Identification of bioactive metabolites and evaluation of in vitro anti-inflammatory and in vivo antinociceptive and antiarthritic activities of endophyte fungi isolated from Elaeocarpus floribundus blume.

ETHNOPHARMACOLOGICAL RELEVANCE: Functional disability associated with rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease is a challenging concern in healthcare systems. Along with environmental factors and epigenetic disorders, multiple pathways are reported as prominent mechanism for the progression of RA symptoms including; pain, swelling and stiffness of joints. Elaeocarpus floribundus Blume has been used as a folklore medicine for RA from ancient times. This plant harbours a suite of endophytic fungi that produce a range of metabolites of potential interest. Thus, for the establishment of a scientific basis for this folklore use, it is essential to find out the involvement, if any, of the endophytic fungi living in this plant and the metabolites they elaborate, for the management of RA. AIM OF THE STUDY: This study was designed to isolate, identify and evaluate the in vitro anti-inflammatory and in vivo antinociceptive and antiarthritic activities of the compounds produced by the endophytic fungi living in different parts of Elaeocarpus floribundus Blume. MATERIALS AND METHODS: Endophytic fungi from different parts of the plant were isolated and cultured for the production of secondary metabolites. Chromatographically fractionated fungal extracts were assessed for anti-inflammatory and antinociceptive activities. For the evaluation of anti-inflammatory activity, in vitro cyclooxygenase (COX1/COX2) and 5-lipoxygenase (5-LOX) inhibitory assays were performed. For the evaluation of in vivo antinociceptive activity, hot plate acetic acid induced writhing, and formalin induced paw licking methods were adopted, whereas complete Freund's adjuvant (CFA) induced poly-arthritic method was adopted for the evaluation of antiarthritic activity. The most effective fraction was analyzed by liquid chromatography-mass spectroscopy (LC-MS) in search of the bioactive extracellular metabolites. RESULTS: Five endophytic fungi viz. Aspergillus fumigatus, Aspergillus niger, Rhizoctonia oryzae, Rhizopus oryzae, and Syncephalastrum racemosum were isolated. COX1/COX2 and 5-LOX inhibitory assays state that the Aspergillus niger fraction possesses the greatest activity against these enzymes of inflammatory process. In vivo antinociceptive showed significant (***P<0.001) reduction of pain in a dose dependent manner. As well, significant (***P<0.001) reduction of paw volume was observed in CFA induce poly-arthritic test. LC/MS analysis of the Aspergillus niger fraction revealed the presence of bioactive compounds including tensyuic acid, hexylitaconic acid, chlorogenic acid, nigragillin, TMC-256C1, asnipyrone B, asperenone, fumaric acid and fusarubin, all having reported pharmacological activities. CONCLUSION: The present study demonstrates that secondary metabolites produced by endophytic fungi living in various parts of Elaeocarpus floribundus Blume had potential to relief pain and inflammation. The endophytes were found to contain multiple biomolecules effective in rheumatoid arthritis. These findings provide a rationale for the folklore use of the plant in the management of rheumatoid arthritis.

Roseomonas elaeocarpi sp. nov., isolated from olive (Elaeocarpus hygrophilus Kurz.) phyllosphere.

An aerobic, Gram-stain-negative, coccobacillus-shaped, non-endospore-forming, pink-pigmented bacterium, designated PN2T, was isolated from an olive leaf. The strain grew at 15-35 °C with an optimum temperature for growth at 30 °C, and at pH 5.0-7.5 with an optimum pH for growth at 6.0. Growth was observed in the presence of up to 1.02 % (w/v) NaCl. The major fatty acids were C19 : 0 cyclo ω8c, C16 : 0 and C18 : 1ω7c. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, unknown aminolipids, an unknown phospholipid and an unknown lipid. The respiratory quinone was ubiquinone-10. The DNA G+C content of strain PN2T was 70.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain PN2T was closely related to members of the genus Roseomonas and shared highest similarity with Roseomonas mucosa ATCC BAA-692T (96.5 %), Roseomonas gilardii subsp. gilardii ATCC 49956T (96.2 %) and Roseomonas gilardii subsp. rosea ATCC BAA-691T (96.2 %). Furthermore, the DNA-DNA relatedness value between strain PN2T and the closest related species R. mucosa ATCC BAA-692T was 27 %. These data allowed the phenotypic and genotypic differentiation of strain PN2T from its closest phylogenetic neighbour (R. mucosa ATCC BAA-692T). Based on phenotypic and genotypic characteristics, strain PN2T is classified as representing a novel species of the genus Roseomonas for which the name Roseomonas elaeocarpi sp. nov. is proposed. The type strain is PN2T ( = BCC 44864T = NBRC 107871T).

Production and characterization of a novel laccase with cold adaptation and high thermal stability from an isolated fungus.

A new white-rot fungus SYBC-L1, which could produce an extracellular laccase, was isolated from a decayed Elaeocarpus sylvestris. The strain was identified as Pycnoporus sp. SYBC-L1 according to the morphological characteristics and ribosomal ITS1-5.8S-ITS2 RNA genomic sequence analysis. The highest laccase activity of 24.1 U ml(-1), which was approximately 40-fold than that in basal medium, was achieved in optimal culture medium in submerged fermentation. The laccase produced by Pycnoporus sp. SYBC-L1 was not only a cold adaptation enzyme with a relative catalytic activity of 30.2% at 0 degrees C but also a high thermostable enzyme. The half-lives at 60, 70 and 80 degrees C were 85.5, 37.2, and 2.6 h, respectively. The laccase could effectively decolorize weak acid blue AS and diamond black PV up to 88% and 74.7%, respectively, within 2 h in the absence of any redox mediators. The results suggested Pycnoporus sp. SYBC-L1 was a potential candidate for laccase production and industrial application.

High-throughput culturing of fungi from plant litter by a dilution-to-extinction technique.

High-throughput bacterial cultivation has improved the recovery of slow-growing and previously uncultured bacteria. The most robust high-throughput methods are based on techniques of 'dilution to extinction' or 'extinction culturing'. The low-density partitioning of CFUs in tubes or microwells exploits the fact that the number of culturable species typically increases as inoculum density decreases. Bacterial high-throughput culturing methods were adapted to fungi to generate large numbers of fungal extinction cultures. The efficiency of extinction culturing was assessed by comparing it with particle filtration and automated plate-streaking. Equal volumes of particle suspension from five litter collections of the New Zealand forest tree Elaeocarpus dentatus were compared. Dilute particle suspensions of litter were pipetted into 48-well tissue culture plates containing 1 mL of agar medium per well. Particle volumes from the same samples were applied to continuous agar surfaces in Omnitray plates by automated streaking, and fungal diversity and richness were measured. The spectrum of isolates was assessed by microscopy and sequencing of the ITS or 28S region of the rRNA gene. Estimates of species diversity between the two methods were comparable, but extinction culturing increased species richness. Compared with plating methods using continuous surfaces, extinction culturing distributes fungal propagules over partitioned surfaces. Intercolony interactions are reduced, permitting longer incubation times, and colony initiation and recovery improved. Effort to evaluate and recover colonies from fungal isolation plates was substantially reduced.

Diversity and distribution of saprobic microfungi in leaf litter of an Australian tropical rainforest.

The diversity and distribution of microfungal assemblages in leaf litter of a tropical Australian forest was assessed using two methods: (1) cultures were isolated using a particle filtration protocol (wet season 2001), and (2) fruit bodies were observed directly on leaf surfaces following incubation in humid chambers (wet and dry season of 2002). Four tree species were studied using both methods, namely Cryptocarya mackinnoniana (Lauraceae), Elaeocarpus angustifolius (Elaeocarpaceae), Ficus pleurocarpa (Moraceae), and Opisthiolepis heterophylla (Proteaceae). An additional two species, Darlingia ferruginea (Proteaceae) and Ficus destruens (Moraceae), were studied using direct observations. In total, fruiting bodies of 185 microfungal species were recorded on leaf surfaces (31-81 species per tree species), and 419 morphotypes were detected among isolates obtained by particle filtration (111-203 morphotypes per tree species). Although the observed microfungal diversity was higher with the particle filtration protocol, both methods concurred with respect to microfungal distributions. The overlap of microfungal species in pair wise comparisons of tree species was low (14-30%), and only 2 and 3% of microfungal species were observed in leaves of all tree species by particle filtration and by direct observations respectively. Multivariate analysis of data from direct observations confirmed the hypothesis that microfungal assemblages are strongly influenced by host phylogeny and are also affected by seasonal and site factors. The importance of host species in shaping microfungal distributions was also supported by the particle filtration data. Several taxa new to science, as well as some widespread saprotrophs, were detected on only one host. The underlying reasons for this affinity remain unclear, but we hypothesise that a number of factors may be involved such as fungal adaptation to plant secondary metabolites or the presence of a biotrophic phase in the fungus' life cycle.