Printer Friendly

Isolation and characterization of bioactive metabolites from Xylaria psidii, an endophytic fungus of the medicinal plant Aegle marmelos and their role in mitochondrial dependent apoptosis against pancreatic cancer cells.


Background: The genus Xylaria has been reported as a rich source of biologically active secondary metabolites. In the present study, an endophytic fungus Xylaria psidii has been isolated from the leaf sample of Aegle marmelos (L.) Corr., characterized on the basis of its morphological features and sequence data for the ITS region (KU291350) of the nuclear ribosomal DNA. Biological screening of ethyl acetate extract of Xylaria psidii displayed a potential therapeutic effect on pancreatic cancer cells.

Hypothesis: This study was designed systematically to explore Xylaria psidii, an endophytic fungus for the identification of biologically active secondary metabolites against pancreatic cancer cells.

Methods: While exploring the bioactive secondary metabolites, a sensitive and reliable LC-MS based dereplication approach was applied to identify four compounds A-D from fungal extract. Further bioactivity guided isolation of fungal extract yielded two major metabolites 1 and 2. The structures of 1 and 2 have been determined by detailed spectroscopic analysis including MS, NMR, IR and UV data and similarity with published data. Xylarione A (1) is new whereas (-) 5-methylmellein (2) is reported for the first time from X. psidii. Both the isolated compounds were screened for their effect on the viability and proliferation against a panel of cancer cell lines (MCF-7. MIA-Pa-Ca-2. NCI-H226, HepG2 and DU145) of different tissue origin.

Results: Compounds 1 and 2 exhibited cytotoxicity against pancreatic cancer (MIA-Pa-Ca-2) cells with [IC.sub.50] values of 16.0 and 19.0 [micro]m, respectively. The cell cycle distribution in MIA-Pa-Ca-2 cells, confirmed a cell cycle arrest at the sub-G1 phase. Cell death induced by 1 and 2 displayed features characteristic of apoptosis. Flow cytometry based analysis of 1 and 2 using Rhodamine-123 displayed substantial loss of mitochondrial membrane potential in a concentration dependent manner by both the compounds.

Conclusion: Results conclude that the isolated compounds 1 and 2 are responsible for the activity shown by crude ethyl acetate extract and may act as potential leads for medicinal chemists for designing more potent analogs.


Xylaria psidii

Endophytic fungi



MIA-Pa-Ca-2 cells


Nature has been an exceptional resource of new biologically active compounds since an amazing chemical diversity is found in the large number of species of plants, animals, marine organisms and microorganisms (Buttler et al., 2014). Despite of decline of pharmaceutical company's interest in natural products and the rise in combinatorial chemistry, natural products still have an important role to play, offering an unthinkable diversity of unique chemical skeletons. In last two decades, plant endophytes have gained attention as a source of secondary metabolites with new chemical structures (Tan and Zou, 2001). Endophytes are microbes (fungi and bacteria) that live symbiotically in the internal healthy tissues of the host plant, without damaging the host (Strobel and Daisy, 2003; Petrini and Petrini, 1985; Wennstrom, 1994;). Some endophytic fungus can produce alike biological active compounds as the host, for example taxol (Stierle et al.,1993), capsaicin (Devari et al., 2014) and piperine (Chithra et al., 2014) and some produce drug like camptothecin (Singh et al., 2013) etc. In addition, these endophytes also produce novel secondary metabolites (Schulz et al., 2002) and sometime very unique/attractive chemical skeletons (Liu et al., 2002). These microbial metabolites have also been reported to be associated with several pharmacological activities such as anti-oxidant, anti-viral, anti-diabetic, anti-biotic, anti-cancer and immunosuppressive etc. (Challinor and Bode, 2015; Gutierrez et al., 2012; Strobel and Daisy, 2003; Koul et al., 2016).

Aegle marmelos (L.) Corr. commonly known as "Bael" in hindi (Kesari et al., 2006) belongs to Rutaceae family, is a well-known Ayurvedic medicinal plant. It is an aromatic, and moderate sized plant that usually found in South East Asia and several parts of India (Sankeshi et al., 2013). It is known to have anti-proliferative, anti-pyretic, anti-oxidant, anti-diarrhoeal, anti-inflammatory, hypoglycemic, anti-cancer and anti-fungal activities (Kumari et al., 2014). The principal components include aegeline, marmin, marmelonin, and xanthotoxin etc. However, very little is discovered about secondary metabolites produced by endophytes sheltered inside the healthy tissues of A. marmelos. In our ongoing program to discover cytotoxic compounds from the endophytic fungi (Devari et al., 2014; Guru et al., 2015), we have recently isolated the endophytic fungus Xylaria psidii from the leaf of Aegle marmelos, taxonomically designated by analysis of the nucleotide sequence of its ITS region. According to previous studies, genus Xylaria is broadly distributed in nature, living inside lichens and host plants as endophytic fungi, and growing on the dead wood as decay fungi. A number of new metabolites have been discovered from the strains of Xylaria sp. which were isolated from the leaf of Curcuma xanthorrhiza (Hammerschmidt et al., 2015), Pythium ultimum (Baraban et al., 2013) and Toona sinensis (Zhang et al., 2014) etc. Xylaria species is found to produce a variety of secondary metabolites of biological importance namely succinic acids (Klaiklay et al., 2012), xanthones (Healy et al., 2004), terpenoids (Deyrup et al., 2007; Smith et al., 2002), cyclopeptides (Wu et al., 2011), lactones (Jimenez-Romero et al., 2008) and cytochalasins (Espada et al., 1997). In our initial screening, crude ethyl acetate extract (EtOAc) of Xylaria psidii, was found to be active against a set of cancer cell lines within the cutoff point set by the National Cancer Institute for cytotoxicity ([IC.sub.50] < 20 [micro]g/ml) (Lee and Houghton, 2005). Four compounds A-D previously reported from genus Xylaria have been identified by LC-MS analysis of the ethyl acetate extract of Xylaria psidii (Xu et al., 2013; Isaka et al., 2012). Further, purification of ethyl acetate extract was achieved by chromatographic methods which leads to the isolation of two major compounds, one novel furanodione, named xylarione A (1), together with a known cyclic lactone. (-) 5-methylmellein (2). The structures of compounds 1 and 2 were established by detailed ID and 2D NMR spectroscopic analysis and comparison of their NMR data with those reported in the literature. In addition to the discovery of new compound 1, this is the first report of the isolation of (-) 5-methylmellein (2) in endophytic fungus Xylaria psidii. In this paper, we report the isolation and structural characterization of the metabolites as well as cytotoxic activity against MCF-7 (breast adenocarcinoma), MIA-PaCa-2 (pancreatic carcinoma), NCI-H226 (non-small cell lung cancer), HepG2 (hepatocellular carcinoma) and DU145 (prostate carcinoma) cell lines using MTT assay. The results indicated that compounds 1 and 2 exhibited best cytotoxicity against pancreatic cancer (MIA-Pa-Ca-2) cells with [IC.sub.50] values of 16.0 and 19.0 pm. respectively. The cell cycle distribution in MIA-Pa-Ca-2 cells, confirmed a cell cycle arrest at the sub-G1 phase. Cell death induced by 1 and 2 displayed features characteristic of apoptosis. Flow cytometry based analysis of 1 and 2 using Rhodamine-123 (Rh-123) displayed substantial loss of mitochondrial membrane potential in a concentration dependent manner by both the compounds.

Materials and methods

General experimental procedure

ID and 2D NMR spectra (with chemical shifts expressed in [delta] and coupling constant in Hertz) were recorded on Bruker 400 MHz instrument and are referenced at [CD.sub.3]OD: 3.31 ppm; CD[Cl.sub.3]: 7.27 ppm. UV spectra were obtained on a Shimadzu UV2600 UV-Vis spectrophotometer. IR spectra were obtained on Perkin Elmer IR spectrophotometer. Melting points were taken on Buchi B-545 melting point apparatus. HR-ESIMS were recorded on an Agilent 1100 LC-Q-TOF mass spectrometer and HRMS-6540-UHD machines. Optical rotations were measured on a Perkin Elmer 241-Polarimeter (c g/100 ml) at 589 nm. HPLC purifications were performed on an Agilent 1260 Infinity II HPLC system with UV detector. HPLC solvents like MeOH and ACN were procured from Merck chemicals and water for extractions and HPLC analysis was obtained from high-purity Milli-Q Advantage A10 water system (Millipore, Molsheim, France). All other chemicals such as 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MIT), were purchased from Sigma-Aldrich Company and used as received. Liquid column chromatography was done using silica gel (60-120 and 230-400 mesh). TLC (Merck) was carried out on precoated silica gel plates 60 [F.sub.254] or RP-18 [F.sub.254] plates with 0.5 or 1 mm film thickness. UV light or anisaldehyde-[H.sub.2]S[O.sub.4] or [H.sub.2]S[O.sub.4]-MeOH reagents were used to visualize spots on TLC plates.

Plant material and isolation of endophytic fungus

Healthy, symptomless leaves of Aegle marmelos were collected from Yamuna Nagar district of Haryana, India. Samples were taken in sterilized bags, kept in icebox, followed by isolation of fungal endophytes within 24 h of collection. Samples were first washed with running tap water followed by triple washing with autoclaved distilled water. Surface sterilization was given by consecutive treatment with 70% ethanol for 60s, 1% sodium hypochlorite (NaOCl) for 60s, and 70% ethanol for 30s, after each treatment sample was rinsed with sterile distilled water. Treated samples were placed on sterilized blotting paper to eliminate the extra moisture. Surface sterilized samples were excised into approximately 6x6mm pieces and placed on the 90 mm petridishes containing Water Agar medium (2% Agar w/v). To avoid the bacterial growth, media was supplemented with 200 [micro]g/ml streptomycin sulfate (HiMedia Laboratories). Effectiveness of the surface sterilization was insured by taking the imprints of treated leaves to consider as negative control. The petridishes were incubated at 28[degrees]C for 3-12 days until the mycelium appeared from the inoculated plant sample. Hyphal growth emerged from the inoculated samples were picked and transferred to potato dextrose agar media, incubated at 28[degrees]C to obtain a pure culture. The endophytic fungal strain Xylaria psidii was submitted to the Col. Sir R. N. Chopra, Microbial Resource Center Jammu (MRCJ), India under accession number MRCJ-295.

DNA extraction, amplification and sequencing

The ZR Fungal/Bacterial DNA MiniPrepTM kit was used to obtain the fungal genomic DNA according to manufacturer protocol. The qualitative and quantitative analysis of genomic DNA was done by NanoDrop 2000. The internal transcribed spacer region was amplified by universal primers ITS5 (forward) and ITS4 (reverse) as described (White et al., 1990). The PCR amplification was carried out in a 40 [micro]l reaction volume with 20 [micro]l of PCR Master Mix 2X (Thermo Scientific), 2 [micro]l of 10 [micro]m each primer, 3 [micro]l of fungal genomic DNA (100 ng), and 13 [micro]l of molecular biology grade water. The thermal cycling program was set as follows: initial denaturation 95[degrees]C for 5 min; 32 cycles of 30 s at 94[degrees]C, 45 s at 58[degrees]C, and 55 s at 72[degrees]C; with final extension of 15 min at 72[degrees]C. The amplified PCR product was checked on 1.8% agarose gel electrophoresis and cleaned up with HiPurA[TM] PCR Product Purification Kit (HiMedia Laboratories) according to manufacturer protocol. The amplified ITS region was sequenced in both directions by ABI Prism 377 DNA sequencer.

DNA sequence assembly and phylogenetic analysis

To taxonomic designate the endophytic fungi; the sequences obtained were assembled and used as query sequence for similarity search by using BLAST algorithm against the nucleotides database maintained at National Center for Biotechnology Information ( The sequence of ITS region was aligned with the most similar reference sequences of the taxa by using the clustalW program. A phylogenetic tree was constructed by using MEGA6 software (Tamura et al., 2013), subsequently analyzed for evolutionary distances by the neighbor joining (NJ) module. The robustness of the clades was estimated with bootstrap analysis by 1000 replications. The assembled contiguous rDNA sequences of the representative isolate was submitted to GenBank database using SEQUIN program under accession no. KU291350.

Inoculum preparation, fermentation and extraction

To prepare the inoculum of the fungal culture, Erlenmeyer conical flasks of capacity 500 ml containing potato dextrose broth (PDB, 200 ml) were used. The flasks were inoculated with fungal agar plugs, kept in shaking incubator at 28[degrees]C with 150 rpm for 3 days. The prepared inoculums (10%) was used to obtain large amount of fermented culture broths in Erlenmeyer flasks (11) containing PDB (350 ml). The flasks were kept in shaking incubator at 28[degrees]C with 150 rpm and dark for 10 days. From 30 Erlenmeyer's flasks, 11.51 fermented culture was obtained. The 11.51 cultures were divided into mycelium and filtrate by filtration. The filtrate was extracted three times repetitively with EtOAc and collected. The mycelia was mashed and extracted with EtOAc (3 x 700 ml) for 18 H. Both the EtOAc extracts were combined and dry using rotavapor.

LC-MS analysis of the extract and isolation

LC-MS is the ideal method for the detection of the known and unknown group of secondary metabolites in natural product extracts. Specifically, LC-MS enables the finding of huge numbers of parent ions present in a crude extract and provide useful information on the chemical framework and reveal the identity of large numbers of compounds. Ethyl acetate extract of Xylaria psidii was analyzed using LCMS, a reversed-phase C18 (RPC18) HPLC, equipped with a photodiode array detector (PDA) and triple quadrupole MS. The first 0-15 min of the run was a gradient from 10 to 50% aqueous (0.1% formic acid)-ACN to 75% ACN in next 5 min, followed by a 75% aqueous (0.1% formic acid) ACN wash for 5 min to complete a total run of 25 min. A flow rate of 0.45mi/min was employed. The column utilized during this experiment was a Merck Chromolith RP 18e, 50 x 4.6 mm, 5 [micro]m pore size. EtOAc extract was then subjected to column chromatography (CC) using silica gel with hexane: ethyl acetate (90:10) as eluent. A portion of the EtOAc soluble fraction (600 mg) was subjected to CC by using silica gel (230-400 mesh, 80g) and eluted with gradient of hexane-ethyl acetate (90:10) to ethyl acetate-methanol (95:05). Sixty-three fractions (40 ml each) were collected, and their composition was checked by TLC, with those showing similar TLC profiles grouped into six fractions (F-l to F-6). Compound 2 (55 mg, (-) 5-methylmellein, Purity >99.5% by HPLC, Fig. S1 a, supplementary material) was obtained from the fraction F-2 at room temperature as a crystalline solid. Fraction F-4, was further purified by semipreparative HPLC using C18 reversed-phase column (Purospher star 5 [micro]m: 10 x 250 mm; 3ml/min: 20-100% C[H.sub.3]CN/[H.sub.2]O over 60 min) to give compound 1 (25.0 mg, xylarione A, Purity > 99.0% by HPLC, Fig. S1b, supplementary material).

Xylarione A (1)

Yellowish powder, m.p. 205-206[degrees]C; [[[alpha]].sub.D.sup.25] +4.7[degrees] (c 1.33, MeOH); UV (MeOH) [[lambda].sub.max] (log [epsilon]) 213 (3.39), 253 (1.79) nm; IR (KBr) [v.sub.max] 2958.1, 2932.4, 2861.2, 1704.3, 1698.5, 1643.2, 1417.5, 1258.2 [cm.sup.-1]; ESIMS: m/z 197.0 [|M+H].sup.+]; HRESIMS: m/z 197.1165 [[M+H].sup.+]; (calcd. for [C.sub.11][H.sub.16][O.sub.3], 196.1178). For [sup.1]H and [sup.13]C NMR spectroscopic data, see Table 1.

(-) 5-methylmellein (2)

White crystalline powder, m.p. 127-129[degrees]C; [[[alpha]].sub.D.sup.25] -103[degrees] (c 1.21, CH[Cl.sub.3]); UV (MeOH) [[lambda].sub.max] (log [epsilon]) 210 (2.25), 243 (3.65), 321 (4.10) nm; 1R (KBr) [v.sub.max] 3250, 3108, 2982.3, 2938.4, 1673.8, 1446.6, 1393.1, 1173.8, 852.6, 843.0, 750.3 [cm.sup.-1]; HRESIMS: m/z 193.0854 [[M+H].sup.+]; (calcd. for [C.sub.11][H.sub.12][O.sub.3], 192.0865). For [sup.1]H and [sup.13]C NMR spectroscopic data, see Table 1.

Cell culture and growth conditions

MCF-7 (breast adenocarcinoma), MIA-Pa-Ca-2 (pancreatic carcinoma), NCI-H226 (non-small cell lung cancer). DU145 (prostate carcinoma) and HepG2 (hepatocellular carcinoma) cells were purchased from European Collection of Authenticated Cell Culture (ECACC). The cells were cultured in RPM1- 1640 or MEM medium containing 1% penicillin-streptomycin and supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were grown at 37[degrees]C in a humidified and 5% C[O.sub.2] environment.

Cell proliferation assay

The cell viability assay was carried out using the colorimetric MTT assay as previously described (Kumar et al., 2013). Briefly, cells from sub-confluent flasks were trypsinized and were seeded on 96 well plate at a density of 1 x [10.sup.4] cells/well. The metabolites were evaluated at various concentrations with a final maximum concentration of 100 [micro]m. After treatment at various time intervals, cells were rinsed and incubated for 4 h with 20 [micro]l of MTT dye (2.5 mg/ml) dissolved in Phosphate buffer saline (PBS). After incubation, the medium was removed using centrifugation at 1500 RPM for 15 min and 150 [micro]l of DMSO were added in each well in order to solubilize the purple formazan crystals. The absorbance was measured at 570 nm using Microplate Reader (BioTek Synergy HT). The percent cell viability was determined and [IC.sub.50] values were calculated using GraphPad prism Software (La Jolla, CA).

Cell cycle analysis

The experiment was performed according to the previously described method (Guru et al., 2015). Briefly, MIA-Pa-Ca-2 cells were treated with different concentrations of metabolites (10-50 [micro]m) for 24 h. After incubation, cells were washed twice with cold phosphate buffer saline (PBS) and fixed in chilled 70% (v/v) ethanol for 30 min. After two washing steps in cold PBS, 400 [micro]g/ml of RNAse A was added and the mixture was incubated for 45 min at 37[degrees]C. Finally, cells were stained with 10 [micro]g/ml propidium iodide (PI) for 30 min in dark and analyzed immediately for DNA contents followed by flow cytometery using FACSCalibur (Becton Dickinson, USA).

Fluorescence microscopy

Fluorescence microscopy was performed to observe the morphological changes during apoptosis as described earlier (Sharma et al., 2015). MIA-Pa-Ca-2 cells were treated with varying concentrations of metabolites for 24 h at 37[degrees]C, washed two times with PBS and fixed with 400 [micro]l cold acetic acid: methanol (1:3, v/v) overnight at 4[degrees]C. Next day, cells were washed, dispensed in 50 [micro]l of the fixing solution, spreaded on glass slide, dried overnight at room temperature. Staining with Hoechst 33,258 (5 [micro]g/ml in 0.01 M citric acid and 0.45 M disodium phosphate with 0.05% Tween 20) was done for 30 min at room temperature. After 30 min, slides were rinsed with distilled water followed by PBS, and mounted with buffered glycerol, covered with glass cover slip and sealed with nail polish. Imaging was performed under fluorescence microscope, to assess any morphological modifications mediated during the course of apoptosis.

Flow cytometric determination of mitochondrial membrane potential (([DELTA][[psi].sub.M])

[DELTA][[psi].sub.M] changes due to apoptosis induced conditions were studied by flow cytometry using Rhodamine-123 as described earlier (Bhushan et al., 2013). Briefly, MIA-Pa-Ca-2 cells were incubated with the varying concentrations of metabolites for 24 h. Rhodamine-123 (5 [micro]m) was added 1 h before to the termination of the experiment. The cells were centrifuged at 3000 rpm for 5 min, collected and washed in PBS. The fluorescence intensity of 10,000 events was analyzed in FL-1 channel on a BD FACSCalibur (Becton Dickinson, USA) flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 525 nm in FITC channel. The decrease in fluorescence intensity due to the loss of mitochondrial membrane potential was analyzed in FL-1 channel.

Statistical analysis

All the results are expressed as means [+ or -] SD or representative of one of the three similar experiments unless otherwise indicated. Statistical analysis was done by using one way ANOVA with Bonferroni post test through GraphPad Prism, p-values * <0.05 were considered statistically significant.

Results and discussion

The fungal strain was identified up to genus level by morphological characteristics (Chacko and Rogers, 1981; Dugan, 2006; Rogers et al., 1992). To taxonomic designate the endophytic fungus upto species level both morphological and molecular characterization were taken in to account. The fungal mycelium was bright white aerial lanose on upper view and the reverse as pale yellow (Fig. S2a and S2b supplementary material), which supports the genus Xylaria. In blast analysis sequences showing similarity higher than 90% were fetched and aligned with the query sequence, using MEGA 6 software (Tamura et al., 2013). The alignment was subjected to neighbor-joining (NJ) analysis to obtain the dendrogram (Fig. 1). The analyzed ITS sequence forms a clade with Xylaria psidii (DQ322158), having 100% bootstrap value, indicating fungal strain as Xylaria psidii.

Xylaria psidii was cultured in PDB for 10 days at 28[degrees]C in shaking condition with 150 rpm. From 30 Erlenmeyer's flasks, 11.51 fermented culture was obtained which was extracted with EtOAc. The extract was concentrated on rotary evaporator for further study. Biological screening of ethyl acetate extract of Xylaria psidii exhibited activity ([IC.sub.50] <20 [micro]g/ml) against a set of cancer cell lines. LC-MS/MS analysis of small portion of ethyl acetate extract showed the existence of four known compounds A-D previously reported from genus Xylaria (Fig. S3 supplementary material). The extract was then subjected to isolation of secondary metabolites by column chromatography (CC) and semipreparative reversed-phase HPLC (C18) to yield novel compound (1), named xylarione A as well as known compound (-) 5-methylmellein (2), which has been isolated for the first time from Xylaria psidii (Fig. 2). Structures of isolated compounds 1 and 2 were established using detailed ID and 2D NMR (Table 1, Fig. S4-S14 supplementary material) and HR-ESIMS spectroscopic analysis.

Compound 1 was obtained as yellowish powder (m.p. 205-206 [degrees]C). ESIMS data showed prominent base peak at m/z 197.0 [[M+H].sup.+] (Fig. S3a supplementary material) which demonstrated that 1 has a molecular weight of 196. The molecular formula was established as [C.sub.11][H.sub.16][O.sub.3] based on prominent signals detected in the HR-ESIMS spectrum at m/z 197.1165 [[M+H].sup.+] which was supported by 1D NMR spectral data. The 1R spectrum showed strong absorption at 1704.3 and 1698.5 [cm.sup.-1] showed the presence of C=0 group in 1 (Fig. S3b supplementary material). The [sup.1]H NMR spectrum (Fig. S4 supplementary material) displayed proton resonance corresponding to one olefinic methine at [[delta].sub.H] 6.84 (1H, t, J = 10.6 Hz), four methylenes between [[delta].sub.H] 2.25-1.38 (m, 8H), two methyls at aH 0.91 (3H, t, J = 6.4 Hz) and 1.29 (3H, d, J = 7.2 Hz), and one methine at [[delta].sub.H] 3.61 (1H, q, J = 7.2 Hz). The [sup.13]C NMR spectrum (Fig. S5 and S6 supplementary material) exhibited presence of total 11 carbon atoms attributable to two carbonyl resonating at [[delta].sub.C] 178.0 (C-2) and 170.2 (C-5). four methylenes at [[delta].sub.C] 29.5 (C-7), 29.4 (C-8), 32.7 (C-9). 23.5 (C-10), two methyls at [[delta].sub.C] 14.33 (C[H.sub.3]-11) and 16.4 (C[H.sub.3]-12), two methines respectively at [[delta].sub.C] 38.9 (C-3) and 145.0 (C-6) and one quaternary carbon at [[delta].sub.C] 134.3 (C-4). Further analysis of 1D NMR collectively with HMBC correlation data revealed that we are dealing with furanodione type moiety. The [sup.1]H-[sup.1]H COSY (Fig S7. supplementary material) spectrum showed signals for two spin systems as shown in Fig. 2. The first spin was determined to a hexylidene moiety, using olefinic methine at [[delta].sub.H] 6.84 as a first point, a sequence of four methylenes at [[delta].sub.H] 2.25, 1.48, 1.38 and a terminal methyl at [[delta].sub.H] 0.91 was confirmed and in the second spin a methyl at [[delta].sub.H] 1-29 correlated to the methine at [[delta].sub.H] 3.61. The HMBC data (Fig. S8 supplementary material) showed useful correlations between C[H.sub.3]-12/C-2, C-3, C-4; H-3/C-2, C-4, C-5, C-6, C-12; H-6/C-3, C-4, C-5, C-7, C-8 and H-7/C-4, C-6, C-9 to establish the structure of 1. From the 2D NOESY data, it was established that H-6 exhibited NOE correlations with C[H.sub.3]-12 placed exocyclic H-6 away from the ring allowed the geometry of the C-6 double bond to be assigned as E. It was further confirmed based on identical chemical shifts of the proton located at the exocyclic double bond (H-6, [[delta].sub.H] 6.84 ppm) with those reported in the literature for related compounds (Hosoe et al., 2010; Ahmed and Langer, 2005; Gill et al., 1993). The absolute configuration was determined to be R for C-12 based on its coupling patterns in the [sup.1]H NMR spectrum together with its optical rotation ([[[alpha]].sub.D.sup.25] +4.7[degrees]), as observed in all previously reported compounds (Hosoe et al., 2010). Finally the structure of 1 was 8established as (E)-4-hexylidene-3-methyldihydrofuran-2,5-dione, a new naturally occurring furanodione named xylarione A. Its structure has not searched in scifinder and chemical abstract neither reported as yet from any natural sources. However compound 1 showed some structure similarities and could be dehydrated cyclised product of methyl xylariate C, previously reported from fungal strain NCY2 (Xylaria sp.) from the medicinal plant Torreya jackii (Hu et al., 2010).

Compound 2 was obtained as a white crystalline powder, mp 127-129[degrees]C. It showed prominent HR-ESIMS signal at m/z 193.0854 [[M+H].sup.+] corresponds to the molecular formula [C.sub.11][H.sub.12][O.sub.3] (Fig. S11 supplementary material). The IR spectrum of 2 showed absorption bands for cyclic lactones (1673.8 [cm.sup.-1]) together with characteristic bands for aromatic ring. The ID ([sup.1]H and [sup.13]C) and 2D NMR spectral analysis of 2 strongly suggested that we are dealing with 8 hydroxy dihydro isocoumarine type structures, which gives violet color upon addition of Fe[Cl.sub.3] (OH) (Claydon et al., 1985). [sup.1]H NMR spectrum (Fig. S12 supplementary material) of 2 showed three ABX type proton signals ([[delta].sub.H] 4.68, m, 1H, H-3; [[delta].sub.H] 2.95, dd, J = 3.4 and 16.8Hz, 1H, H-4a and [[delta].sub.H] 2.72, dd, J = 11.6 and 16.8 Hz, 1H, H-4b); two AB type proton signals ([[delta].sub.H] 7.28, d, J=8.4 Hz, 1H, H-7; [[delta].sub.H] 6.81. d, J = 8.4 Hz, 1H, H-6) and two methyl signals at [[delta].sub.H] 2.20, s, 3H, 5-Me and [[delta].sub.H] 1-55, d, J = 6.0 Hz, 3H, 3-Me respectively. In addition, one proton singlet at [[delta].sub.h] 10.99 assigned to 8-Ar-OH. The [sup.13]C NMR data (Table 1, Fig. S13 and 14 supplementary materials) exhibited presence of 11 carbons attributable to two methyls, three methines, one methylene and five quaternary carbons. Further analysis of the [sup.13]C NMR data confirmed signals for one carbonyl at [[delta].sub.C] 170.3 (C-1), six phenyls [[delta].sub.C] 124.9 (C-5), 137.9 (C-6), 115.6 (C-7), 160.4 (C-8), 108.0 (C-9), 137.0 (C-10), one oxy methine c 75.4 (C-3) and three aliphatic signals [[delta].sub.C] 31.8 (C-4), 20.8 (5-C[H.sub.3]) and 18.0 (3- C[H.sub.3]). Based on observed data above together with comparison with literature, compound 2 was established to be (-) 5-methylmellein (Ballio et al., 1966). The comparison of proton-proton coupling constants, optical rotation together with [sup.1]H and [sup.13]C chemical shifts of 2 inferred the same relative configuration as that of previously isolated from other sources such as Penicillium paxilli (Claydon et al., 1985) and Cephalosporium sp. (Bi et al., 2007). To the best of our knowledge, (-) 5-methylmellein (2) is isolated for the first time from Xylaria psidii.

Isolated compounds 1 and 2 were evaluated against a panel of cancer cell lines. Cytotoxicity assays revealed that both the secondary metabolites were cytotoxic in their [micro]m doses, in the tested cell lines (Table 2). Compounds 1 and 2 exhibited no significant activity against MCF-7 (breast adenocarcinoma), NCI-H226 (non-small cell lung cancer), HepG2 (hepatocellular carcinoma) and DU145 (prostate carcinoma) when compared to the standard drugs flavopiridol. Thus further experimentations were done on MIA-Pa-Ca-2 cells, since both the compounds had best cytotoxicity with [IC.sub.50] values of 16.0 and 19.0 um, respectively. They also demonstrated different cytotoxicity potential ([IC.sub.50]) at different time points as depicted in Fig. 3. This experiment was done to determine the [IC.sub.50] value of both the secondary metabolites at 48 h time point, to further study the cell cycle, MMP loss and microscopic analysis.

In order to assess the pro-apoptotic activity of compounds 1 and 2, the extent of cell death in MIA-Pa-Ca-2 cells was analyzed using flow cytometry via determination of sub-G1 cell population by propidium iodide (PI) staining. As shown in Fig. 4, the regions displayed with various colors demonstrate % population at different phases of the cell cycle. MIA-Pa-Ca-2 cells exposed to both the secondary metabolites for 24 h exhibited a dose-dependent increase in sub-G1 fraction, which may consist both apoptosis and debris fraction implying together the extent of cell death (Fig. 4). The sub-G1 apoptotic population was found to be 5, 8 and 14% following 10, 30 and 50 [micro]m of (-) 5-methylmellein treatment compared to control (untreated cells--1%), where as it was 2, 7 and 40% at 10, 30 and 50 [micro]m for 1.

The above results of apoptosis induction from cell cycle analysis were further confirmed by analyzing nuclear morphological changes in cells using fluorescence microscopy. Both the compounds at 10, 30, and 50 [micro]m concentrations induced apoptotic morphological changes in cells, such as nuclear condensation, membrane blebbing and evolvement of apoptotic bodies. These apoptotic morphological changes were found to be concentration dependent while nuclei of untreated cells were intact and having normal morphology. These results confirmed that apoptotic cell morphology was induced, upon treatment with both the compounds in Mia-Pa-Ca-2 cells (Fig. 5).

The MMP ([DELTA][psi]M) loss in MIA-Pa-Ca-2 cells after treatment with both the compounds was studied by using rhodamine-123 (a dye which is reduced by healthy mitochondria into fluorescent probe and that fluorescence is measured by flow cytometer in FL-1 channel). Apoptosis induction by Xylarione A and 5-methylmellein was through intrinsic apoptotic pathways, which was confirmed by MMP loss, as a result of mitochondrial damage.

The untreated control cells were functionally active with high rh-123 fluorescence. Exposure of cells to the compounds resulted in mitochondrial damage, shown by decrease in rh-123 fluorescence. (-) 5-methylmellein induced MMP loss in concentration dependent manner, i.e. 10% at 10 [micro]m and 20% at 50 [micro]m (Fig. 6). Whereas, in case of xylarione A, MMP loss was about 7% and 26% at 10 [micro]m and 50 [micro]m, respectively. The loss of mitochondrial membrane potential ([DELTA][psi]M) was greatly due to the widening of mitochondrial permeability transition pores (PTP), resulting in the leakage of proapoptotic proteins from mitochondria to cytosol.


The isolated endophytic fungal strain was identified as Xylaria psidii from its ITS region sequence analysis and morphological characteristics. The study was further aimed to isolate the secondary metabolites from Xylaria psidii and to determine the cytotoxic potential of isolated compounds against a panel of human cancer cell lines. The in vitro cytotoxic effects of compounds 1 and 2 were measured by MIT assay. The isolated secondary metabolites 1 and 2 displayed significant anti-proliferative activity in pancreatic cancer cells (MIA-Pa-Ca-2) and found to be non toxic in normal human breast epithelial fR2 cells. The cell cycle analysis demonstrated that 1 and 2 have cytotoxic effects on the cells in a concentration dependent manner which was inferred by increase in sub-Gl population. Additionally, fluorescence microscopy demonstrated that the nuclei of untreated MIA-Pa-Ca-2 cells showed homogeneous fluorescence with no segmentation and when treated with various concentrations of both the metabolites characteristic changes of apoptosis such as nuclear condensation, membrane blebbing and formation of apoptotic bodies. To better understand the mechanism MMP assay was done because mitochondrion plays an important role in inducing apoptosis. Rhodamine 123 (a cationic fluorophore) was used to monitor the mitochondrial membrane potential due to its accumulation inside the cells. The energization of mitochondria induces quenching of Rh-123 fluorescence and the rate of fluorescence decay is directly proportional to mitochondrial membrane potential (Baracca et al., 2003). Both the compounds (1 and 2) lead to loss of mitochondrial membrane potential in a concentration dependent manner. These results provide the confirmation that the isolated compounds 1 and 2 are responsible for the activity shown by extract and may act as potential leads for medicinal chemists for designing more potent analogs.


Article history:

Received 8 June 2016

Revised 13 July 2016

Accepted 17 July 2016

Conflict of interest

The authors declare that there are no known conflicts of interest associated with this publication.

Statement of human and animal rights

This article does not contain any animal studies performed by the authors.


This work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi (Grant No. BSC-0117 PMS1, BSC-0108 and MLP-1009). DA, NS, VS, SG, MK and SS thankfully acknowledge the support in the form of research fellowships by Council of Scientific and Industrial Research (CSIR), New Delhi. The manuscript bears IHM Communication No. IIIM/1940/2016.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.07.004.


Ahmed, Z., Langer, P., 2005. Synthesis of natural pulvinic acids based on a [3 + 2] cyclization-Suzuki cross-coupling strategy. Tetrahedron 61, 2055-2063.

Ballio, A., Barcellona, S., Santurbano. B., 1966. 5-methylmellein, a new natural dihydroisocoumarin. Tet. Lett. 7, 3723-3726.

Baraban, E.G., Morin, J.B., Phillips, G.M., Phillips, A.J., Strobel, S.A., Handelsman, J., 2013. Xyolide, a bioactive monenolide from an Amazonian endophytic fungus, Xylaria feejeensis. Tet. Lett. 54, 4058-4060.

Baracca, A., Sgarbi, G., Solaini, C., Lenaz, G., 2003. Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through [F.sub.0] during ATP synthesis. Biochim. Biophys. Acta-Bioenergetics 1606, 137-146.

Bhushan, S., Kakkar, V., Pal, H.C., Guru, S.K., Kumar, A., Mondhe, D.M., Sharma, P.R., Taneja, S.C., Kaur, I.P., Singh. J., Saxena, A.K., 2013. Enhanced anticancer potential of encapsulated solid lipid nanoparticles of TPD: a novel triterpenediol from Boswellia serrata. Mol. Pharm. 10. 225-235.

Bi, Y.M., Bi, X.B., Fang, A., Zhao, Q.R., 2007. Metabolites from the fungus Cephalosporium sp. AL031. Arch. Pharm. Res. 30, 267-269.

Buttler, M.S., Robertson, A.A.B., Cooper, M.A., 2014. Natural product and natural product derived drugs in clinical trials. Nat. Prod. Rep. 31, 1612-1661.

Chacko, J.R., Rogers, D.J., 1981. Cultural characteristics of some species of Xylaria. Mycol 73. 415-428.

Challinor, V.L. Bode. H.B., 2015. Bioactive natural products from novel microbial sources. Ann. N. Y. Acad. Sci. 1354. 82-97.

Chithra, S., Jasim, B., Sachidanandan, P., Jyothis, M., Radhakrishnan, E.K., 2014. Piperine production by endophytic fungus Colletotrichum gloeosporioides isolated from Piper nigrum. Phytomedicine 21. 534-540.

Claydon, N., Grove, J.F., Pople, M., 1985. Elm bark beetle boring and feeding deterrents from Phomopsis oblonga. Phytochemistry 24. 937-943.

Devari, S., Jaglan, S., Kumar, M., Deshidi, R., Guru, S., Bhushan, S., Kushwaha, M., Gupta, A.P., Gandhi, S.G., Sharma, J.P., Taneja, S.C., Vishwakarma, RA., Shah, B.A.. 2014. Capsaicin production by Alternaria alternala, an endophytic fungus from Capsicum annum: LC-ES1-MS/MS analysis. Phytochemistry 98. 183-189.

Deyrup, S.T., Gloer, J.B., O'Donnell, K., Wicklow, D.T., 2007. Kolokosides A-D: Triterpenoid glycosides from a Hawaiian Isolate of Xylaria sp. J. Nat. Prod. 70, 378-382.

Dugan, F.M., 2006. The Identification of Fungi: an Illustrated Introduction with Keys, Glossary, and Guide to Literature. American Phytopathological Society Press, St. Paul.

Espada, A., Rivera-Sagredo, A., De La Fuente, J.M., Hueso-Rodriguez, JA., Elson, S.W., 1997. New cytochalasins from the fungus Xylaria hypoxylon. Tetrahedron 53. 6485-6492.

Gill, G.B., James, G.D., Oates, K.V., Pattenden, G., 1993. The synthesis of 5-ylidenepyrrol-2 (5 H)-ones from maleimides and from pyrrol-2-(5 H)-ones. J. Chem. Soc., Perkin Trans. 1, 2567-2579.

Guru, S.K., Pathania, AS., Kumar, S, Ramesh, D., Kumar, M., Rana, S., Kumar, A.. Malik, ?., Sharma, P.R., Chandan, B.K., Jaglan, S., Sharma, J.P., Shah, B.A., Tasduq, S.A., Latoo, S.K., Faruk, A., Saxena, A.K., Vishwakarma, R.A., Bhusan, S., 2015. Secalonic acid-D represses HIF-lor/VEGF mediated angiogenesis by regulating the Akt/mTOR/p70S6K signaling cascade. Cancer Res 75. 2886-2896.

Gutierrez, R.M., Gonzalez, A.M., Ramirez, A.M., 2012. Compounds derived from endophytes: a review of phytochemistry and pharmacology. Curr. Med. Chem 19. 2992-3030.

Hammerschmidt, L, Ola. A., Muller, W.E.G., Lin, W., Mandi, A., Kurtan, T., Proksch, P.. Aly, A.H., 2015. Two new metabolites from the endophytic fungus Xylaia sp. isolated from the medicinal plant Curcuma xanthorrhiza. Tet. Lett. 56, 1193-1197.

Healy. P.C., Hocking. A., Tran-Dinh, N., Pitt, J.I., Shivas, R.C., Mitchell, J.K., Kotiw, M.. Davis, R.A., 2004. Xanthones from a microfungus of the genus Xylaria. Phytochemistry 65. 2373-2378.

Hosoe, T., Gloer, J.B., Wicklow, D.T., Raja, HA., Shearer, CA., 2010. New nonadride analogues from a freashwater isolate of an undescribed fungus belonging to the order Pleosporales. Heterocydes 81, 2123-2130.

Hu, Z.Y., Li, Y.Y., Lu, C.H., Lin, T., Hu. P., Shen, Y.M., 2010. Seven novel linear polyketide from Xylaria sp. NCY2. Helv. Chim. Acta 93, 925-933.

Isaka, M., Srisanoh, U., Sappan, M., Kongthong, S., Srikitikulchai, P., 2012. Eremophilane and eudesmane sesquiterpenoids and a pimarane diterpenoids from the wood-decay fungus Xylaria sp BCC 5484. Phytochem. Lett 5, 78-82.

Jimenez-Romero, C., Ortega-Barria, E., Arnold, A.E., Cubilla-Rios, L, 2008. Activity against Plasmodium falciparum of lactones isolated from the endophytic fungus Xylaria sp. Pharm. Biol. 46. 700-703.

Kesari, A.N., Gupta, R.K., Singh, S.K., Diwakar, S., Watal, G., 2006. Hypoglycemic and antihyperglycemic activity of Aegle marmelos seed extract in normal and diabetic rats. J. Ethnopharmacol 107, 374-379.

Klaiklay, S., Rukachaisirikul, V., Sukpondma, Y., Phongpaichit, S., Buatong, J., Bussaban, B., 2012. Metabolites from the mangrove-derived fungus Xylaria cubensis PSU-MA34. Arch. Pharm. Res. 35. 1127-1131.

Koul, M., Meena, S., Kumar, A., Sharma, P.R., Singamaneni, V., Hassan, S.R., Hamid, A.. Chaubey, A., Prabhakar, A., Gupta, P., Singh, S., 2016. Secondary metabolites from endophytic fungus Penicillium pinophilum induce ROS-mediated apoptosis through mitochondrial pathway in pancreatic cancer cells. Planta Med 82. 344-355.

Kumar, S., Guru, S.K., Pathania, A.S., Kumar, A., Bhushan, S., Malik, F., 2013. Autophagy triggered by magnolol derivative negatively regulates angiogenesis. Cell. Death Dis. 4. e889.

Kumari, K.D.K.P., Weerakoon, T.C.S., Handunnetti, S.M., Samarasinghe, K., Suresh, T.S., 2014. Anti-inflammatory activity of dried flower extracts of Aegle marmelos in Wistar rats. J. Ethnopharmacol 151, 1202-1208.

Lee, C.C., Houghton, P., 2005. Cytotoxicity of plants from Malaysia and Thailand used traditionally to treat cancer. J. Ethnopharmacol. 100. 237-243.

Liu, J.Y., Liu, C.H., Zou. WX. Tian, X., Tan, R.X., 2002. Leptosphaerone, a metabolite with novel skeleton from Leptosphaeria sp. IV403. an endophytic fungus in Artemisia annua., Helv. Chim. Acta 85, 2664-2667.

Petrini, L. Petrini, 0., 1985. Xylariaceous fungi as endophytes. Sydowia 38. 216-234.

Rogers, J.D., Ju, Y.M., Hemmes, D.E., 1992. Hypoxylon rectangulosporum sp. nov., Xylaria psidii sp. nov. and comments on taxa of Podosordaria and Stromatoneurospora. Mycol 84 (2). 166-172.

Sankeshi, V., Kumar, P.A., Naik, R.R., Sridhar, G., Kumar, M.P., Copal, V.V.H., Raju, T.N.. 2013. Inhibition of aldose reductase by Aegle marmelos and its protective role in diabetic cataract. J. Ethnopharmacol 149, 215-221.

Schulz, B., Boyle, C. Draeger, S., Rommen, A.K., Krohn, K., 2002. Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol. Res. 106, 996-1004.

Sharma, R., Guru, S.K., Jain, S.K., Pathania, A.S., Vishwakarma. RA., Bhushan, S., Bharate, S.B., 2015. 3-(2, 6-Dichloro-benzyloxy)-11-oxo-olean-12-ene-29-oic acid, a semisynthetic derivative of glycyrrhetic acid: synthesis, antiproliferative, apoptotic and anti-angiogenesis activity. Med. Chem. Comm 6, 564-575.

Singh, S., Bukkambudhi, R.G., Gudasalamani, R., Uma, S.R., Manchanahally, B.S., 2013. Endophytic fungi from Miquelia dentata Bedd., produce the anti-cancer alkaloid, camptothecin. Phytomedicine 20, 337-342.

Smith, C.J., Morin, N.R., Bills, G.F., Dombrowski, A.W., Salituro, G.M., Smith, S.K.. Zhao, A., MacNeil, D.J., 2002. Novel sesquiterpenoids from the fermentation of Xylaria persicaria are selective ligands for the NPY Y5 Receptor. J. Org. Chem 67, 5001-5004.

Stierle, A., Stroble, G., Stierle, D., 1993. Taxol and taxane production by Taxomyces andreanae. an endophytic fungus of Pacific yew. Science 260. 214-216.

Strobel, G., Daisy, B., 2003. Bioprospecting for microbial endophytes and their natural products. Microbiol. Mol. Biol. Rev. 67, 491-502.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729.

Tan, RX, Zou, WX, 2001. Endophytes: a rich source of functional metabolites. Nat. Prod. Rep. 18, 448-459.

Wennstrom, A., 1994. Endophyte--the misuse of an old term. Oikos 71, 535-536.

White, T.J., Bruns, T.D., Lee, S.B., Taylor, J.W., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Taylor, J.W. (Ed.). PCR Protocols: a Guide to Methods and Applications. Academic Press Inc. USA. pp. 315-322.

Wu, W., Dai, H., Bao, L. Ren, B., Lu, J., Luo, Y., Guo, L. Zhang, L., Liu, H., 2011. Isolation and structural elucidation of proline-containing cyclopentapeptides from an endolichenic Xylaria sp. J. Nat. Prod. 74, 1303-1308.

Xu, H.N., Zhi, H.Z., Xiao, Y.Z., Xin, H.L, Shu, H.Q,. 2013. Polyketides from a marine derived fungus Xylariaceae sp. Mar. Drugs 11. 1718-1727.

Zhang, Q,, Xiao, J., Sun, Q.Q., Qin, J.C., Pescitelli, G., Gao, J.M., 2014. Characterization of cytochalasins from the endophytic Xylaria sp. and their biological functions. J. Agric. Food. Chem 62. 10962-10969.

Divya Arora (a,d), Nisha Sharma (a,d) Venugopal Singamaneni (b), Vishal Sharma (a,d), Manoj Kushwaha (a), Vidushi Abrol (a), Santosh Guru (c), Sonia Sharma (c,d), Ajai Prakash Gupta (a), Shashi Bhushan (c,d),*, Sundeep Jaglan (a,d),*, Prasoon Gupta (b,d),*

(a) Quality Control & Quality Assurance Division, CSIR-Indian Institute of Integrative Medicine. Canal Road, Jammu 180001, India

(b) Natural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India

(c) Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India

(d) Academy of Scientific & Innovative Research (AcSIR), CSIR, New Delhi, 110025, India

Abbreviations: 1D/2D NMR. One or Two Dimensional Nuclear Magnetic Resonance; [IC.sub.50], Half maximal inhibitory concentration; pm. Micro molar, IR. Infrared; H RMS, High-Resolution Mass Spectrometry; ESIMS. Electrospray Ionization Mass Spectrometry; LC-Q-TOF, Liquid Chromatography Quadrupole Time-of-flight; HPLC. High Pressure Liquid Chromatography; MTT, 3-(4, 5 -dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; TLC, Thin Layer Chromatography; UV, Ultraviolet; DNA, Deoxyribonucleic acid; ITS. Internal transcribed spacer: PCR. Polymerase chain reaction; BLAST. Basic Local Allignment Search Tool; NCBI. National Centre for Biotechnology Information; MEGA. Molecular Evolutionary Genetics Analysis; rDNA, Ribosomal DNA; NCI, National Cancer Institute; DCM, Dichloromethane. RPMl, Rosewell Park Memorial Institute; MEM. Minimum Essential Medium; DMSO. Dimethyl sulfoxide; RNAse. Ribonuclease; BD FACS. Becton Dickinson Fluorescence-activated cell sorting; FITC, Fluorescein isothiocyanate; SD, Standard deviation; ANOVA. Analysis of variance; PDA. Potato Dextrose Agar; CC, Column chromatography; ATCC, American Type Culture Collection; MW. Molecular weight; COSY. Correlation Spectroscopy; NOESY. Nuclear Overhauser effect spectroscopy; HMBC, Heteronuclear Multiple Bond Correlation; MMP. Mitochondrial membrane potential; Rh-123. Rhodamine-123.

* Corresponding authors. Fax: 91 191 2569019.

E-mail addresses: (S. Bhushan). (S. Jaglan). (P. Gupta).

Table 1
NMR spectroscopic data for compounds 1 and 2.

Pos.            1 (a)
                (mult., J in Hz)   [[delta].sub.c]

2                                  178.0
3               3.61 (q. 7.2)      38.9
4 a             --                 134.3
4 b             --                 --
5               --                 170.2
6               6.84 (t, 10.6)     145.0
7               2.25 (m)           29.5
8               1.48 (m)           29.4
9               138 (m)            32.7
10              138 (m)            23.5
11              0.91 (t, 6.4)      14.3
12              129 (d. 7.2)       16.4

Pos.            2 (b)
                (mult., J in Hz)        [[delta].sub.c]

1                                       1703
3               4.68 (m)                75.4
4 a             2.95 (dd. 3.4, 16.8)    31.8
4 b             2.72 (dd. 11.6, 16.8)   --
5               --                      124.9
6               6.81 (d.8.4)            137.9
7               7.28 (d, 8.4)           115.6
8               --                      160.4
9               --                      108.0
10              --                      137.0
11              --                      --
12              --                      --
Ar-OH           10.99 (s)               --
3-C[H.sub.3]    1.55 (d. 6.0)           18.0
5-C[H.sub.3]    2.20 (s)                20.8

Recorded in (a) C[D.sub.3]OD. (b) CD[Cl.sub.3] at 400 MHz
(TMS as internal standard), chemical shifts, multiplicity
and coupling constants (J. Hz) were assigned by 1D NMR data.

Table 2
Cytotoxicity of isolated secondary metabolites (1 and 2)
in different cancer cell lines (a).

Compound                IC50, [micro]m, in Different
                        Human Cancer Cell line

                        MCF-7               MIA-Pa-Ca-2

Xylarione A (1)         18 [+ or -] 1.08    16 [+ or -] 1.09
5-methyl mullein (2)    22 [+ or -] 1       19 [+ or -] 1.77
Flavopiridol            1.7 [+ or -] 0.08   0.4 [+ or -] 0.03

Compound                IC50, [micro]m, in Different
                        Human Cancer Cell line

                        NCI-H226           HepG2

Xylarione A (1)         25 [+ or -] 1.98   37 [+ or -] 1.99
5-methyl mullein (2)    23 [+ or -] 1.65   19 [+ or -] 1.16
Flavopiridol            1 [+ or -] 0.06    2 [+ or -] 0.1

Compound                IC50, [micro]m, in Different
                        Human Cancer Cell line

                        DU145              fR2 (Normal)

Xylarione A (1)         22 [+ or -] 1.12   79 [+ or -] 2.12
5-methyl mullein (2)    20 [+ or -] 1.77   76 [+ or -] 2.2
Flavopiridol            2 [+ or -] 0.1     >50

(a) Data are Mean [+ or -] SD (n = 3).


Please note: Some tables or figures were omitted from this article.
COPYRIGHT 2016 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2016 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Arora, Divya; Sharma, Nisha; Singamaneni, Venugopal; Sharma, Vishal; Kushwaha, Manoj; Abrol, Vidushi
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Nov 15, 2016
Previous Article:Target molecules in 3T3-L1 adipocytes differentiation are regulated by maslinic acid, a natural triterpene from Olea europaea.
Next Article:The anthraquinones rubiadin and its 1-methyl ether isolated from Heterophyllaea pustulata reduces Candida tropicalis biofilms formation.

Terms of use | Privacy policy | Copyright © 2021 Farlex, Inc. | Feedback | For webmasters