Bangladesh Journal of Pharmacology Volume: 13; Number 4; Year 2018 Cite this article as: Shylaja G, Sasikumar K, Sathiavelu A. Antimycobacterial and anti-oxidant potential of the bio- active metabolite isolated from the endophytic fungus Daldinia eschscholtzii. Bangladesh J Pharmacol. 2018; 13: 330-32. Antimycobacterial and anti-oxidant poten- tial of the bioactive metabolite isolated from the endophytic fungus Daldinia eschscholtzii Sir, Endophytic fungi from the plant are considered as one of the predominant resources to produce novel bioactive metabolites with many therapeutic potentials, used since the production of taxol (an anti-cancer drug) obtained from the endophytic fungus Taxomyces andreanae (Gouda et al., 2016). In this study, endophytic fungi Daldinia eschscholtzii was isolated from the ornamental plant Mussaenda luteola (Family: Rubiaceae) since this plant was reported with many therapeutic properties like cytotoxicity, anti- inflammatory, antimicrobial and many others with predominant phytochemicals such as iridoids, triter- penes and phenols (Shylaja and Sathiavelu, 2017). Daldinia sp. belongs to the class ascomycetes and order xylariales constantly accompany with certain genera of host plants as an endophyte but also appear on other woody substrates. This genus is considered as a warehouse of bioactive metabolites with major therapeutic potentials. The immune suppressive compound dalesconol A and B polyketides, daeschol A, helicascolide A, dalesconol C and dalmanol A are some of the compounds obtained from D. eschscholtzii (Yuyama et al., 2013). Some other metabolites isolated from this species include selesconol, isoindolinones, dihydroisocoumarins, benzofuran lactones with thera- peutic properties like immunosuppressive, antioxidant, cytotoxic and anti-HIV respectively. This fungus had been investigated as an endophyte from the plant Pogostemoncablin and identified bioactive metabolites such as eschscholin A, 3,5-dihydroxy-2-methyl-4H- chromen-4-one etc. (Liu et al., 2017). The aim of the present study was to isolate and characterize the secondary metabolite from the endophytic fungus D. eschscholtzii and evaluation of anti-oxidant and antimycobacterial potential. The endophytic fungus was isolated from the leaf segment of the plant M. luteola (Petrini, 1986) and it was identified as D. eschscholtzii by 18S rRNA sequencing (Guo et al., 2000). The 25 days grown culture was filtered and extracted with ethyl acetate and the filtrate (2 L) was dried to obtain a crude extract (620 mg). It was subjected to a modified solvent-solvent fractiona- tion method (Mtunzi et al., 2017; Shylaja et al., 2018) for isolation of secondary metabolites. Initially, the crude extract was partitioned with petroleum ether (26 mg), chloroform (242 mg) and methanol (147 mg). The chloroform fraction was dried and purified further by the column chromatography using chloroform solvent alone. Then the resultant fraction was washed again with petroleum ether, chloroform and methanol in the ratio of 3:3:1 to obtain a soluble fraction and insoluble precipitate. The precipitate was dissolved in chloroform and eluted in the column chromatography to yield compound 1 (46 mg). The structure elucidation of the compound 1 was analyzed based on the spectral data obtained from FT-IR, MS and 1H and 13C NMR. The compound 1 was isolated as oily yellow substance, and the FT-IR spectrum shows the absorption maxima νmax at 3292, 2926, 2854 which revealed the presence of OH and CH stretch, 1689 showed C=O, the peak at 1195 -1331 showed the presence of C-O (aromatic ester). The νmax from 503-947 revealed the presence of C-H. The molecular ion peak at m/z 503.992 [M+H]+ by HRESIMS indicated the molecular formula of com- pound 1 was C33H58O3 (Calcd. 502.824 g/mol). The 13C and 1H NMR spectra of the compound 1 showed signals for a tetrasubstituted benzene. The peaks at δC 160.09 and δC 156.96 were due to the presence of two conjugated ketone (C=O) at C-1 and C-4 positions of the benzene ring respectively. A methoxy group (CH3O-) at the C-5 position and free methyl group at C-2 position showed peaks at δC 22.69 and δC 21.48 respectively. A long linear alkyl chain (pentacosyl, C-25) at a C-3 position of benzene rings exhibited peak range from δC 24.73 to δC 45.58 and free methyl group from the long alky chain (C-31 position) exhibited signal at δC 21.37. The olefinic carbons in the aromatic benzene ring at C- 2, C-3, C-5 and C-6 represented peaks ranging from δC 117.06 to δC 123.99. In 1H NMR, the free methyl (–CH3) group at positions C-31(3H) and C-32 (3H) showed signals at δH 0.842 and δH 0.872 respectively. The proton signal at δH 2.196 was due to the methoxy group (CH3O) at position C-5 (3H). The methylene group (- CH2) from linear alkyl chain at position C-8 to C-30 (2H) exhibited signal range from δH 0.948 to δH 2.883. The olefinic proton at C-6 (1H) of aromatic ring showed signal from δH2.926. Based on the spectral data obtained A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2018; 13: 330-332 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v13i4.38593 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor and comparing with the previous report that the compound 1 was characterized as 5-methoxy-2-methyl- 3-pentacosyl-1,4 benzoquinone (Qin and Liu, 2004) and the structure is given in Figure 1. Earlier studies show that three new homologous series metabolites of 3-alkyl-5-methoxy-2-methyl-1,4-benzo- quinones, with chain lengths varies of C21 to C23 and many other benzoquinones metabolites were isolated from the fungus D. concentrica (Stadler et al., 2014; Qin and Liu, 2004). The antimycobacterial potential of the compound 1 was determined using resazurin-based microtitre assay (REMA) which is used to study the metabolism and sustainability of microorganisms (Martin et al., 2005). Resazurin dye is a blue color oxido -reduction reagent which gets oxidized and turns to pink color due to the metabolism of growing microorganisms (Rivoire et al., 2007). The various concentration of isolated compound 1 was tested for antimycobacterial potential against Mycobacterium tuberculosis H37Rv strain. The results revealed that compound 1 showed the significant inhibition compared to the standard drugs and compound 1 had the MIC value of 25 μg/mL (Figure 2). The compound 1 was also screened for its anti-oxidant potential using the DPPH free radical scavenging assay (Yadav et al., 2014). The result showed maximum inhibition of 80.2 ± 1.3% at 100 μg/mL of concentration (Table I). This is the first report on the isolation of Daldinia sp. from the plant M. luteola. The results of the present study conclude that compound 1 is 5-methoxy-2-methyl -3-pentacosyl-1,4 benzoquinone isolated from the fungus D. eschscholtzii which may be a prominent resource of the pharmacologically active lead molecule for the development of antimicrobial drug. The authors acknowledge the laboratory facility and other supports of the Vellore Institute of Technology University, India. Gunasekaran Shylaja, Kandasamy Sasikumar and Arunachalam Sathiavelu School of Biosciences and Technology, Vellore Institute of Technology University, Vellore 632014, Tamilnadu, India. Corresponding author: email: asathiavelu@vit.ac.in References Gouda S, Das G, Sen SK, Shin HS, Patra JK. Endophytes: A treasure house of bioactive compounds of medicinal importance. Front Microbiol. 2016; 7: 1538. Guo B, Dai JR, Ng S, Huang Y, Leong C, Ong W, Carte BK. Cytonic acids A and B: Novel tridepside inhibitors of hCMV protease from the endophytic fungus Cytonaema species. J Nat Prod. 2000; 63: 602-04. Liu HX, Tan HB, Li SN, Chen YC, Li HH, Zhang WM. Two new metabolites from Daldinia eschscholtzii, an endophytic fungus derived from Pogostemoncablin. J Asian Nat Prod Res. 2017; 19. Martin A, Palomino JC, Portaels F. 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Three new homologous 3-alkyl-1,4- Bangladesh J Pharmacol 2018; 13: 330-332 331 Table I DPPH free radical scavenging potential of the compound 1 % Inhibition of DPPH free radical Concentration (µg/mL) Compound 1 Standard 25 68.7 ± 0.6 76.5 ± 1.8 50 72.5 ± 0.4 81.6 ± 2.2 75 75.0 ± 0.4 85.6 ± 2.0 100 80.2 ± 1.3 90.0 ± 1.6 Figure 1: Structure of the compound 1 (5- methoxy-2-methyl- 3-pentacosyl-1,4 benzoquinone) Figure 2: Antimycobacterial potential of endophytic fungal metabolite C1 from D. eschscholtzii benzoquinones from the fruiting bodies of Daldinia concentrica. Helv Chim Acta. 2004; 87: 2022–24. Rivoire N, Ravololonandriana P, Rasolonavalona T, Martin A, Portaels F, Ramarokoto H, Razanamparany VR. Evaluation of the resazurin assay for the detection of multidrug- resistant Mycobacterium tuberculosis in Madagascar. Int J Tuberc Lung Dis. 2007; 11: 683-88. Shylaja G, Sasikumar K, Sathiavelu A. 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Acta Amaz. 2013; 43: 1-8. 332 Bangladesh J Pharmacol 2018; 13: 330-332 DatePrinted: This article was downloaded by you on: Nov 07, 2018