On the action and mechanism of withaferin-A from Withania somnifera,a novel and potent melanin dispersing agent in frog melanophores

The present work was carried out to determine the effects of lyophilized root extracts of Withania somnifera along with pure withaferin-A, on the isolated skin melanophores of frog, Rana tigerina which are disguised type of smooth muscle cells and offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The lyophilized extract of W. somnifera and its active ingredient withaferin-A induced powerful dose-dependent physiologically significant melanin dispersal effects in the isolated skin melanophores of R. tigerina, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of W. somnifera and its active ingredient withaferin-A got highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of W. somnifera and withaferin-A is mediated by cholino-muscarinic like receptors having similar properties.     

On the action and mechanism of withaferin-A from Withania somnifera, a novel and potent melanin dispersing agent in frog melanophores

The present work was carried out to determine the effects of lyophilized root extracts of Withania somnifera along with pure withaferin-A, on the isolated skin melanophores of frog, Rana tigerina which are disguised type of smooth muscle cells and offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The lyophilized extract of W. somnifera and its active ingredient withaferin-A induced powerful dose-dependent physiologically significant melanin dispersal effects in the isolated skin melanophores of R. tigerina, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of W. somnifera and its active ingredient withaferin-A got highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of W. somnifera and withaferin-A is mediated by cholino-muscarinic like receptors having similar properties.     

Mediation of cholino-piperine like receptors by extracts of Piper nigrum induces melanin dispersion in Rana tigerina tadpole melanophores

Aim: The present study was carried out to determine the effects of lyophilized dried fruit extracts of Piper nigrum and pure piperine on the tadpole melanophores of frog Rana tigerina which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The nature of specific cellular receptors present on the neuro-melanophore junction and their involvement in pigmentary responses has been explored.

Material: Effects of lyophilized extracts of P. nigrum and pure piperine were studied on the isolated tail melanophores of tadpoles of the frog R. tigerina as per the modified method.

Results: The extract of P. nigrum and its active ingredient piperine caused significant melanin dispersal responses leading to darkening of the tail melanophores, which were completely antagonized by atropine and hyoscine. These per se melanin dispersal effects were also found to be markedly potentiated by neostigmine an anticholinesterase agent.

Conclusion: It appears that the melanin dispersal effects of the extracts of P. nigrum and pure piperine leading to skin darkening are mediated by cholinergic muscarinic or piperine-like receptors having similar properties.     

Berberine-induced pigment dispersion in Bufo melanostictus melanophores by stimulation of beta-2 adrenergic receptors

Abstract

Reduced production of melanin by decreased or the absence of melanocytes leads to various hypopigmentation disorders, and the development of melanogenetic agents for photoprotection and hypopigmentation disorders is one of the top priority areas of research. Hence, the present study was carried out to elucidate the ability of berberine, a principal active ingredient present in the roots of the herb Berberis vulgaris to stimulate pigment dispersion in the isolated skin melanophores of the toad Bufo melanostictus. In the present study, mean melanophore size index of the isolated skin melanophores of B. melanostictus was assayed after treating with various concentrations of berberine. A marked melanin dispersion response leading to skin darkening was observed in the isolated melanophores of toad in response to berberine, which was found to be mediated through beta-2 adrenergic receptors. The physiologically significant dose-related melanin dispersion effects of berberine per se were found to be completely abolished by propranolol, which is a specific beta-2 adrenergic receptor blocker. These per se melanin dispersal effects were also found to be markedly potentiated by isoprenaline, which is a specific beta-adrenoceptor agonist. The results indicate that berberine causes a tremendous, dose-dependent, physiologically significant pigment dispersing in the isolated skin melanophores of B. melanostictus.     

Effect and mechanism of action of resveratrol: a novel melanolytic compound from the peanut skin of Arachis hypogaea

Purpose: The present work was carried out to determine the effects of ethanolic extracts of Arachis hypogaea and its active ingredient resveratrol on the isolated tail melanophores of the Bufo melanostictus to find the mechanism of skin lightening at the cellular level.

Methods: 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.

Results: Significant skin lightening activity of the extract of A. hypogaea and its active ingredient resveratrol was observed on the tail melanophores of tadpole. The pigment cells responded by distinct aggregation leading to skin lightening, this 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 propanolol (beta blocker) and partially blocked by prazosin (alpha blocker) and were also found to be highly potentiated by reserpine.

Conclusion: These studies suggest that the active ingredient of A. hypogaea such as resveratrol can act as a sympathomimetic compound and induce aggregation of melanophores of tadpole B. melanostictus via the induction of beta type of the adrenoceptors. The present study opens new vistas for the use of A. hypogaea and its active ingredient, resveratrol for its clinical application as a nontoxic melanolytic compound for the treatment of hyperpigmentation.     

Effect of cyclic AMP and cytochalasin B on tissue cultured melanophores of Xenopus laevis

The effects of cytochalasin B or low concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1–10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.     

Gamma radiation effects on growth and yield attributes of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L. with reference to enhanced production of psoralen

The present work describes radiation-induced effects on vegetative, reproductive traits and psoralen content in Psoralea corylifolia L. The effects of gamma radiation on Psoralea seeds were investigated by exposing seeds with doses of 2.5, 5, 10, 15 and 20 kGy at dose rate of 1.65 kGyh⁻¹ and studying the plant growth at three developmental stages: preflowering, flowering and post flowering (seed to seed) after irradiation. Irradiation with lower doses of gamma rays significantly improved vegetative traits while higher doses proved depressing for same parameters. Similar trend was followed in reproductive traits. Psoralen, showed highest concentration in seeds (7.56%) at 20 kGy and lowest in control roots (0.23%). Increment in psoralen was striking for higher gamma doses applied. These long-term changes in plant development may be attributed to alteration in plant genome induced by irradiation. The results show in depth development stimulation and enhancement of secondary metabolite in Psoralea corylifolia L. following low and high dose treatment respectively depicting the potential of gamma rays in plant biotechnology and metabolomics.     

Effects of radix polygoni multiflori components on tyrosinase activity and melanogenesis

Radix Polygoni multiflori is a herb used effectively to prevent graying and treat skin depigmentation diseases in traditional Chinese medicine but its active ingredients have not been discovered yet. In this investigation, we tested six compounds isolated from Radix Polygoni multiflori, to discover the active component on melanogenesis. Three experiments were performed in the present investigation: mushroom tyrosinase activity, melanin content B16 cell proliferation assay. Among all the six components tested, THSG showed the most potent effects on tyrosinase activation and melanogenesis; it was shown to be a potent tyrosinase activator and a melanogenesis stimulator in this study. On the other hand, we found that gallic acid significantly inhibited tyrosinase and, in addition, anthraquinones were cytotoxic to melanoma cells. They were both harmful to melanogenesis. Therefore, we propose that THSG acts as the active ingredient of Radix Polygoni multiflori on melanogenesis.     

Cellular Aspects of the Control of Physiological Color Changes in Amphibians

SYNOPSIS. The present paper is concerned mainly with the melanin-dispersingeffect of melanocyte-stimulating hormones (MSH's) on the skinmelanophores of amphibians. In addition, some of the more recentevidence for the unihumoral theory of the control of color changeis reviewed. The mechanism of dispersion of melanin is stillunknown, but evidence is accumulating that the action of MSHmay be mediated by an increase in the melanophoric content ofadenosine 3', 5'-monophosphate (cyclic AMP). For example, cyclicAMP has a specific, reversible melanin-dispersing effect onthe melanophores of the isolated skin of R. pipiens and Xenopuslaevis. It also has a reversible "melanophore—expanding"effect on the tissue—cultured embryonic melanophores ofthe spotted salamander, Ambystoma maculatum. The effect of cyclicAMP on melanophores of R. pipiens does not require sodium butis inhibited by hypertonicity. Finally, new evidence is presented that confirms that the melanin-dispersingeffect of catecholamines on melanophores of X. laevis is mediatedby beta adrenergic receptors,because it is blocked by the highlyspecific ß—blocking agent, propranolol. On theother hand, the melanin-aggregating effect of catecholamineson amphibian melanophores appears to be mediated by alpha adrenergicreceptors. There is even a possibility that the effects of catecholaminesare also mediated through a control of cyclic AMP levels inmelanophores, with beta adrenergic stimulation producing anincrease in cyclic AMP levels, followed by dispersion of melanin,and alpha adrenergic stimulation producing a decrease in cyclicAMP levels, followed by aggregation of melanin.     

Fibronectin-induced migration of melanophores in vitro in scales of medaka,Oryzias latipes

Summary The effects of fibronectin on melanophores were examined in two mutant strains of medaka, Oryzias latipes: mm (BmmR), which has condensed melanophores and normal dendritic melanophores; and cm (BcmR), which has condensed melanophores. When medaka scales were cultured in the presence of fibronectin, melanophores of the wild type and dendritic melanophores of the mm mutant changed their shape and migrated, whereas melanophore migration was rarely seen in the absence of fibronectin. Melanophores of the cm mutant and condensed melanophores of the mm mutant did not migrate even in the presence of fibronectin. When melanophores of the wild type and mm mutant were condensed by adrenalin, they did not migrate. On the other hand, when melanophores of the cm mutant were dispersed by theophylline, they were able to migrate. These results indicate that fibronectin induces the migration of melanophores and that dispersion of melanin granules may be requisite for such migration.     

A strategy for screening microbial strains with lipolytic specificity toward monoacylglycerols

For easy obtaining the microorganisms with lipolytic specificity toward monoacylglycerols, we developed a simple and effective method to isolate the objective strains. This method employed a nile-blue agar-plate culture containing mono- and tri- acylglycerols for microorganism screening and selected the desired microorganisms by analysis of free fatty acid contents in lipid extracts obtained from culture broth. Using this strategy, we successfully isolated one mold strain with superior lipolytic ability for the hydrolysis of monoacylglycerols. The mold was identified and designated as Paecilomyces nostocoides NTU-FC-LP01. The lyophilized mycelia of the isolated mold used as a biocatalyst showed high specificity toward monoacylglycerols rather than di- and tri- acylglycerols. Furthermore, the lyophilized mycelia catalyzed the monoolein synthesis through the direct esterification of oleic acid and glycerol. It indicated that the lyophilized mycelia of the present P. nostocoides NTU-FC-LP01 could be used as a natural immobilized biocatalyst for the glycerol/oleic acid esterification to produce monoolein.     

Role of calcium in melanosome aggregation withinLabeo melanophores

In isolated scale melanophores ofLabeo rohita the melanosome aggregating effect of K⁺ was inhibited in Ca²⁺ deprived medium. Moreover, the Ca²⁺-antagonists, verapamil and lanthanum inhibited the action of K⁺ in concentration dependent manner. The elevation of extracellular Ca²⁺ could compromise the verapamil induced inhibition in a concentration dependent manner. The cation Ca²⁺ appeared to be required only for K+ -induced aggregation and not melanosome aggregationper se, as in this fish adrenaline and melanin concentrating hormone effectively caused aggregation of melanosomes in Ca²⁺ free medium     

Analyzing the responses of saccharin in context with melatonin receptors on the melanophores of the fish Labeo rohita (Ham.)

The hormone melatonin regulates the biological clock and assist in various other physiologies of vertebrates. Present work is intended to check the affinity of saccharin towards the melatonin receptors and the possible role of saccharin interference in the melatonin physiology. The present in vitro study is based on the working model of isolated scale melanophores in the dorso-lateral region of Labeo rohita. The pigment cells were incubated in the agonist and the antagonists within a limited time frame and subsequently their Melanophore Size Index (MSI) were calculated. The inferences were drafted through the observed signal transduction upshots in pigment translocations within the melanophores. Saccharin, in a wide dose range, has consistently induced a concentration-related aggregation similar to the aggregatory effect as shown by melatonin on the melanophores. Binding of saccharin with the receptors and eliciting its aggregatory effect is partially dependent on the release of neurotransmitters. The aggregatory effects were found to be significantly blocked by luzindole, K185, and prazosin, which are the potent melatonin receptor blockers, at the higher concentrations of saccharin. Hence, all the three subtypes of melatonin receptors viz. MT₁, MT₂, and MT₃ are participating in saccharin-mediated aggregations. Blocking by neomycin shows that Ca²⁺ ions are very crucial in dispensing the aggregatory effect of the sweetener. This research demands that an intensive and careful thorough study should be made about saccharin, specifically its effects upon melatonin physiology, before its unwarranted use as the food ingredients for human use.     

Biotic elicitor enhanced production of psoralen in suspension cultures of Psoralea corylifolia L.

Cell cultures of Psoralea corylifolia L. were established from the leaf disk derived callus. The effect of different biotic elicitors prepared from the fungal extract (Aspergillus niger and Penicillium notatum), yeast extract and chitosan with different concentrations was studied. The increased synthesis of psoralen in 16-day old cell cultures under 16 h of light and 8 h of dark period was studied. Elicitation of psoralen in A. niger elicitor treated cells was found 9-fold higher over control cells. Treating the cells with P. notatum, yeast extract and chitosan elicitors lead to four to seven-fold higher psoralen accumulation over control cells. The extract of A. niger at 1.0% v/v increased the significant accumulation of psoralen (9850 μg/g DCW) in the cultured cells. Our study clearly shows that all the elicitors had the potential to increase the accumulation of psoralen but the A. niger elicitor at 1.0% v/v induced maximum accumulation.     

Vitiligo,Psoralen, and Melanogenesis: Some Observations and Understanding

Since the etiology of vitiligo is still unknown, we searched for some abnormal biochemical parameters, if any, in subjects with vitiligo. Higher urinary excretion of indole metabolites in vitiliginous patients have been noted, in association with higher dioxygenase, superoxide dismutase, and tyrosine aminotransferase activity in their serum. Similar results have also been found in an animal model, Bufo melanostictus, during induced tyrosinase inhibition. Treatment with psoralen can reverse the parameters, except tyrosine aminotransferase, to a normal level. Although psoralens are not the magic bullet for the therapy of vitiligo, they are still being used as a chemotherapeutic agent against vitiligo on a major scale to date. Tryptophan was found to participate in the pathway of melanogenesis, as a precursor as well as a positive regulator of tyrosinase. Its behavior in this regard is much more similar to the conventional substrates tyrosine and dopa (dihydroxyphenylalanine). In consideration of combined participation of tyrosine and tryptophan in the synthesis of melanin and its breakdown, the possible influence of different enzymatic reactions, like mono-oxygenase, dioxygenase, and deamination, has been suggested.     

Penicillium in pre-harvest corn in Valencia (Spain) II. Study of the enzymatic and toxigenic capacities of the species

This paper presents data on plate assays of enzymatic activity and antibacterial effects of species of Penicillium isolated in samples of pre-harvest corn.In general, the species tested present high levels of amylolytic and lipolytic properties, while the proteolytic, urease, DNAse and cellulolytic levels are lower.The presence of ochratoxin A in the raw extracts of P. variabile, rubratoxin B in those of P. purpurogenum, and citrinin in those of P. steckii was detected by thin layer chromatography techniques against standards. Toxicity tests against brine shrimp larvae and 12 microorganisms indicated great variability among the extracts.The possibility of using the production of enzymes and mycotoxins in the classification of some groups of Penicillium is studied.     

Enhancement by fibronectin of spreading of isolated melanophores of the medaka,Oryzias latipes

Summary Cell spreading of isolated melanophores in medium containing fibronectin was observed in the wild type and two mutants of the medaka, Oryzias latipes. Isolated and cultured melanophores of the wild type and the mm mutant were different in appearance from those within scales but dendritic in shape and with fully dispersed pigment granules. Isolated melanophores of the cm mutant were stellate with dispersed pigment granules, whereas in scales the pigment granules are condensed. In the presence of fibronectin, spreading of cultured melanophores of wild type and cm mutant was observed. Spreading of melanophores from the mm mutant was observed only among dendritic melanophores, but not among condensed melanophores. The increase of spreading was inhibited by antibody against fibronectin. To test the involvement of cytoskeletal elements, colchicine, vinblastine or cytochalasin B were added to the culture medium; spreading did not increase, even in the presence of fibronectin. These results suggest that fibronectin-induced melanophore spreading is correlated with the state of pigment granule dispersal and that microtubules and microfilaments may play a role in the mechanism of spreading.Department of Zoology NJ-15, University of Washington, Seattle, Wa 98195, USA.     

oca2 regulation of chromatophore differentiation and number is cell type specific in zebrafish

We characterized a zebrafish mutant that displays defects in melanin synthesis and in the differentiation of melanophores and iridophores of the skin and retinal pigment epithelium. Positional cloning and candidate gene sequencing link this mutation to a 410‐kb region on chromosome 6, containing the oculocutaneous albinism 2 (oca2) gene. Quantification of oca2 mutant melanophores shows a reduction in the number of differentiated melanophores compared with wildtype siblings. Consistent with the analysis of mouse Oca2‐deficient melanocytes, zebrafish mutant melanophores have immature melanosomes which are partially rescued following treatment with vacuolar‐type ATPase inhibitor/cytoplasmic pH modifier, bafilomycin A1. Melanophore‐specific gene expression is detected at the correct time and in anticipated locations. While oca2 zebrafish display unpigmented gaps on the head region of mutants 3 days post‐fertilization, melanoblast quantification indicates that oca2 mutants have the correct number of melanoblasts, suggesting a differentiation defect explains the reduced melanophore number. Unlike melanophores, which are reduced in number in oca2 mutants, differentiated iridophores are present at significantly higher numbers. These data suggest distinct mechanisms for oca2 in establishing differentiated chromatophore number in developing zebrafish.     

Production of psoralen by in vitro regenerated plants from callus cultures of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L.

Psoralea corylifolia is an attractive, endangered annual producing various bioactive compounds of medical importance. This plant contributes to Indian pharmaceutical and cosmetic industries for the production of commercial medicines, Ayurvedic skin care ointments and soap. The influence of various plant growth regulators (PGRs) and additives on high-frequency rapid adventitious shoot regeneration from transverse thin cell layer (tTCL) hypocotyl explants of P. corylifolia was investigated. Organogenic callus was obtained in tTCL hypocotyl explants on Murashige and Skoog (1962) medium supplemented with 15 μM naphthaleneacetic acid (NAA) and 3 μM benzylaminopurine (BA). The highest adventitious shoot regeneration (107.5 shoots per explant) was achieved in culture when transferred to half-strength solid MS medium. The regenerated shoots were rooted and the plantlets successfully acclimatized in moistened (1/8-MS basal salt solution with 3 μM indole-3-butyric acid (IBA), 1 μM 2-isopentenyladenine (2iP) and 100 mg l⁻¹ Bavistin (BVN)); garden soil, farmyard soil and sand (2:1:1, v/v/v). The acclimatized plants produced flowers in the growth chamber. When planted in the field these plants set viable seed. The psoralen content in different tissues of ex vitro and naturally-grown plants was determined by high-performance liquid chromatography (HPLC). The highest psoralen content was recorded in seeds from naturally-grown (6.48 μg g⁻¹ DW) and ex vitro plants (6.46 μg g⁻¹ DW). This system can be used for rapid mass propagation of P. corylifolia, for conservation strategies, and to produce phytomedicines.