Ravindra H. Patil

Endophytic Mycoflora of Indian Medicinal Plant, Terminalia arjuna and their Biological Activities

Studies were undertaken to isolate, identify and evaluate the biological activities of endophytic mycoflora of Indian medicinal plant, Terminalia arjuna. A total of 20 isolates of endophytic fungi were obtained from the leaves, twigs and bark tissues of the Terminalia arjuna. Out of 20, six isolates exhibited promising antibacterial, antifungal and anti-inflammatory activities when cultivated at shake flask level. The selected isolates were identified on the basis of morphology and ITS gene sequencing. Three isolates, designated as TA BA 1, TA L1 and TA L2 were identified as Aspergillus flavus whereas; the remaining three endophytic fungi were identified as Diaporthe arengae (TA TW2), Alternaria Sp. (TA TW1) and Lasiodiplodia theobromae (TA BA2). Aspergillus flavus was found as the predominant endophyte in leaves and bark tissues of the plant. The crude extract of the test isolates showed considerable antimicrobial activity against common human bacterial ( Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Salmonella abony and Bacillus subtilis ) and fungal ( Candida albicans, Aspergillus niger and Penicilium sp.) pathogens. The extract of Diaporthe arengae (TA TW2) significantly reduced the concentration of DPPH free radical as percent DPPH scavenging activity was found to be highest (69.56%) in comparison with other isolates. The % inhibition of hemolysis of RBCs was found to be highest (82.85%) with Diaporthe arengae (TA TW2) in comparison (83.26%) with standard drug (Ibuprofen). Among all, the extract of the Diaporthe arengae (TA TW2) showed excellent biological activities and hence was subjected to further characterization. The phytochemical investigation of the extract revealed the presence of terpenoids as the major phytoconstituents which was supported by TLC and UV spectroscopic studies. The results indicate that the isolated endophytes could be the valuable source of these bioactive molecules with diverse biological activities. The bioactivities may be attributed to the terpenoids present in the endophytic extract.

Chapter 5 – Bioactive Secondary Metabolites From Endophytic Fungi: A Review of Biotechnological Production and Their Potential Applications

New viral diseases, multidrug-resistant microorganisms, and infections by uncommon pathogens are emerging as new threats to human, plant, and animal health. Therefore, there is continuous need of discovery and development of new, safer, and effective drugs to combat these diseases. In the recent past, our drug discovery strategies heavily relied on microorganism where nearly 80% of the worlds antibiotics have their origins. These microorganisms are mainly isolated from the soil and most of the worlds soils have been screened out for the search of new microbes. Therefore, there is an urgent need to explore new microbial habitats for the microorganisms that produce novel bioactive compounds. “Endophytes” are the microorganisms which are present inside the living tissues of the plant by establishing a variety of relationships such as symbiotic, mutualistic, or parasitic with their host plants. In their relationship with the host plant, endophytes contribute to the plant by producing a plethora of substances that provide protection and survival benefits to the host plant. Endophytic fungi have been recognized as a novel source of bioactive secondary metabolites with diverse biological activities such as novel antibiotics, antimycotics, immunosuppressant, anticancer compounds, and so on to name a few. Their bioactive potential was first recognized when worlds first multibillion dollar anticancer drug, paclitaxel (Taxol), was obtained from Taxomyces andreanae, an endophytic fungus isolated from the yew plant, Taxus brevifolia. However, it has been estimated that less than 1% of endophytic microorganisms are currently known, suggesting that a vast majority of them are yet to be discovered. Thus, endophytic microbes represent a potential source for the discovery of new and useful compounds, or new platforms for the organic synthesis of such compounds for the benefit of mankind. Moreover, plants located in the area of biodiversity hotspots of India, such as Eastern Himalaya, Western Ghats, and Andaman and Nicobar Island are believed to be host to novel endophytes with diverse chemical potential. The objective of the present article is to review the current state of information on the endophytes and their hosts, advances in isolation techniques, mode of cultivation, culture conditions, and biological activities of metabolites derived from endophytic microorganisms.

Data on the cost effective surface sterilization method for C.carandas (L.) seeds and callus induction from aseptic seedling.

Surface sterilization of explant is an important and most sensitive step in plant tissue culture. Inappropriate concentrations of sterilants have lethal effect in cell division and it restricts growth and development of explant. Therefore, suitable concentration, combinations and duration of exposure of sterilant is essential to raise in vitro cultures successfully. This data demonstrates use of various sterilizing agents for aseptic plantlet germination from seed of Carissa carandas (Apocynaceae). The present dataset provides information in support of cost-effective explant sterilization potential of benzalkonium chloride containing commercial bleach (Lizol) and its comparison with traditionally used surface sterilants in plant tissue culture i.e. 0.1% HgCl2 alone and in combination with 70% alcohol. The data on callogenic response using MS medium supplemented with plant growth regulators is also shared.

Rapid Chromatographic Determination and Structural Confirmation of β-Hydroxy Acid Form of Lovastatin in the Fermentation Broth of Aspergillus Terreus PM03

Lovastatin, a fungal secondary metabolite, competitively inhibits 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase, which catalyzes the rate limiting step in the biosynthesis of cholesterol. Preparative thin-layer chromatography (TLC) followed by RP-HPLC was used for determination of lovastatin in the fermentation broth of Aspergillus terreus PM03. The TLC separated bands were cut, recovered, and analyzed by HPLC. The proposed HPLC method used a mobile phase comprising 1 : 1 mixture of acetonitrile and deionized water at a flow rate of 2 mL/min and spectrophotometric detection at a wavelength of 238 nm. The chromatograph revealed a single, sharp and symmetric peak at a retention time of 6.8 min that corresponded to an open β-hydroxy acid form and was comparable to the peak of standard β-hydroxy acid form of lovastatin with respect to both retention time and optical absorption at 238 nm. The data of UV–VIS and FTIR spectroscopy confirmed the presence of lovastatin in the purified extract. The structure of the obtained product was also confirmed by LC-MS and NMR analyses.

Endophytes: Potential Source of Therapeutically Important Secondary Metabolites of Plant Origin

Use of plants and plant-derived metabolites for human health and well-being is as old as human civilization. The plant kingdom contains an estimated 400,000–500,000 different species and each plant produces a number of secondary metabolites which enables them to withstand various biotic and abiotic stresses. The plant secondary metabolites such as alkaloids, steroids, flavonoids, terpenoids, etc., are known to have a number of biological activities. Moreover, because of their natural origin, the plant-derived metabolites are increasingly preferred for therapeutic applications all over the world. However, the overharvesting of plants for biologically active secondary metabolites is rapidly diminishing the valuable treasure of medicinal plants. Endophytes are the microbial symbionts which live in the internal tissues of plants and mimic the chemistry of the host plant. Because of their huge diversity and ability to produce a range of metabolites similar to host plant, they have attracted significant attention of scientific community all over the world. The plant- and endophyte-derived metabolites which have attracted sufficient research in last decade include compounds with antioxidant, antihypercholesterolemic, antidiabetic, and anticancer activities. The present article reviews the current state of research on biologically active metabolites from plant and endophytic fungi. The work carried out in our lab on bioprospecting of endophytic fungi for molecules with antihypercholesterolemic potential is also included.