions used. The highest degree of aggregation from the control value of 4.2± 0.05 to 1.08 ± 0.03 was induced by 6.4×10-5 g/ml of resveratrol per se. The powerful mela- nin aggregating effects of resveratrol were also found to be completely blocked by propranolol (beta blocker) and praozosin (Alpha blocker), speci!c adrenergic antago- nists in pre selected concentrations of 4×10-5g/ml each. In response to the highest concentration of 6.4×10-5 g/ml of extract of A. hypogaea, in presence of propanolol and

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partially blocked by prazosin, the MMSI was found to be

3.76± 0.03 and 1.34± 0.03respectively ( Fig. 2). Reser- pine also potentiated the pretreated melanin aggregat- ing effects of both, the extract of A. hypogaea and pure resveratrol, which were completely blocked by 4×10-5 g/ ml of propranolol and Prazosin (Fig. 2).

The effects of extract of A. hypogaea, in varying con- centrations have been studied on the isolated dorsal skin melanophores of the adult toad, B. melanostictus. In the present study the effect of extract of A. hypogaea and its active ingredient resveratrol induced a physiologically as well as statistically signi!cant melanophore aggre- gating effect, leading to lightening on the isolated dorsal skin melanophores of toad, B. melanostictus. These pow- erful melanophore aggregating effects of plant extract and its pure active ingredient were found to be signi!- cant in all the concentrations (1 × 10-6 to 6.4 × 10-5 g/ml) and were found to be completely blocked by propranolol

(Ε blocker) and partially blocked by prazosin ( blocker) which are adrenergic receptor antagonists.

In the present investigation it is shown that there is a complete involvement of beta adrenergic receptors

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and partial involvement of alpha adrenergic receptors in bringing about the aggregation of dorsal skin melano- phores of Bufo melanostictus. The data of the present study, that the melanin aggregating effects on the isolated melanophores of the toad, B. melanostictus by extract of A. hypogaea were completely blocked by propranolol (beta blocker) and partially blocked by Prazosin (alpha blocker), are in full corroboration with the !ndings of Ali and Peter, (1994) who found that adrenaline caused a powerful aggregation of all the melnophores in con- centrations ranging from 1× 10-7 to 6.4 × 10-6 g/ml. The powerful melanin aggregating effects of adrenaline was blocked by alpha and beta adrenergic receptors (phenoxy- benzamine and propranolol) indicating that the integu- mental melanophores of Rana tigerina contain a mixed population of alpha and beta adrenergic receptors. Grove, (1969) had reported that in the melanophores of Phoxi- nus some beta receptors were also present along with the dominantly present alpha adrenergic receptors.

The present !ndings are quite similar to the ear- lier !ndings of Trendeleburg, (1963) stated that reser- pine caused the hypersensitivity to adrenaline and nor adrenaline in mammalian smooth muscle by depletion of the neurotransmitter substance. Darabi et al., (1976) reported that the effects of reserpine on the sensitivity to catecholamines and adrenoceptor antagonists pro- pranolol and azapetine were studied on the fundus strips of the rat. The results obtained in this study would sup- port the view that reserpine may increase the sensitivity of beta receptor to isoprenaline and adrenaline by the mechanism which may be independent of the uptake of catecholamines in the sympathetic nervous system.

These data clearly substantiate that the seed skin extract of A. hypogaea and its active ingredient, res- verartrol, induced powerful, dose dependent, physiologi- cally signi!cant melanin aggregating effects in the iso- lated dorsal skin melanophores of B. melanostictus that were completely blocked by propranolol and partially blocked by prazosin. As such, the melanin aggregating effects of extract of A. hypogaea and its active ingre- dient, resveratrol, were highly potentiated by reserpine. It appears that the melanin aggregating effects of the extracts of A. hypogaea and pure resveratrol leading to skin lightening are mediated by adrenergic receptors.

CONCLUSION

It is concluded that extract of A.hypogaea and its active ingredient resveratrol induced powerful dose dependent physiologically signi!cant melanin aggregating effects in the dorsal skin melanophores of B. melanotictus. These studies suggest that the active ingredient of Arachis hypogaea such as resveratrol can act as a sympathomimetic

compound and induce aggregation of dorsal skin melano- phore leading to skin paling of the Bufo melanotictus, via induction of both type of the adrenoceptors beta and alpha. It appears that the melanin aggregating effect of the extract of A. hypogaea and resveratrol leading to skin lightening via the stimulation of beta adrenoceptors.

The present study is signi!cant in relation to the role of adrenaline as a neurotransmitter substance and the nature of adrenergic receptors as one of the causative factors of melanolysis. Further studies are required to be done to ascertain the role of resveratrol as an adrenergic modulator in other species of vertebrates along with more speci!c agonists and antagonists. Thus in the present work, resveratrol has been found to be a novel stimulator of beta adrenergic receptors leading to inhibitory effect of melanin aggregation causing paling of the skin, thus opening new pharmacological vistas for this compound.

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