Chromatographic fingerprinting of epiphytic fungal strains isolated from Withania somnifera and biological evaluation of isolated okaramine H.

Medicinal plants are "goldmines" of natural products, and continue to provide key scaffolds for drug development. They have immense therapeutic potential, encapsulating a plethora of metabolites within them, which have yet to be explored. Withania somnifera (L.) Dunal is one such medicinal plant known since time immemorial for its therapeutic activity in the Ayurveda system of medicine. Studies have revealed Nature's marvel of these medicinal plants harbouring endophytic and epiphytic microorganisms from phyllosphere to rhizosphere. Chromatographic fingerprinting was carried out using HPTLC and HPLC on five epiphytic strains isolated from the leaves, stem and fruits of Withania somnifera. Out of five filamentous fungi, one fungus identified as Aspergillus aculeatus S20 was well explored. An indole alkaloid, okaramine H, was isolated using systematic chromatographic investigation at a retention time of 26.278 min showing λmax at 206, 236, 284 and 370 nm. Confirmation was achieved using NMR and mass spectrometry (MS) as analytical techniques. Structure elucidation was done by studying the fragmentation pattern using MS/MS and an accurate mass was determined using HR-ESI-QTOF-MS showing m/z of 521.2546 [M + H]+. The percentage purity of isolated okaramine H was found to be >90. Well known for its insecticidal activity, okaramine H was explored for its antileishmanial activity against the Leishmania donovani parasite for the first time. Under in vitro conditions, the compound showed an inhibitory effect on Leishmania donovani promastigotes with an IC50 of 147 µg mL-1.

Development of Mupirocin-Impregnated Bacterial Cellulosic Transdermal Patches for the Management of Skin Infection.

The present study reports the production of bacterial cellulose (BC) membranes using Komagataeibacter hansenii for the development of transdermal wound healing patches. BC-based transdermal patches were developed by impregnating varied concentrations of antibiotic mupirocin and characterized by SEM, FTIR, TGA, and DSC to study the interaction of BC with antibiotic. Developed patches were evaluated for antimicrobial activity, in vitro drug release study, in vivo efficacy, and acute dermal toxicity studies. The antibacterial activity of mupirocin-impregnated patches (mup-BC) showed an inhibition zone from 26.16 ± 0.76 to 35.86 ± 0.61 mm. The in vivo efficacy of mup-BC patches on the superficial abrasion mouse model infected with MRSA 15187 was determined. A single application of the mup-BC (Batch-3) showed a significant decrease up to 2.5 log10 colony-forming units (CFUs) in the infected skin. Acute dermal toxicity study showed no notable sign of toxicity. Pharmacokinetic study indicated that an application of mup-BC (Batch-3) showed a peak plasma concentration of around 1.5 µg/mL mupirocin. The overall preparation, ease of application, and efficacy results clearly indicate that the patches developed in the present study find immense application in the healthcare sector, especially for the treatment of burn or dermal wound infections.

Fungal production of kojic acid and its industrial applications.

Kojic acid has gained its importance after it was known worldwide that the substance functions primarily as skin-lightening agent. Kojic acid plays a vital role in skin care products, as it enhances the ability to prevent exposure to UV radiation. It inhibits the tyrosinase formation which suppresses hyperpigmentation in human skin. Besides cosmetics, kojic acid is also greatly used in food, agriculture, and pharmaceuticals industries. Conversely, according to Global Industry Analysts, the Middle East, Asia, and in Africa especially, the demand of whitening cream is very high, and probably the market will reach to $31.2 billion by 2024 from $17.9 billion of 2017. The important kojic acid-producing strains were mainly belongs to the genus Aspergillus and Penicillium. Due to its commercial potential, it continues to attract the attention for its green synthesis, and the studies are still widely conducted to improve kojic acid production. Thus, the present review is focused on the current production processes, gene regulation, and limitation of its commercial production, probable reasons, and possible solutions. For the first time, detailed information on the metabolic pathway and the genes involved in kojic acid production, along with illustrations of genes, are highlighted in the present review. Demand and market applications of kojic acid and its regulatory approvals for its safer use are also discussed. KEY POINTS: • Kojic acid is an organic acid that is primarily produced by Aspergillus species. • It is mainly used in the field of health care and cosmetic industries. • Kojic acid and its derivatives seem to be safe molecules for human use.

Morphologically dissimilar spores of Aspergillus sojae exhibit genomic homogeneity but the differential in Kojic acid accumulation.

We reported that Aspergillus sojae (SSC-3), an indigenous isolate from rice husk, is a potent kojic acid producer. During optimization, it was observed that under static fermentation conditions, this fungal strain produces two dissimilar morphological green and yellow spores, i.e., SSC-3(Y) and SSC-3(G). Furthermore, these different spore types differ in color, morphology, and in kojic acid metabolite accumulation, with green spores producing 12.87 g/l and yellow spores producing 8.63 g/l of kojic acid on the 12th day of fermentation. To understand if there is a genetic basis for the difference in morphology and metabolite accumulation characteristics, sequencing of internal transcribed spacer regions (ITS) and RAPD analysis from both the spore were carried out. Our study revealed that though the spores are dissimilar with respect to morphology and metabolite accumulation profile, they are genetically homogenous. This suggests that there could be epigenetic differences in these spore types, which may be explored in detail in further studies.

Conversion of amino acids to aryl/heteroaryl ethanol metabolites using human CYP2D6-expressing live baker's yeast.

Different natural aromatic/heterocyclic l-amino acids were biotransformed into aryl/heteroaryl ethanol metabolites via oxidative deamination, decarboxylation and reduction cascades using live baker's yeast cells producing intracellular human CYP2D6 enzyme. Among the three yeast strains expressing 3 different CYP2D6 variants, CYP2D6(2) (i.e. CYP2D6 wild-type) provided the best result under neutral pH conditions at RT. We have successfully converted six natural amino acids into their corresponding alcohols, having one carbon atom less, with moderate yields. Some of the downstream products like tryptophol and tyrosol induced the pTrKB (Tropomyosin receptor kinase B) activation pattern similar to that of BDNF (brain-derived neurotrophic factor), thereby depicting potential antidepressant activity. Control experiments and molecular modelling studies revealed that this tandem bio-transformation probably happens via a pyruvate intermediate. This study establishes that CYP2D6-expressing live yeast cells can be a powerful tool for the enzymatic C-N, C-C bond cleavage of amino-acids.

Consistent production of kojic acid from Aspergillus sojae SSC-3 isolated from rice husk.

A consistent kojic acid producing fungal strain has been isolated from rice husk using glucose-peptone medium. The isolate was identified as Aspergillus sojae SSC-3 on 18S rDNA analysis. A. sojae was capable of producing substantially good amount of kojic acid, however the production was varying from batch to batch. In order to obtain consistent, repeated and high levels of kojic acid, monospore isolation procedures was adopted. The highest production of kojic acid obtained was 12 ± 2 g/L in 120 h with sucrose (10%) and yeast extract (0.5%) as carbon and nitrogen source respectively. The process was scale up to 10 L fermenter size which repeatedly resulted in the production of 18 ± 2 g/L of kojic acid in 96 h. Kojic acid was recovered (> 82%) from the fermentation broth with > 99% purity. Best to our knowledge this is the first report were kojic acid production is reported from Aspergillus sojae strain.

Biotransformation of Chrysin to Baicalein: Selective C6-Hydroxylation of 5,7-Dihydroxyflavone Using Whole Yeast Cells Stably Expressing Human CYP1A1 Enzyme.

Naturally occurring polyphenolic compounds are of medicinal importance because of their unique antioxidant, anticancer, and chemopreventive properties. Baicalein, a naturally occurring polyhydroxy flavonoid possessing a diverse range of pharmacological activities, has been used in traditional medicines for treatment of various ailments. Apart from its isolation from natural sources, its synthesis has been reported via multistep chemical approaches. Here, we report a preparative-scale biotransformation, using whole yeast cells stably expressing human cytochrome P450 1A1 (CYP1A1) enzyme that allows regioselective C6-hydroxylation of 5,7-dihydroxyflavone (chrysin) to form 5,6,7-trihydroxyflavone (baicalein). Molecular modeling reveals why chrysin undergoes such specific hydroxylation mediated by CYP1A1. More than 92% reaction completion was obtained using a shake-flask based process that mimics fed-batch fermentation. Such highly efficient selective hydroxylation, using recombinant yeast cells, has not been reported earlier. Similar CYP-expressing yeast cell based systems are likely to have wider applications in the syntheses of medicinally important polyphenolic compounds.