Abstract: The present study was carried out to determine the effects of extracts of Citrus reticulata and its active ingredient hesperidin on the isolated tail melanophores of the Bufo melanostictus to find the mechanism of skin lightening at the cellular level. The tail melanophores of the tadpole B. melanostictus, were assayed using the mean melanophore size index and their responses were recorded in presence of various concentrations of the plant extract and its active ingredient along with specific antagonists and potentiator. Significant skin lightening activity of the extract of C. reticulata and hesperidin was observed on the tail melanophores of tadpole. The pigment cells responded by distinct aggregation leading to skin lightening, the effect was reversible, as re-immersion in physiological saline made the melanophores return to their normal intermediate state. These melanin aggregating effects were completely blocked by prazosin (alpha blocker) and were also found to be highly potentiated by reserpine. These studies suggest that the active ingredient of C. reticulata such as hesperidin can act as a sympathomimetic compound and induce aggregation of tail melanophore of tadpole Bufo melanostictus via induction of the alpha adrenoceptors. The present study opens new vistas for the use of C. reticulata and its active ingredient, hesperidin for its clinical application as a non toxic depigmenting compound for the treatment of hyperpigmentation.
INTRODUCTION
Melanin containing pigment cells melanophores or melanocytes which originate from the neural crest, mimic the properties of smooth muscles. These cells, present in the skin of vertebrates, including human beings, allow rapid as well as slow color changes in response to external stimuli. Intracellularly receptor mediated responses of these pigment cells lead to skin darkening or skin lightening which is controlled by either nerves alone or by hormones or by a combination of both (Fujii, 1969, 2000; Bagnara and Hadley, 1973; Aspengren et al., 2003). Consequently receptors of different nature have been implicated in pigmentary responses of various animal species such as adrenergic, cholinergic and histaminergic, showing aggregation or dispersion of melanophores in vertebrates leading to skin paling or darkening, respectively (Kasukawa and Fujii, 1985; Fuji et al., 1982; Ali et al., 1995, 1998; Peter et al., 1996).
The melanophore model has been pharmacologically used since the past many years and has yielded fruitful influences which are being used for the therapeutic applications of pigmentary disorders (Aspengren et al., 2008). For excessive and abnormal hyperpigmentation of skin, development of new whitening agents without toxic implications from medicinal plants has been one of the recent areas of dermatological research (Smit et al., 2009).
The “return to nature” trend of recent years had been accompanied by a booming interest in whitening agents from natural products (Parvez et al., 2007). Few medicinal plants have been reported to posses melanolytic activity causing depigmentation. The methanol extract of the bark of Machilus thunbergii has showed much stronger melanin biosynthesis inhibitory activity than arbutin (Li et al., 2003). In order to develop new skin whitening agents, plant material have also been evaluated for inhibition of melanogenesis (Roh et al., 2004; Akazawa et al., 2006; Arung et al., 2006; Lee et al., 2006; Chang et al., 2007).
Citrus flavonoid hesperidin have shown a wide range of therapeutical properties for medical and clinical applications such as anti-inflammatory, antihypertensive, diuretic, analgesic and hypolipidemic activities (Galati et al., 1994, 1996; Monforte et al., 1996). Despite the use of hesperidin in several ailments cited above there are no studies on its use as a melanolytic agent. Except for the work of Zhang et al. (2007), who reported that citrus flavonoid hesperidin induced melanolysis in mammalian cultured melanocytes.
Up till now it is not clear that whether any plant material or its active ingredients is known to cause depigmentation via any of the receptor types involved such as adrenergic, histaminergic and tryptaminergic. An understanding of the benefits of natural and botanical extracts provides opportunities to develop new clinical products to address pigmentation problems. Incorporation of the role of receptors in the skin lightening response and its mechanism at cellular level has not been done. Hence, the present research work has been undertaken where effects of active ingredients of Citrus reticulata have been studied on the tail melanophores of B. melanostictus in order to assess the nature and role of the cellular receptors involved and their stimulation by the active ingredients of C. reticulata.
MATERIALS AND METHODS
Collection and identification of Citrus reticulata: Plant materials C. reticulata were purchased from local market and were authenticated by Dr. S.S. Khan of Botany, Department Saifia Science College Bhopal. The voucher specimen No. J/R 15 was deposited at the Herbarium of the Faculty of Botany, Department Saifia Science College Bhopal (M.P.) India.
Preparation of extract of Citrus reticulata: Citrus reticulata (Rutaceae) fruits known as orange were purchased from local market and were authenticated by Dr. S.S. Khan of Botany, Department Saifia Science College Bhopal. Extract was prepared according to the method of Choi et al. (2007), where the fruit peels were dissected and dried in shed, ground with a mortar mill. The powdered peels were used for extraction. 10 g of the powdered peels were extracted for 3 days with 200 mL of ethanol-water (7:3, v/v) at room temperature. The extract was decanted and the remaining residue was extracted again with 100 mL of ethanol-water (7:3). The combined extract was evaporated in Vacuum evaporator. The extract was re-dissolved in distilled water for in vitro studies using different concentration. The pure hesperidin (80%) CAT No. (Molecular weight) from Sigma-Aldrich, USA was used in the present study. A stock solution of pure hesperidin (1 mg mL-1) was prepared in 0.02% dimethylsulphoxide (DMSO) and used in further dilutions.
Studies on tail melanophores of B. melanostictus: B. melanostictus tadpoles of one spawn were collected from a small pond during the rainy season of 2009 and kept in large aquaria. Prior to the experiments, the tadpoles were allowed to acclimatize to laboratory conditions for two days. Diseased, injured or lethargic tadpoles were removed and only active, uniformly sized tadpoles were used. For in vitro studies the whole tail pieces were used, tail were cut off from these tadpoles and 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 bicarbonate 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 7-10 min with frequent shaking. Tail pieces containing about 50-100 melanophores, the responses of control as well as of those melanophores which were incubated in 10 mL ARS saline containing various concentrations of ethanolic extracts of A. hypogaea and resveratrol along with their specific antagonists were measured accordingly to the method of Bhattacharya et al. (1976). This is a modified method of Hogben and Slome (1931), where actual diameter (length X breadth) of ten randomly selected melanophores was recorded using Leitz Ocular Micrometer, calibrated previously with 10x10 magnification. The value thus obtained was then multiplied by the unit of micrometer which was 15 μm. Thereafter the arithmetical mean was calculated. This was the mean melanophores size index (MMSI).Statistical data analyses are presented as Mean±SEM (standard error of the mean) and n represents the number of dose concentrations (treated) used for a particular experiment. Comparisons were made between treated and control groups by use of Student’s t test. All data were analyzed using GraphPad Prism software (UK) p<0.05 indicates statistically significant difference.
Statistical analysis: Statistical data analyses are presented as Mean±SEM (standard error of the mean) and n = 7 represents the number of dose concentrations (treated) used for a particular experiment. Comparisons were made between treated and control groups by use of Student’s t-test. All data were analyzed using GraphPad Prism software (UK) p<0.05 indicates statistically significant difference.
RESULTS
Effect of extracts of A. hypogaea per se along with α adrenergic antagonist (prazosin) and its potentiator (reserpine) on the isolated tail melanophores of B. melanostictus: It was found that extract of C. reticulata per se induced a dose dependent melanin aggregation response in the isolated tail melanophores of B. melanostictus, where all the melanophores had become perinuclear making the skin pale in color. In response to the maximal concentration of 6.4x10-5 g mL-1 of extracts of C. reticulata, the MMSI of the melanophores had become 1.07±0.1 (0.0001) from a control value of 3.86±0.06.
| Fig. 1: | The dose response curve for the melanophore aggregating
effect of ethanolic extract of Citrus reticulata (per se)
(♦) show effect on the tail melanophores of B. melanostictus. The
complete blocking effects of specific antagonist prazosin (4x10-5 g mL-1, (▲) show effect against Citrus reticulata extract aggregated melanophores are also shown. Closed circles show (●)
the effect of reserpine (4x10-5 g mL-1) on the dose
response curve ethanolic extract of Citrus reticulata. propranolol
(4x10-5 g mL-1, (■) shows no blocking effect.
RI signifies the MMSI after the reimmersion of tail pieces in Amphibian
Ringer Saline after repeated washings. Abscissae: Doses of C. reticulata and antagonists in g mL-1. Ordinate: responses of melanophores
(MMSI). Vertical bars represent the standard error of mean; p signifies
the level of significance |
|
After repeated washings and Reimmersion (RI) of the C. reticulata treated melanophores in normal saline, it was found that the powerful melanin aggregation effects of the extract got completely vanished, as the MMSI had become 3.56±0.07, almost near the control values of 3.86±0.06 (Fig. 1). It was found that the powerful per se melanophore aggregation effects of extracts of C. reticulata were completely blocked by 4x10-5 g mL-1 of prazosin (α blocker). In response to the highest concentration of 6.4x10-5 g mL-1 of extract of C. reticulata in presence of prazosin the MMSI was found to be 3.56±0.07 and 1.1±0.04 whereas the same without pretreatment with was 1.07±0.1 (Fig. 1).
Tail pieces containing melanophores of B. melanostictus were subjected to reserpine treatment with a constant dose of (4x10-5), after which they were further treated with increasing concentrations of extracts of C. reticulata (1x10-6 to 6.4x10-5 g mL-1). It was found that the per se melanin aggregating effects of C. reticulata extracts got highly potentiated by 4x10-5 g mL-1 of reserpine. The MMSI had become 0.73±0.05 (0.11) in comparison to per se (1.07±0.1) treatment of the tail melanophores with extracts of C. reticulata. It was also found that the potentiated melanophore aggregation effects of C. reticulata by reserpine were also blocked by 4x10-5 g mL-1 of prazosin (Fig. 1).
Effect of standard hesperidin per se on the isolated tail melanophores of B. melanostictus along with α adrenergic antagonist (Prazosin) and its potentiator (reserpine): In order to further validate the exact role of C. reticulata extract and its active ingredients in inducing melanin aggregation leading to lightening of melanophores of B. melanostictus, the tail melanophores were incubated with hesperidin in concentrations ranging from 1x10-6 to 6.4x10-5 g mL-1. It was observed that hesperidin induced a physiologically significant and reversible melanophore aggregation in all concentrations used. The highest degree of aggregation from the control value of 3.85±0.07 to 0.72±0.03 (0.0001) was induced by 6.4x10-5 g mL-1 of hesperidin per se. The powerful melanin aggregating effects of hesperidin were also found to be completely blocked by prazosin (Alpha blocker), specific adrenergic antagonist in pre selected concentrations of 4x10-5 g mL-1 each.
| Fig. 2: | The dose response curve for the melanophore aggregating
effect of Hesperidin (hesperidin, per se) (♦) show effect on
the tail melanophores of the complete blocking effects of specific antagonists
prazosin (4x10-6 g mL-1, (▲) show effect against
hesperidin aggregated melanophores are also shown. (●) show the effect
of reserpine (4x10-5 g mL-1) on the dose response
curve resveratrol. propranolol (4x10-5 g mL-1, (■)
shows no blocking effect. RI signifies the MMSI after the re immersion of
tail pieces in ARS after repeated washings. Abscissae: Doses of hesperidin
in g mL-1. Ordinate: responses of melanophores (MMSI). Vertical
bars represent the standard error of mean; p signifies the level of significance |
|
In response to the highest concentration of 6.4x10-5 g mL-1 of extract of C. reticulata, in presence of prazosin, the MMSI was found to be 3.12±0.11 (Fig. 2). Reserpine also potentiated the pretreated melanin aggregating effects of pure hesperidin, the MMSI was found to be 0.55±0.02 (0.0001) which was completely blocked by 4x10-5 g mL-1 of Prazosin (Fig. 2).
DISCUSSION
In the present investigation, the ethanolic extract of C. reticulata and standard hesperidin both in dose ranges of 1x10-6 to 6.4x10-5 g mL-1 induced powerful melanin aggregation in the tail melanophores of Bufo melanostictus, leading to paling of the skin. Pure hesperidin showed more potency in melanophore aggregation than the extract of C. reticulata. Interestingly the physiologically significant dose related melanin aggregation effects caused by extract of C. reticulata and standard hesperidin per se were found to be completely blocked by prazosin, a specific alpha adrenergic receptor antagonist.
These data strongly indicate that in the tail melanophores of Bufo melanostictus, the aggregation of melanin granules leading to lightening of the skin which is induced by hesperidin is mediated by alpha adrenoceptors making it the new and novel adrenoceptor agonist. It appears that the active compound of C. reticulata, hesperidin has a powerful effect at the neuro melanophore junction of the tadpole tail melanophores, thereby increasing the endogenous content of adrenaline which consequently induced pigment aggregation via activation of adrenergic receptors. There is hardly any report in the literature showing the activation of adrenergic receptors by plant extracts particularly C. reticulata and its active ingredient, hesperidin in any of the smooth muscle cells including the pigmented cells, the melanophores. As far it is known, this is the first report of its kind where both ethanolic extracts of C. reticulata and hesperidin have been found to stimulate the dominantly present alpha adrenergic receptors in the pigment cell system of B. melanostictus, leading to skin lightening.
The present findings suggest the dominant presence of alpha adrenergic receptors, which mediate pigment aggregation in the tail melanophores of B. melanostictus. These findings are in agreement with the observation of Karlsson et al. (1989) in Labrus ossifagus, Abrao et al. (1991) in Synbranchus marmoratus, Burton and Vokey (2000) in Pleuronectes americanus.
It appears that hesperidin, the active ingredient of A. hypogaea, induces similar inhibitory effects leading to melanophore aggregation, which are quite interesting from the characteristic property of the compound hesperidin, as it is known to cause vasoconstriction of the systemic blood vessels of the toad. Similarly Intravenous injection of hesperidin had increased the contractility of the in situ rabbit heart. Intravenous injection of the decoction to anesthetized rabbits produced tonic contraction of the in situ uteri; the condition was reversed after 15 min (Weiliang et al., 1998).
In light of the above reports, the data of the present findings suggest that the active ingredients of C. reticulata particularly hesperidin stimulate the abundantly present adrenergic receptors of tadpole tail melanophores leading to the lightening effect. Data of the present findings also point to the novel role of hesperidin as a new sympathomimetic compound. Similar to that of adrenaline and related agonists (phenylephrine) which have been earlier found on other species (Goodman et al., 2006). The present study is significant 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 hesperidin as an adrenergic modulator in other species of vertebrates along with more specific agonists and antagonists.
CONCLUSION
It is concluded that extract of C. reticulata and its active ingredient hesperidin induced powerful dose dependent physiologically significant melanin aggregating effects in the tail melanophores of B. melanostictus, which were completely blocked by Prazosin. The per se melanin aggregating effects of extract of C. reticulata and its active ingredient hesperidin got highly potentiated by reserpine. It appears that the melanin aggregating effect of the extracts of C. reticulata and pure hesperidin leading to skin lightening is mediated by adrenergic like receptors having similar properties.