Isolation of Fungal Strains: An Experimental Study

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Endophytic microorganisms are bacteria or fungi that live inside plant tissues at any moment of their life cycle, without causing damage or disease symptoms to their hosts (deSouza et al. 2011; Sunkar & Nachiyar 2011). They have been found in every plant species examined to date and recognized as the potential products for exploitation in medicine, agriculture and industry. Endophytes are ubiquitous with rich biodiversity. It is noteworthy that of nearly 300.000 plant species that existed on the earth, each individual plant is the host to one or more endophytes. The opportunity to find new and targeting natural products from interesting endophytes microorganisms among myriads of plants in different niches and ecosystem is remarkable (Strobel et al. 2005; Anitha et al. 2011).
The plant Tinaspora crispa (Family Menispermaceae) occupies a very important place in the field of medicinal plants and is widely used as a traditional medicine. Tinaspora crispa (locally named “brotowali” in Indonesia) is a small herb which grows widely in temperate and tropical part of Asia. More specifically, the plant is widely found in tropical and subtropical Philippines, Indonesia, Malaysia, Thailand, India, China, and Vietnam. The plant is also known by its numerous synonyms, viz., Menispermum crispum Linn., Tinaspora cordifolia F. Vill., Tinaspora tuberculata, and Tinaspora rumphii (Mohammed et al. 2012; Elfita et al. 2013).
It is widely used in the traditional medicinal practice of peoples living in Malaysia, Indonesia, and Thailand to treat ailments like fever, jaundice, hyperglycemia, wounds, intestinal worms and skin infections. Moreover, T. crispa is also used to treat tooth and stomach ache, coughs, asthma and pleurisy. In several studies, T. c...

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... 500 MHz (1H) and 125 MHz (13C) on JEOL JNM ECA-500 spectrometer).

RESULTS
Isolation, Purification, and Extraction of Fungal Strains.
Two fungi were isolated from the roots of brotowali The strains were identified as Trichoderma sp. and Aspergillus sp. by the Sekolah Ilmu dan Teknologi Hayati, Institut Teknologi Bandung. The broth culture was filtered to separate the filtrate and mycelia and extracted twice by partition with an equal volume of ethyl acetate. The organic solvent was evaporated to dry under vacuum to yield 6.2 g of crude extract. All comparative Thin Layer Chomatograpic (TLC) analyses, developed in the following solvents: n-hexane-EtOAc (5:5 v/v); n-hexane-acetone (6:4); and n-hexane-chloroform (3:7), showed two major compounds. The isolation of these two lactones from ethyl acetate extract of Trichoderma sp from brotowali is described in Figure 1.
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