Exploration of anti-Malassezia potential of Nyctanthes arbor-tristis L. and their application to combat the infection caused by Mala s1 a novel allergen.

BACKGROUND: Malassezia commensal yeasts along with multitude of antigens have been found to be associated with various skin disorders including Pityriasis versicolor (PV). Amongst them Mala s1, a 37 kDa protein has been proved to be a major allergen reacting with a large panel of sera. However, there exists no therapeutic alternative to combat such problems in form of plant based natural compounds. The purpose of this study is in the first place, to determine the anti-Malassezia activity of Nyctanthes arbor-tristis L. (NAT) ethanolic leaf extract through turbidimetric growth curves, disruption of plasma membrane and secondly, it aims to present in silico validation of its active constituents over Mala s1a novel allergen. METHODS: The antifungal susceptibility 50 % ethanolic extract of NAT was determined by broth microdilution method according to CLSI guidelines. Further MICs and IC50 were determined spectrophotometrically using the software SoftMax® Pro-5 (Molecular Devices, USA). Active constituents mediated disruption of plasma membrane was studied through flowcytometry by permeabilization of fluorescent dye Propidium Iodide (PI). Antioxidant activity of the extract was determined using the DPPH stable radical. Molecular validation of fungal DNA from the extract was observed using PCR amplification. In silico analysis of its active constituents over Mala s1 was performed using HEX software and visualized through Pymol. RESULTS: The anti-Malassezia potential of NAT leaf extracts reflected moderate MIC 1.05 µg/µl against M. globosa, while least effective against M. restricta with MIC 1.47 µg/µl. A linear correlation coefficient R (2) = 0.866 was obtained in case of M. globosa while minimum was observed in M. restricta with R (2) = 0.732. The flow cytometric data reveal ~ 75 % cell death when treated with active constituents ß-Sitosterol and Calceolarioside A. The docking confirmations and the interaction energies between Mala s1 and the active constituents (ß-Sitosterol and Calceolarioside A) from extracts showed an effective binding which suggests Mala s1 as efficient allergen for site specific targeting. CONCLUSIONS: This study revealed that Nyctanthes arbor-tristis L. (NAT) extracts possess high anti-Malassezia potential which is driven mainly by disruption of plasma membrane. Also in silico validation and molecular modeling studies establishes Mala s1 as a novel allergen that could be a potential target in disease treatment. Our results would also provide a foundation for the development of new therapeutic approach using NAT extract as lead compound with high antioxidant property as an added trait for skin care.

Pharmaco-Phylogenetic Investigation of Methyl Gallate Isolated from Acacia nilotica (L.) Delile and Its Cytotoxic Effect on NIH3T3 Mouse Fibroblast.

Present exploration deals with the therapeutic perspective of methyl gallate isolated from the leaf extract of Acacia nilotica (L.) Delile in contrast to food-borne bacterial pathogen's viz., Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus with their evolutionary succession. The extract was subjected to phytochemical analysis and isolated compound was identified as methyl gallate using UV-vis, IR and NMR spectra. It was found most potent against K. pneumoniae with its minimum inhibition concentration (MIC) of 0.32 mg/ml and minimum bactericidal concentration (MBC) at 0.62 mg/ml. The correlation of MIC values with an evolutionary succession assists the relationship between their genetic and toxic properties. The cytotoxic pursuit of methyl gallate was additionally assessed over NIH3T3 mouse fibroblast by Neutral red (NR) uptake, MTT cell proliferation assay and did not disclose any relevant influence on cell viability as well as cell proliferation. As such, the methyl gallate extracted from the leaf of A. nilotica holds massive antibacterial aptitude and hands out towards a new paradigm for food and pharmaceutical industries.

Antidermatophytic Activity of the Fruticose Lichen Usnea orientalis.

In the present study, the new biological sources in the form of lichen Usnea orientalis Motyka was screened for its antidermatophytic potential. Six species of dermatophytes were chosen on the basis of their prevalence for antidermatophytic assays, and the Clinical Laboratory Standard Institute (CLSI)-recommended broth microdilution procedure was used to detect the efficacy of extract against dermatophytes. Thin layer chromatography of lichen extracts reveals the presence of two secondary metabolites viz. salazinic acid and usnic acid. U. orientalis extract exhibited promising antidermatophytic activity against all tested pathogens. Amongst all tested pathogens, Epidermophyton floccosum exhibited most susceptibility towards extract, whereas Trichophyton mentagrophytes exhibited the least susceptibility. Topical application of U. orientalis extract might be helpful in the cure of dermal infections.

Management of cosmetic embarrassment caused by Malassezia spp. with fruticose lichen Cladia using phylogenetic approach.

During anti-Malassezia screening of plants by CLSI broth microdilution method, Cladia aggregata (Swartz) Nyl. (family Cladoniaceae), a fruticose lichen from Sikkim (northeast Himalayan region), has been found effective at minimum inhibitory concentrations (mg/mL) of 2.72, 0.63, and 1.28 against yeast-like fungi namely, M. furfur, M. globosa and M. sympodialis, respectively. These test pathogens are responsible for pityriasis versicolor (PV) and seborrheic dermatitis (SD) in humans. We tried to establish the reason for variable MICs against various Malassezia spp. using bioinformatical tools, thereby reducing the cost of the experimentation. This is the first report on anti-Malassezia activity of C. aggregata and thus can serve as a potential source for the development of cosmaceuticals.

Studies on antidermatophytic activity of waste leaves of Curcuma longa L.

During antidermatophytic screening of some essential oils, Curcuma longa L. exhibited the strongest antifungal activity, completely inhibiting the mycelial growth of ringworm, caused by the fungi- Microsporum gypseum and Trichophyton mentagrophytes. The essential oil from leaves of Curcuma longa was fungicidal at 2.5 µl/ml at which it tolerated heavy doses of inoculum. The fungicidal activity of the oil was thermostable up to 80 °C and self life up to 24 months in storage. The oil also showed a broad fungitoxic spectrum, inhibiting the mycelial growth of other fungi, viz., Epidermophyton floccosum, M. nanum, T. rubrum, T. violaceum. Moreover, up to 5 % concentration it did not exhibit any adverse effect on mammalian skins. The oil has been formulated in the form of an ointment, 1 % w/v and subjected to topical testing on patients of the Out Patient Department (OPD) at Moti Lal Nehru Medical College, Allahabad. Patients were selected on the basis of KOHpositive results and diagnosed tenia corporis. After the second week of treatment, all patients were KOH- negative. At the end of medication, 75 % of patients recovered completely while 15 % showed significant improvement from the disease. The ointment thus, can be exploited commercially after ongoing successful clinical trials. Relationship of the dermatophytes to the toxicity of the oil vis-a-vis phylogeny using molecular data of the pathogens have also been discussed.

Parathyroid hormone gene polymorphism and sporadic idiopathic hypoparathyroidism.

The pathogenetic mechanisms involved in the development of sporadic idiopathic hypoparathyroidism are currently under investigation. Although autoantibodies against the calcium-sensing receptor (CaSR) have been implicated to play a role, these could be demonstrated in only 49% of a group of 51 patients with sporadic idiopathic hypoparathyroidism that we previously studied. Therefore, we investigated 49 of these patients further, regardless of their antibody status, and looked for mutations in the section of the PTH gene sequence that coded for prepro-PTH as well as the 3'-untranslated region (3'-UTR) of the gene, which is believed to be involved in the stability of its mRNA. We also examined the relationship between the clinical manifestations of the disease and the occurrences of two commonly observed single nucleotide polymorphisms (SNPs) in the PTH gene. In 49 of the patients with idiopathic hypoparathyroidism and in 55 healthy controls, the SNPs were characterized by restriction analysis using DraII and BstBI enzymes. In a subset of these patients, exons 2 and 3 of the PTH gene (n = 37) and its 3'-UTR region (n = 40) were also sequenced. No mutations were observed in the segment of the PTH gene coding for the signal peptide, prohormone, or the 3'-UTR region. However, three well described SNPs were observed: 1) an A-->G substitution in intron 1 in 35.1% of the patients; 2) a G-->A substitution in intron 2, characterized by BstBI, in one or both alleles in 27%; and 3) a C-->A substitution at codon 52 (CGA) of exon 3, characterized by DraII, in one or both alleles in 59.7% of the patients. There was no significant difference in the frequency of occurrence of these SNPs between the patient and the control groups. Furthermore, the mean age at onset of symptoms, body mass index, frequency of cataract, tetany, convulsion, basal ganglia calcification, serum calcium, inorganic phosphorus, and intact PTH were not significantly different between patients with and without the above-described SNPs. Thus, the data from this report demonstrate that in patients with sporadic idiopathic hypoparathyroidism, neither the clinical manifestations nor the biochemical indexes of the disease are related to the occurrence of mutations or SNPs in the PTH gene. Because neither patient nor control samples exhibited any variations in the sequence of their 3'-UTR regions, it is unlikely that mRNA instability is a factor in the pathogenesis of the disease. Additional studies are required to investigate the role of other genes and autoantigens that may be involved in the genesis of idiopathic hypoparathyroidism.

Parmelia cirrhatum: a potential source of broad spectrum natural antifungal.

During antifungal screening of some plants, the water extract of lichen, Parmelia cirrhatum was found to be most effective against tested pathogenic fungi at the range of 60-80 microL/mL concentrations. The extract showed heavy doses of inoculum potential at 80 microL/mL and the antifungal activity of the extract did not expire up to 24 months of storage. The extract did not showed any irritation on mammalian skin up to 10% concentration. Thus, the water extract of Parmelia cirrhatum can be used a potential source of natural antifungal against fungal infections.