ConclusionIn conclusion we report i

Conclusion
In conclusion we report isolation of an endophytic fungus capable
of producing capsaicin upto three generations, which may find
application as an alternative source for its large scale production.
Upscaling of the fermentation broth resulted in the isolation of
compounds 2 and 3, of which 3 was found new to the literature.
Notably, we present an approach employing a fast and sensitive
(MRM) LC-ESI-MS/MS method for the separation and quantification
of capsaicin and compound 3. The developed multimode
procedure can easily be utilized as analytical tool for analysis of
1 and 3 in the presence of other possible constituents. Compound
3 displayed significant anti-proliferative activity against different
human cancer cell lines and was found to induce apoptosis evidenced
by Hoechst staining and loss of mitochondrial membrane
potential. Further studies are underway to optimize conditions
for large scale production of capsaicin and isolate other metabolites
from this fungus. Additional studies on 3 are aimed at carrying
out structural modifications to optimize its activity and elucidating
more details of its molecular mode of action.
Experimental
General experimental procedures
1H and 13C NMR spectra in DMSO-d6 were recorded on 400 MHz
spectrometers with TMS as an internal standard. Chemical shifts
are expressed in parts per million (d ppm); J values are given in
Hertz. Reagents and solvents used were mostly AR grade. Silica
gel coated aluminum plates were used for TLC. HRMS were recorded
on UHD LC/MS Q-TOF. RPMI-1640, 3-(4,5-dimethylthiazole-
2-yl)-2,5-diphenyltetrazolium bromide (MTT), Rhodamine
123, penicillin-G, Fetal calf serum (FCS), Phosphate buffer saline
(PBS), citric acid, streptomycin, L-glutamine, Hoechst-33258, and
pyruvic acid were all high-quality analytical and molecular biology
grade.
Plant materials and sampling
Mature fruits of a healthy and symptomless one year old plant
of C. annum (Family: Solanaceae) were collected from Jammu (32
430 5800 N, 74 480 3200 E), India. Fruit samples were taken to laboratory
in an icebox, stored in sterilized polybags at 4 C and processed
within 24 h of collection.
Isolation and maintenance of endophytic fungus
To eliminate the epiphytic microbes, the fruit samples of C.
annum were first washed with tap water and processed for isolation
of endophytic fungi. The samples were washed thrice with
autoclaved distilled water and further surface sterilized by consecutive
treatment with 70% ethanol for 1 min, 1% sodium hypochlorite
(NaOCl) for 90 s, after each treatment sample was rinsed with
sterile distilled water. The wet samples were placed on sterilized
filter paper to eliminate the extra moisture. Surface sterilized
sample was cut into 3  3 mm pieces using sterilized surgical
blades and placed on petridishes containing three different media,
potato dextrose agar (Infusion from potatoes 200 g L1, dextrose
20.0 g L1, agar 15.0 g L1) pH 5.6 ± 0.2, yeast malt agar, (yeast extract
4.0 g L1, malt extract 10.0 g L1, glucose 4.0 g L1) 5.5 ± 0.2
and water agar medium (2% Agar w/v) in petridishes (90 mm in
diameter). To avoid the bacterial growth each media was supplemented
with 200 lg/ml streptomycin sulfate. The petridishes were
incubated at 28 C for 3–15 days until the mycelium appeared from
the inoculated sample. The hyphal growth was picked from sample
inoculated on water agar, transferred to potato dextrose agar media
and incubated at 28 C to obtain a pure culture. For long term
preservation the pure endophytic fungal isolate was stored at
4 C (lyophilized) and 80 C in 10% (v/v) glycerol, documented
with accession number MRCJ-10 in the institutional microbial
repository.
DNA extraction, PCR amplification and sequencing of fungal ITS1-5.8SITS2
region
The genomic DNA was extracted by using ZR Fungal/Bacterial
DNA MiniPrep™ kit according to manufacturer’s protocol. The
quality and quantity of genomic DNA was checked by NanoDrop
2000. PCR amplification of ITS1-5.8S rDNA-ITS2 regions was performed
using universal primers ITS5: 50-GGAAGTAAAAGTCGTAACAAGG-
30 and ITS4: 50-TCCTCCGCTTATTGATATGC-30 (White et al.,
1990). PCR amplification was performed in a 50 ll reaction volume
with 25 ll of PCR Master Mix Promega Corp., Madison, WI), 2.50 ll
of 10 lM each primer, 3 ll of fungal genomic DNA (100 ng) and
17 ll molecular biology grade water. The thermal cycling program
were as follows: initial denaturation 95 C for 5 min; 35 cycles of
30 s at 95 C, 45 s at 56 C, and 60 s at 72 C; and a final extension
of 10 min at 72 C. The PCR product was cleaned with UltraClean™
PCR Clean-up DNA purification kit according to manufacturer’s
protocol.
Phylogenetic analysis of endophytic fungus
The amplified ITS region was sequenced in both directions by
ABI 377 DNA sequencer using the Big Dye Terminator v3.1 cycle
sequencing kit, following manufacturer’s instruction. To identify
the endophytic fungus, the sequences obtained were used as query
sequence for similarity search by using BLAST algorithm against
the database maintained at NCBI (http://www.ncbi.nlm.nih.gov)
and the FC-46 strain was taxonomic designated as Alternaria alternata.
The ITS1-5.8S rDNA-ITS2 sequence of FC-46 isolate was
aligned with the most similar reference sequences of the taxa by
using the CLUSTAL W program. A phylogenetic tree was constructed
using software MEGA5 (Tamura et al., 2011), subsequently
analyzed for evolutionary distances by the neighbor
joining method. The robustness of clades was estimated by bootstrap
analysis with 1000 replications. The contiguous rDNA sequences
of the representative isolate was submitted to GenBank
database using SEQUIN program with accession no. KC989106.
Cultivation, fermentation and preparation of extracts
An inoculum of A. alternata was prepared in Erlenmeyer flasks
(500 ml) with potato dextrose broth (PDB) media (150 ml) incubated
for 5 days in shaking (150 rpm) at 28 C. Batch fermentation
was performed in Erlenmeyer flasks (1L) on PDB, pH 5.5. Each flask
containing 300 ml broth was inoculated with 10 percent of inoculum
prepared and incubated at 28 C in dark with shaking at
200 rpm for 7 days. After completion of batch fermentation the
broth was harvested and further processed for preparation of extracts
following the National Cancer Institute’s protocol (McCloud,
2010) i.e., homogenized whole broth was transferred to a separating
funnel with an equal volume of DCM. Following shaking and
separation of layers, the DCM phase was withdrawn, the organic
solvent removed by rotary evaporation. The organic extract was
then subjected to column chromatography on silica gel with ethyl
acetate:hexane (10:90) as eluents.
Alternariol-10-methyl ether (3). Colorless solid, IR (neat) tmax
3400.86, 1658.39 cm1. HRESIMS (m/z) 273.0756 [M+H]+ (calcd
for [C15H12O5]+ 273.0685).