Protoplasma (2014) 251:1089–1098 DOI 10.1007/s00709-014-0617-0

ORIGINAL ARTICLE

Isolation, structure determination, and antiaging effects of 2,3-pentanediol from endophytic fungus of amada and docking studies

Sudeep Tiwari & Sailendra Singh & Pallavi Pandey & Shilpi K. Saikia & Arvind Singh Negi & Shailendra K. Gupta & Rakesh Pandey & Suchitra Banerjee

Received: 22 November 2013 /Accepted: 16 January 2014 /Published online: 11 February 2014 # Springer-Verlag Wien 2014

Abstract An endophytic fungus was isolated from the rhi- (22.2 %) higher survival percentage was recorded in compar- zomes of Curcuma amada (), which was iden- ison to untreated control. The compound 1, however, lacked tified as Fusarium oxysporum on the basis of its morpholog- potential antimicrobial activity, indicating the plausible rami- ical and molecular characters. Chromatographic separation fication of the position of OH group in such bioactive mole- and spectroscopic analysis of the fungal metabolite (chloro- cules. In silico evaluation of these molecules against common form extract) led to the identification of one pure compound as well as unique targets corroborated better antiaging poten- having molecular formula C5H12O2, i.e., 2,3-pentanediol (1). tial of 1 in comparison to that of 2. The results for the first time Activity analysis of compound 1 demonstrated improved indicated that the utilization of the endophytic fungi of antiaging (antioxidant, thermotolerance) properties against C. amada could, thus, be a possible source for obtaining Caenorhabditis elegans, in comparison to a similar, commer- non--based bioactive compounds having broader thera- cially available molecule i.e., 1,5-pentanediol (2). The effec- peutic applications pertaining to age-related progressions. tive (lower) concentration of 1 significantly showed (28.6 %) higher survival percentage of the worms under thermal stress Keywords Antiaging . Caenorhabditis elegans . Curcuma (37ºC) compared to its higher concentration (25.3 %), while amada . Endophytic fungus . Fusarium oxysporum . similar trends were followed in oxidative stress where 2,3-pentanediol . 1,5-pentanediol . Molecular docking . Oxidative stress Handling Editor: Jan Raoul De Mey Sudeep Tiwari and Sailendra Singh contributed equally to this work. Introduction S. Tiwari : S. K. Saikia : R. Pandey (*) Microbial Technology and Nematology Division, Central Institute of Endophytes are microorganisms that colonize intercellularly Medicinal and Aromatic (CSIR-CIMAP), Lucknow, Uttar Pradesh 226015, India or intracellularly within healthy plant tissues and consequently e-mail: [email protected] represent a harmonious symbiotic relationship without caus- ing any apparent harm or disease symptoms to the host plants : : * S. Singh P. Pandey S. Banerjee ( ) (Chandra 2012). In recent years, fungal endophytes, especial- Plant Biotechnology Division, Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, Uttar Pradesh 226015, ly those from medicinal plants, have drawn worldwide atten- India tion as they have not only mimicked the biosynthetic potential e-mail: [email protected] of their host plant but have also demonstrated potentials to synthesize unrelated bioactive molecules that find wide- A. S. Negi Medicinal Chemistry Division, Central Institute of Medicinal and ranging applications in medicine, agriculture, and industry Aromatic Plants (CSIR-CIMAP), Lucknow, Uttar Pradesh 226015, (Zhou et al. 2010;Linetal.2011; Bhagat et al. 2012; India Suryanarayanan et al. 2012). Advent of modern biotechno- logical tools has documented a far-reaching predictable prac- S. K. Gupta “ Department of Bioinformatics, Indian Institute of Toxicology and tical utility of economically important plant-inhabiting endo- Research (CSIR-IITR), Lucknow, Uttar Pradesh 226015, India phytic fungi” in industrial production of bioactive substances 1090 S. Tiwari et al. as part of their mutualistic relationships (Guo et al. 2008). in vivo bioactivity analysis of the fungal metabolite. Besides There is an increasing recognition of the vast repertoire of evaluating the antimicrobial property of the compound, anti- biomolecules produced by diverse group of fungal endo- oxidant and antiaging potentials of the same under thermal/ phytes, which can either be categorized as biologically active oxidative stress conditions had also been investigated and/or chemically novel molecules or can be broadly classi- employing a popular experimental model—Caenorhabditis fied as alkaloids, terpenoids, quinones, lignans, phenols, ste- elegans, which mimics the age-related physiological process- roids, isocoumarins, etc. (Lin et al. 2011; Bhagat et al. 2012). es of mammals, including humans (Guarente and Kenyon Numerous studies on isolation and categorization of endo- 2000; Wilson et al. 2006). phytic diversity from various medicinally important plant The present study reports Fusarium oxysporum as the species are progresssing, in order to unravel their relatively major endophytic fungi of C. amada through its isolation unstudied capability of synthesizing novel natural products for and molecular characterization, which yielded a distinctive therapeutic and industrial applications (Guo et al. 2008; fungal metabolite-2,3-pentanediol (1) that demonstrated both Bhagat et al. 2012;Chandra2012). It is however noteworthy in vivo and in silico antiaging potentials. that among the vast plant kingdom consisting of the nearly 300,000 plant species, only a handful of plant species has been studied with regard to their colonized endophytic populations, Materials and methods which leaves ample opportunity to find new and interesting endophytic microorganisms relating to the unstudied plant 1H, 13C and 2D nuclear magnetic resonance (NMR) spectra systems for isolating potential bioactive molecules similar to were recorded using Brukner Advance 300 DRX spectrometer that of the source plant and/or some “unnatural natural and the chemical shifts (δ) were expressed in ppm with products”or new metabolites having therapeutic/commercial reference to TMS as internal standard. ESI-MS data were potentials (Gunatilaka 2006; Aly et al. 2011; Bhagat et al. obtained in Shimadzu LC-MS system after dissolving com- 2012). pound in acetonitrile. Curcuma amada Roxb. (Family: Zingiberaceae), popularly known as mango ginger, is one such globally acclaimed Fungal isolation species of the genus Curcuma that stands next to turmeric in terms of its diverse healing properties owing to its potential to In vivo-grown C. amada rhizomes of single-plant sibling lines synthesize several major bioactive compounds, such as were collected from Chappra district of Bihar, India, and were curcumin, demethoxycurcumin, bisdemethoxycurcumin, di- planted at the research fields of CSIR-CIMAP (26°16′,N, terpene labda-8(17), and 12-diene-15,16-dial (Singh et al. 80°46′,E), Lucknow, India. After 2 months of acclimatization, 2010). Although several earlier studies have revealed percep- the fresh rhizome portions were aseptically collected. The tible bioactivities of the isolated endophytic assemblages of rhizomes were washed thoroughly under running tap water several other Curcuma species, namely Curcuma longa L. and subsequently surface sterilized with 70 % ethanol and

(Maehara et al. 2011), Curcuma wenyujin (Wang et al. 0.1 % HgCl2 for 5 min, followed by 3–4 thorough washing 2012b), and Curcuma phaeocaulis (Bian et al. 2012), no with sterilized distilled water. The sterilized rhizomes were similar report is yet available with respect to the endophytes dried on sterile filter papers, dissected into small (3–5mm) of C. amada, which has been represented by a sole study pieces, and then placed on Potato Dextrose Agar (PDA) without any assigned bioactivity (Banu and Kumar 2009). medium supplemented with antibiotics (100 μg/ml streptomy- This information clearly indicates that functional identifica- cin) followed by incubation at 27±2 °C for 3–10 days. The tion of the most prevalent endophytic microorganisms of plates were methodically examined for fungal growth, and the C. amada coupled with activity-linked characterization of fungal hyphae spreading out from the explants were isolated the major chemical components of the same is the need of as single-fungal strain (coded as CIM-3) which was subse- the hour in view of their future applications. quently incubated on PDA for 7 days at 27±2 °C. Previous research indicated free-radical scavenging or an- tioxidant properties of C. amada metabolites (Policegoudra Fungal identification et al. 2007), but similar type of activity of the compound from the endophytic fungus of this plant is not yet explored. Genomic DNA of the exponentially growing culture of CIM-3 Moreover, many of the molecules of natural origin with anti- was extracted according to the manufacturer’s protocol using oxidant properties that scavenge ROS have attained a central the Zymo research (ZR) Fungal/Bacterial DNA MiniPrepTM stage in antiaging research (Huang et al. 2007; Shukla et al. Kit. Fungal 5.8s rDNA-ITS region was amplified from the 2012). In the background of this information, the present extracted genomic DNA by using the fungal domain specific study was designed to isolate, identify, and chemically char- primers of ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)and acterize the major endophytic fungi of C. amada coupled with ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) (White et al. Microbe based antiaging potentials of 2,3-pentanediol 1091

1990). Each 50 μl of the PCR reaction mixture comprised of negative: Salmonella typhi (MTCC733), Klebsiella 5 μl of 10× PCR buffer, 2 μlof100μMdNTPs,2μl(5pM) pneumonia (MTCC109), Escherichia coli (MTCC723)]. All each of the forward and the reverse primers, 4 μlfungalDNA the bacterial strains were provided by Microbial Type Culture as template, and 0.4 μl of Taq polymerase. PCR was carried Collection Centre (MTCC), Institute of Microbial Technology out through an initial denaturation step at 94 °C for 5 min, (IMT), Chandigarh, India. The impregnated paper disc followed by 35 cycles of 94 °C for 1 min, 50 °C for 50 s, and (Whattman) with different test concentrations (100 μg/disc, 72 °C for 60 s, followed by a final extension at 72 °C for 200 μg/disc, 500 μg/disc) were placed over the seeded plates 5 min. PCR was performed using a thermal cycler (G-Storm- and incubated for 24 h at 37 °C. The activity was measured in GSI, Gene Technologies Ltd, Essex, UK). The amplification terms of zone of microbial growth inhibition (Hi Antibiotic of ITS region was confirmed by running the amplicons on Zone Scale) through Himedia scale. 1.5 % agarose gel. The PCR-amplified product (100 μl) was cleaned using QIAquick spin column (Qiagen, New Delhi, Antiaging activity India) according to manufacturer’s instructions and sequenced using ABI 3130 xl analyzer (Applied Biosystems, USA). C. elegans strain N2 Bristol (wild type) strain of C. elegans The nucleotide sequence was subjected to homology and E. coli OP50 were procured from the Caenorhabditis search using Blast tool (www.ncbi.nlm.nih.gov/org) and was Genetics Center (CGC), University of Minnesota submitted to Sequin (GenBank, NCBI) as the accession (Minneapolis, MN, USA). The bacterial culture was main- number (JQ713567). The phylogenetic analysis was per- tained on Luria agar (LA) plates, and the nematode strain was formed by neighbor-joining in MEGA5, using 1,000 boot- routinely cultured and maintained at 20 °C on nematode strap replicates. The strain showing nucleotide homology to F. growth medium (NGM) seeded with E. coli OP50 bacteria oxysporum has been deposited at IMTECH, Chandigarh, (Sulston and Brenner 1974). India, as accession no. MTCC 11547. Comparative effects of (1) and (2) on C. elegans The effects Fermentation, extraction, and isolation of isolated fungal compound-1 with respect to that of com- mercially available molecule, i.e., 1,5-pentanediol (2) were The fungal culture, growing in potato dextrose broth (PDB) at evaluated for different antiaging parameters of C. elegans.For 27±2 °C, were harvested from 4th to 18th day of incubation at all the assays, worm eggs were synchronized in different 2 days interval and were regularly analyzed for compound concentrations of test compound. The above concentrations isolation. After incubation, the culture filtrate from each ex- of compounds were chosen as no obvious toxic effect was perimental flask was filtered through muslin cloth to remove observed at these concentrations. After 3 days of synchroni- mycelia. Filtered medium (250 ml) was fractionated with zation, the worms (L4, young adults) were transferred to the organic solvents of increasing polarity viz. chloroform and treated NGM plates. ethyl acetate in triplicates. These fractions were dehydrated on dried Na2SO4 and concentrated in vacuo (Buchi Rotavapor Life span assay Age-synchronized L1 worms were treated R-144). All the extracts were subjected to TLC analysis (silica with the test compound concentrations (0, 0.01, 0.1, and gel 60F254 Plate) using the optimized solvent systems 1 %) till their L4 stage. Hundred (n=100) pretreated (L4) MeOH/CHCl3 (5 : 95). The product was isolated through worms were transferred to fresh NGM plates having test preparative TLC (silica gel 60 F254 plate) and was elucidated compound supplementation as (0, 0.01, 0.1, and 1 %). The by various spectroscopies, NMR experiments, and plates having no supplementation (0 %) were termed as con- electrospray mass spectrometry. trol plates. To block the progeny development, 50 μM5- fluorodeoxyuridine (FUdR; Sigma, St. Louis, MO, USA) Biological activity assay was added to the NGM plates and the worms were allowed to grow at 20 °C. The worms were transferred after every Test assay for antimicrobial activity alternate day to ensure the proper supplementation of the test compound. The survival of the worms was observed daily The isolated compound was tested for antibacterial activity under a stereoscopic microscope (Leica S8AP0, Heerbrugg, against selected pathogenic microbes through disc diffusion Switzerland). Worms (L4) were scored daily for survival by assay as described earlier (Bauer et al. 1966) with slight touch-provoked method (Lithgow et al. 1995)untilallnema- modifications. The minimum inhibitory concentration (MIC) todes had died. There were five replicates for each treatment. of compound (1) was determined as per McFarland Standard The experiment was repeated thrice independently. Mean and 0.5 against the selected bacteria [Gram positive: maximum life span values were analyzed and statistical sig- Staphylococcus aureus (MTCC96), Streptococcus mutans nificance (p<0.05) of the survival curves was demonstrated (MTCC890), Bacillus subtilis (MTCC121); and Gram by log-rank test using Kaplan–Meier software. The survival 1092 S. Tiwari et al. curves of control when compared with treated concentrations server (a web-accessible database that currently stores over showed a significant modulation in life span of wild-type N2 7,000 receptor-based pharmacophore models covering 1,627 worms (p≤0.05). The data apart from life span was statistical- drug target information, 459 of which are human protein ly analyzed by one-way ANOVA using Assistat 7.6 beta targets) was selected for this purpose (Liu et al. 2010). statistical assistance software. Differences between the data PharmMapper server predicts the molecular targets of any were considered significant at p≤0.05. given compound using pharmacophore mapping approaches, where the spatial arrangement of various features that are Pharyngeal pumping assay To investigate the effect of (1) essential for a molecule to interact with specific targets are and (2) on pharyngeal pumping of the worms, movement of analyzed before final scoring. the pharynx terminal bulb was recorded according to the To predict the potential antiaging targets for the two com- protocol described previously (Shukla et al. 2012). pounds, the PharmMapper results were further mapped with the data available on Human Ageing Genomic Resources Intracellular ROS detection assay To determine intracellular (HAGR). HAGR includes a curated GenAge database for all ROS level in treated and control worms, a non-fluorescent dye the genes related to human ageing and longevity-associated

H2DCF-DA (Sigma, MA, USA) was used to measure the genes in other model organisms (Wuttke et al. 2012). Only level of fluorescence generated through its interaction with those proteins which had homologous proteins in C. elegans intracellular peroxides converting it into a fluorescent com- were considered as potential targets for antiaging properties of pound i.e., 2′7′-dichlorofluorescein (DCF) (Royall and (1) and (2). Ischiropoulos 1993). Fluorescent readings were measured using SpectraMax M2 multimode microplate reader, (Molecular Devices) at 485 nm excitation and 530 nm emis- Results sion. The observations were recorded every 20 min for 2½h at 37 °C. Screening and identification of the endophytic fungus

Stress resistance assay In order to evaluate the effects of (1) A total of 12 endophytic fungi were obtained from the and (2) on worms’ resistance against oxidative stress, an rhizome of C. amada, which showed analogous growth intracellular ROS generator, Paraquat (Sigma, St. Louis, after 3 days of culture initiation (Fig. 1). On the basis of MO, USA) was used (Zhang et al. 2009). From the above- morphological analysis, most of them corresponded with said synchronized worms, adult day-2 worms were transferred Fusarium spp. To confirm the identity through molecular to 10 mM paraquat and assayed for survival after 72 h of techniques, the PCR product was sequenced and Blast continuous exposure. Stress tolerance of the worms were analysis of 18S rRNA gene (CIM-3) was performed. determined as illustrated earlier (Wilson et al. 2006) with Sequences having maximum similarities were retrieved, minor modifications. For thermal stress analysis, adult day-1 aligned with that of isolate JQ713567. The alignment worms with and without treatments were placed at 37 °C and subjected to neighbor-joining analysis indicated the fun- survival of worms was recorded after every hour by touch- gus as F. oxysporum (Fig. 2) [The fungal strain CIM-3 provoked method (Lithgow et al. 1995).

Statistical analysis

Significant differences between the life span were determined using Kaplan–Meier analysis (MedCalc software). Data apart from life span was statistically analyzed by one-way ANOVA using Assistat 7.6 beta statistical assistance software. Differences between the data were considered significant at p≤0.05.

Molecular docking

Computational approaches were applied to predict the molec- ular targets for these two compounds. The molecular docking study was performed to identify the binding efficacy of both the compounds with target receptor using CDOCKER proto- Fig. 1 Growth of endophyte isolate (Fusarium oxysporum)ofC. amada col available in Accelrys Discover Studio 3.5. PharmMapper rhizome on PDA Microbe based antiaging potentials of 2,3-pentanediol 1093

Fig. 2 Phylogenetic tree constructed from the 18S rRNA gene of strain CIM-3 and related organisms using neighbor-joining algorithm. Accession numbers of corresponding sequences are given in parenthesis and scale bar corresponds to nucleotide substitutions per nucleotide position

(JQ713567) has been deposited at IMTECH, Chandigarh experiments, 1H-1H COSY experiment cross peaks between MTCC No (11547) for preservation and future use]. H1/H2, H4/H5, H3/H4 and correlations between H1/C2, H2/ C1, H3/C2, H4/C2-C3, H5/C3-C4 in HMBC spectrum indi- Structural determination of the compound cated structure of 1 as 2,3-pentanediol (Fig. 3).

Compound (1) was obtained as colorless oil. Its molecular Antibacterial activity formula was determined to be C5H12O2 by ESI-MS (m/z 104 [M]+) and further confirmed by high-resolution mass The MIC of compound (1) against the tested pathogenic bac- (HRESIMS; 143.1101 [M+K]+). 13C NMR showed the pres- teria was found to be negative with all the tested concentrations ence of five carbon atoms. DEPT 90 and 135 experiments indicating no possible antibacterial activity of the same. clearly indicated presence of two methines (CH) at δ71.24 and

72.89 ppm, one methylene (CH2)atδ30.07 ppm, and two Antiaging activities 1 methyls (CH3)atδ17.20 and 19.66 ppm. In HNMR,boththe methyl protons appeared at δ1.11 and 1.13 as broad singlets Life span assay (Table 1). The methylene proton was at δ1.23, while the methane protons were at δ3.34 and δ3.77 as broad multiplet Life span of C. elegans was significantly enhanced with all the and broad singlet, respectively. More deshielded position C3 tested (0.01 %, 0.1 %, and 1 %) concentrations of (1). The and C4 methines were indicated as oxygenated. 13C NMR and mean and maximum life span showed significant increase at HSQC experiments confirmed both the methyl carbons at 20 °C in comparison to that of (2) (Fig. 4a, b and Table 2). δ17.20 and 19.66, methylene carbon at δ30.07, and methine carbons more deshielded again at 71.24 and 72.89 due to In vivo antioxidant assay attachment of hydroxyls. Finally, two-dimensional NMR Free radicals play an important role in aging and age-related diseases in all living systems (Finkel and Holbrook 2000; 1 13 Table 1 H (300 MHz, CDCl3), C (75 MHz, CDCl3), and DEPT 135 and 90 NMR spectral data of compound 1 along with ESIMS and 1 CH 1 CH OH HRESIMS 3 2 2 2 Position δCa DEPT δHb HMBCa Mass CHOH CH2 135 3 CHOH 3 CH2 + 119.66CH3 1.13, bs C2 ESI MS=104 [M ] 2 72.89 CHOH 3.34, bm C1 HRESIMS=143.1101 4 CH2 4 CH2 3 71.24 CHOH 3.77, bs C2 [M+K]+ 5 CH3 5 CH2OH 430.07CH2 1.23, bs C2,C3 For C5H12O2

517.20CH3 1.11, bs C4,C3 Compound 1 (2,3-pentanediol) Compound 2 (1,5-pentanediol) a Fig. 3 Molecular structure of the isolated endophytic fungal metabolite Recorded at 75 MHz compound-1 (2,3-pentanediol) and commercially available glycol b Recorded at 300 MHz compound-2 (1,5-pentanediol) 1094 S. Tiwari et al.

Shukla et al. 2012). It was observed that in comparison to the non-treated control and compound (2) supplementation, the compound (1) at 0.01 and 0.1 % concentration rendered 18.3 and 27.9 % reduction of ROS generation, respectively, in wild- type worms (Fig. 5). Since 1 % concentration was not showing significant difference from 0.1 % (even when indicated in life span assay: Table 2), it was not included for further study.

Stress resistance

In the present study, it was observed that both the test concen- trations of (1) significantly (p<0.01) increased the mean life span of the wild-type worms when challenged to thermal stress at 37 °C (Fig. 6).Thelifespanwithbetterperforming concentration (0.01 %) of (1) under such thermal stress was found to be 28.6 % higher (survival) than that of untreated control and 7.3 % higher than that of the corresponding concentration of compound (2). Similarly, the compound (1) at the same concentration showed 22.2 % higher survival rate under oxidative stress (paraquat) as compared to the control and 4.5 % higher than that of the corresponding concentration of compound (2), respectively (Fig. 7).

Effect on pharyngeal pumping

Pharyngeal pumping is the movement of the pharynx muscles which normally deteriorates with age. In order to investigate Fig. 4 Effect of 1 (a)and2(b) on the life span of C. elegans.The whether compound (1) and (2) affects this physiological be- synchronized L1 larvae were transferred to NGM plates in the absence havior, pharyngeal pumping was recorded in C. elegans fed (control 0 %) or presence (treated 0.01, 0.1, and 1 %) of test concentra- with different concentrations of these two compounds. No tions, and worms were allowed to develop to adulthood. Worms were scored daily for survival until all nematodes had died. The survival curves significant difference was observed in the pumping rates of of control when compared with treated concentrations showed a signifi- treated and non-treated worms, which rules out the possible cant modulation in life span of wild-type N2 worms (p≤0.05) (A). involvement of dietary restriction in life span enhancement. Statistical significance (p<0.05) of the survival curves was demonstrated by log-rank test using the Kaplan–Meier survival analysis. The data, apart from life span was statistically analyzed by one-way ANOVA using Molecular docking Assistat 7.6 beta statistical assistance software. Differences between the data were considered significant at p≤0.05 The potential proteins involved in the aging-related processes in C. elegans, which are either targeted by compound (1), (2),

Table 2 Effect of compound 1and 2 on the life span of C. elegans at 20 °C

Strains Treatment (%) Mean life span±SE % change P value Max. life span±SE Min. life span±SE

N2 Control 19.74±0.23 27.00±1.00 10.00±0.58 0.01 % (1) 24.69±0.32 25.08 <0.0001*** 33.00±0.58 13.67±0.34 0.1 % (1) 22.55±0.28 14.22 <0.0001*** 32.34±0.34 13.00±0.58 1 % (1) 22.51±0.29 14.00 <0.0001*** 30.67±0.34 12.67±0.58 0.01 % (2) 22.91±0.41 16.05 <0.0001*** 31.00±0.58 10.00±0.58 0.1 % (2) 21.76±0.51 10.24 0.0010 30.00±0.00 12.60±0.34 1 % (2) 21.83±0.40 10.59 0.0002 30.00±0.00 13.30±0.34

The life span assay was done with wild-type N2 at 20 °C. Worms were treated with different test concentrations of compound 1 and 2 from L1 stage till the completion of the assay. The mean life span was calculated as the average number of days worms survived in each test concentration ***P<0.0001 Microbe based antiaging potentials of 2,3-pentanediol 1095

Fig. 5 Effect of 1 and 2 on ROS level in C. elegans.Graphwasplottedas Fig. 7 Effect of compound 1 and 2 on paraquat induces oxidative stress relative change in ROS compared to control as 100 %. Errors bars on wild-type N2 worms. Errors bars represent the standard error of mean represent the standard error of mean (SEM). Statistically significant at (SEM). Statistically significant at *p<0.05, **p<0.01 *p<0.05, **p<0.01 or both are shown in Table 3. Both these molecules shared two Discussion common protein targets, i.e., Mitogen-activated protein kinase 14 (MAPK14) and 3-phosphoinositide-dependent protein ki- Study of the symbiotic microorganism from C. amada rhi- nase 1 (PDPK-1) as revealed in Table 2. The molecular zomes resulted into the first-time isolation of an endophytic docking study was performed to identify the binding efficacy fungus, characterized as F. oxysporum with potential to syn- of both the compounds with target receptor using CDOCKER thesize a major bioactive compound, identified as 2,3- protocol available in Accelrys Discover Studio 3.5. In case of pentanediol (1). Noticeably, the population composition and 1, the CDOCKER interaction energy with MAPK14 was ecological distribution of endophytic fungi in Zingiberaceae −26.48 kcal/mol which was much lower than the interaction family revealed the maximum occurrence of Fusarium energy observed with (2) (−16.83 kcal/mol) indicating better (51.0 %) followed by that of Gibberella (17.6 %) as the efficacy of the former. The interaction of both the compounds symbiotic microorganism in the rhizomes of C. longa in the binding cavity of MAPK14 is shown in Fig. 8. (17.6 %) (Lang et al. 2010). Furthermore, F. oxysporum endo- Similarly, the interaction energy of compound-1 with phytes had also been isolated earlier from several other me- PDPK-1 was found to be much lower in (−16.54 kcal/mol) dicinal plant species, which produced diverse metabolite with in comparison to that of (2) (−9.17 kcal/mol), suggesting anticancer and antimicrobial activities (Kour et al. 2008;Cui superiority of the former. Moreover, the isolated (1) demon- et al. 2012). It is interesting to note that instead of the plant- strated very high specificity for caspase-3 and vitamin D3 related metabolites, the present study reports production of a receptor and shared the same binding cavity as that of vitamin different class of compound by the isolated endophyte of D(Table3), indicating its role in controlling the expression of C. amada, which further corroborates earlier observations antiaging genes. (Wang et al. 2012a). It is evident from literature that the endophytes of C. amada has been less explored, and only a single report is available reporting the isolation of an unknown endophyte, which did not reveal any antimicrobial activity (Banu and Kumar 2009). Moreover, the production of diol type of compounds from any fungal endophyte has not been reported so far, although a sole study reported the formation of (1) through reduction of 2,3- pentanedione by 14 out of 21 tested microorganisms of which one belongs to the genus Fusarium (i.e., F. poae CBS 317.73) (Taylor and Mottram 1996). Then again, (1) has been reported to be one of the constituents in the leaf gel extract (LGE) of Aloe greatheadii var. davyana (Botes et al. 2008). The present observation therefore stands out in identifying for the first time a distinctive form of F. oxysporum as the most prevalent Fig. 6 Effect of compound 1 and 2 on thermal stress on wild-type N2 worms. Errors bars represent the standard error of mean (SEM). Statis- endophyte of C. amada with the potential to synthesize an tically significant at *p<0.05, **p<0.01 uncommon metabolite in the form of 2,3-pentanediol (1). 1096 S. Tiwari et al.

Table 3 Molecular targets for 2 and 1 involved in aging-related process and adhesives (Walker and Smith 1996). Diols had also been in C. elegans very beneficially used as solvent, enhancer, and water-binding Protein involved in aging-related process Compound Compound substance which had effectively been blended with numerous 2 1 known pharmaceutical constituents for topical use (Faergemann et al. 2005). Likewise, various other diols belonging to the C4 Mitogen-activated protein kinase 14 ✓✓ family (specially 2,3-butanediol) are gaining global attention ✓✓ 3-phosphoinositide-dependent protein kinase 1 due to their significant implications in drugs, cosmetics, lotion, ✓ Cyclin A2 etc., which has already initiated their commercial production ✓ Daf-2 from diverse microorganisms (Ji et al. 2011). ✓ C-abl oncogene 1, receptor tyrosine kinase Noticeably, although compound-2 has demonstrated nota- ✓ Mitogen-activated protein kinase 8 ble influences as efficient antimicrobial agent against both ✓ Fibroblast growth factor receptor 1 antibiotic-susceptible and resistant bacteria, herpes virus, and ✓ Glutathione reductase fungi (Faergemann et al. 2005; Sundberg and Faergemann Protein-L-isoaspartate (D-aspartate) O- ✓ 2008), the biological activity of 1 is still unexplored. Even methyltransferase Phosphoenolpyruvate carboxykinase 1 ✓ though both the molecules share an almost similar structure (soluble) with slight difference in terms of the position of the hydroxyl Vitamin D3 receptor ✓ group only, there exists a vast difference in their physiological Caspase-3 ✓ properties. Interestingly, unlike (2), the compound (1) did not Protein involved in aging-related process Compound Compound reveal any antimicrobial activity against the presently 2 1 employed assay organisms, reiterating the significance of the Mitogen-activated protein kinase 14 + + change in the position of OH groups in terms of the physio- 3-phosphoinositide-dependent protein kinase 1 + + logical activities of the two concerned molecules as evidenced Cyclin A2 + − earlier (Ultee et al. 2002; Cushnie and Lamb 2005). Daf-2 + − Compound (1) demonstrated much better life span extension C-abl oncogene 1, receptor tyrosine kinase + − (25.08 %, Table 2) in comparison to untreated control and Mitogen-activated protein kinase 8 + − showed 9.03 % higher survival percentage to corresponding Fibroblast growth factor receptor 1 − + concentration of (2); similarly,sametrendswereobservedin Glutathione reductase − + thermotolerance with 7.3 % higher survival rate of worms when Protein-L-isoaspartate (D-aspartate) − + compared to that with better performing concentration of (2). In O-methyltransferase several earlier studies, increased thermotolerance has been not- Phosphoenolpyruvate carboxykinase 1 − + (soluble) ed to be closely associated with the improved longevity and Vitamin D3 receptor − + other age-related parameters (Lithgow et al. 1995). Eventually, Caspase-3 − + (1) also showed significant antioxidant potential through de- creasing the ROS level, which substantiates its role as antiaging molecule, as oxidative stress has been recognized to be a key The compound (1) of the present study belongs to the broad factor for regulating the life span in both C. elegans and humans group of bioactive compounds, known as diols/glycols, which (Finkel and Holbrook 2000). The present observation corrobo- plays a crucial role as antimicrobial, antifreezing, plasticizer, rates several previous studies that have demonstrated positive

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