Potato dextrose agar (PDA), rose Bengal chloramphenicol agar (RBCA) and potato dextrose broth (PDB) were purchased from Himedia Laboratories Pvt. Ltd (Mumbai, India). Mercuric chloride, sodium hypochlorate, chloramphenicol, chloroform, methanol, tetrahydrofuran, ethidium bromide, hexadecyltrimethylammonium bromide (CTAB), ethylenediaminetetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) and cyclohexanone, were procured from Sigma-Aldrich (St. Louis, MO, USA). TLC Silica Gel 60 F254 was procured from Merck KGaA (Darmstadt, Germany). Baeyer–Villiger monooxygenases (BVMOs), from recombinant E. coli, glycerol stock (ECS-Mo01, ECS-Mo02, ECS-Mo03, ECS-Mo04, ECS-Mo05 and ECS-Mo06) were procured from Enzymicals AG (Enzymical, Greifswald, Germany). Pure calebin-A with minimum 99% assay was obtained from the analytical department of Sami Labs Limited, Bangalore, India (Majeed et al. 2015) and used as standard throughout the study. Curcumin C3 Complex® contained minimum 95% curcuminoids (~ 75% curcumin, 20% demethoxycurcumin and ~ 3.5% bisdemethoxycurcumin) was obtained from Sabinsa Corporation, 20 Lake Drive, East Windsor, NJ, USA 08520. Endophytic fungus Ovatospora brasiliensis strain, MTCC 25236 was isolated from C. caesia rhizome and deposited in the Microbial Type Culture Collection and Gene Bank (MTCC) (Chandigarh, India).
Extraction, isolation and identification of calebin-A from the C. caesia rhizome
Fresh rhizomes were cleanly washed with deionizer water, sliced and dried at 50 °C in a hot air oven for 24 h. Fine powder (1.0 kg) was stirred with ethyl acetate (B.P = 77 °C), for three times of 30 min each. Final volume of 1 L of extract was added to rotary evaporator to obtain a solvent free concentrate. Solvent-free ethyl extract powder 25 g was loaded in the silica gel column chromatography and then chloroform and methanol (8:2) was used as elute solvent followed by methanol with increasing polarity. All the collected fractions were subjected to HP-TLC and HPLC for confirmation of calebin-A.
Isolation of endophytic fungi from C. caesia rhizome
Healthy rhizomes of C. caesia were collected from the nursery of Sami Labs Limited, Bangalore, India. The plant rhizomes were analyzed and verified as C. caesia by the taxonomist and the voucher specimen was stored at the storage department of Sami Labs Limited with the voucher number SAMI/14/R002. The rhizomes were washed gently several times with water to remove soil and adherent particles and then dried with sterile blotting paper. Samples were sterilized by soaking into 70% alcohol for 1 min followed by sodium hypochlorate (5.3%) treatment for 5 min. Finally, the rhizomes were soaked in 0.25% of mercuric chloride (HgCl2) for 30 s followed by thorough rinsing with sterile distilled water to remove traces of mercuric chloride and other treatment agents and then dried using sterile blotting paper. The rhizomes were cut horizontally and vertically into small pieces using sterile blade and then carefully placed on potato dextrose agar (PDA) plate containing chloramphenicol (50 µg/ml). The plates were then incubated at 28 ± 1.0 °C for 7 to 14 days with regular monitoring for the fungus growth. The hyphal tip which grew out from the plant tissue was carefully transferred to the RBCA plates and then incubated for 7 days at 28 ± 1.0 °C. The purity of the isolated endophytic fungus was determined by the colony morphology and microscopic examination.
Physiological profile of endophytic fungus
The pure isolated endophytic fungus strain EPE-10 was grown on RBCA and subjected to the microscopic examination (Eclipse CI, Nikon Instruments Inc, Japan). Photographic images were captured using Nikon DS Ri2 attached to a Nikon Eclipse Ci microscope. The images were processed on Nikon basic essential software. Furthermore, pure colonies were picked up and subjected to biochemical characterization based on sugar fermentation pattern in basal broth medium as per the standard method described earlier (Majeed et al. 2016).
DNA extraction and phylogenetic analysis
Endophytic fungus strain EPE-10 was grown on PDA and the genomic DNA of the pure endophytic fungus strain EPE-10 was extracted using CTAB following the protocol of Graham et al. (1994) with slight modification. Quality of DNA was evaluated on 1.0% agarose gel and a single band of high-molecular weight DNA was observed (Fig. 7). Polymerase chain reaction was performed with primer pairs targeted to the 18S rRNA gene using the standard protocol suggested by the manufacturer and a single discrete PCR amplicon band of ~ 500 bp was observed when resolved on agarose. The PCR amplicon was purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with NS1 (5′-GTAGTCATATGCTTGTCTC-3′) and NS4 (5′-CTTCCGTCAATTCCTTTAAG-3′) primers using BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on ABI 3730XL Genetic Analyzer (Applied Biosystems). Consensus sequence of the PCR amplicon was generated from forward and reverse sequence data using aligner software (Kimura 1980). The 18S rDNA region sequence was used to carry out Basic Local Alignment Search Tool (BLAST) with the database of National Center for Biotechnology Information (NCBI) GenBank® (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Based on maximum identity score first ten sequences were selected and aligned using multiple alignment software program Clustal W (https://www.genome.jp/tools-bin/clustalw). Distance matrix was generated and the phylogenetic tree was constructed using MEGA 7 (Kumar et al. 2016).
Biotransformation of curcumin into calebin-A
Freshly grown endophytic fungus strain EPE-10 was inoculated to PDB (500 ml) and incubated at 37 °C for 5 days in shaker incubator (Scigenics Biotech, Chennai, India) with 140 rpm. After incubation, Curcumin C3 Complex® (50 mg dissolved in 20 ml of DMSO) was added to flask and incubated at 37 °C for 5 days in shaker incubator with 140 rpm. At 24 h interval, 100 ml of the sample was withdrawn from the flask followed by the addition of ethyl acetate (300 ml) with continuous mixing for 30 min using separating funnel (Borosil, Mumbai, India). The suspension was kept still in a stand for 30 min for separation of aqueous and organic layers. Top organic layer was carefully collected in a fresh tube and concentrated to dryness at 45 °C under vacuum using rotary evaporator (Heidolph, Schwabach, Germany). The sample was further dissolved in 20 ml of methanol. Controls respectively without adding curcumin into the medium and without adding the fungus to the medium were also used and processed similar to above. The presence of calebin-A in the extracts was identified and quantified using HPTLC, HPLC and LC–MS techniques as described below.
High performance thin layer chromatography (HPTLC)
The preliminary identification of curcumin and calebin-A was performed by using HPTLC system (Camag, Muttens, Switzerland) comprising of Camag Linomate V semiautomatic sample applicator and Linomat syringe (100 μl). The stationary phase was TLC silica gel plates (Merck Millipore, 60 F254) where, 2 μl of each samples were loaded and developed using solvent system chloroform:methanol (98:2). Using scanner 3 (Camag), the plate was scanned at 280 nm with deuterium illumination. The images were captured on Camag reprostar 3 with win CATS software (ver. 1.4.3.6336) and compared the Rf value with the standard calebin-A.
High performance-liquid chromatography (HPLC)
The identification and quantification of curcumin and calebin-A was performed by using Shimadzu Class Vp series HPLC system, equipped with a DAD detector (SPD-M10A Vp), binary gradient pump (LC20 AD) and, C18 column (250 × 4.6 mm, 5 µm particle size). The solvent system used for mobile phase was tetrahydrofuran: 0.6% citric acid in water (40:60) at a flow rate of 1.0 ml/min with column temperature 25 °C. The injection volume was 20 µl. Identification and quantification of the curcumin and calebin-A was done by comparing the retention time and characteristic absorption spectra from the DAD with those of the authentic standards. Data acquisition and analysis were carried out using Shimadzu LC Solution version 1.25. Samples were taken in triplicates for the analysis.
Liquid chromatography coupled with mass spectrophotometer (LC–MS)
Analysis of curcumin and calebin-A were performed by using liquid chromatography Thermo–Finnigan surveyor coupled to electrospray ionization on a triple Quad mass spectrometer (Thermo–Finnigan LCQ Advantage Max) equipped with degasser, binary pump, auto sampler, and column heater. For analysis, 4 μl of the extract was injected. The auto sampler was cooled at 10 °C. Chromatographic separation was achieved using C18 column (250 × 4.6 mm, 5μ particle size, Thermo, BDS) with flow rate 0.3 ml/min at 25 °C. The isocratic solvent system was acetonitrile and 0.1% acetic acid in water (40:60). Electrospray ionization was performed in the negative ion mode using helium gas at a pressure of 5 psi for the nebulizer with a flow of 5 l/min and a temperature of 300 °C. The sheath gas temperature was 250 °C with a flow rate of 11 l/min. The capillary was set at 3500 V and the nozzle voltage was 500 V.
Baeyer Villiger monooxygenases (BVMO) enzyme activity
Endophytic fungus O. brasiliensis MTCC 25236 was cultivated in PDB medium, containing 0.5 mg/ml of Curcumin C3 Complex® (dissolved in 25 mg/ml of DMSO). The culture was incubated for 72 h at 37 ± 0.5 °C at 140 rpm. After every 24, 48 and 72 h of interval, culture was centrifuged (10,000×g for 10 min) to remove cells and then supernatant was collected separately. Cell free supernatant was used as a crude extracellular enzyme complex to determine the BVMO activity. In another set of experiment, the above collected cell pellet was washed three times with sterile phosphate buffer (0.1 M PBS, pH 7.5) containing 4 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich). The washed cells were re-suspended in 10 ml phosphate buffer (50 mM; pH 7.5), and then were sonicated (for 45 s each cycle with 80% amplitude in cold condition) using ultrasonic homogenizer (Sartorius AG, Göttingen, Germany). The cell homogenate was centrifuged (10,000×g) at 4 °C and the clear supernatant was used as crude intracellular enzyme complex BVMOs activity. The in vitro BVMO activity was measured by monitoring the NADPH consumption (which is a must for BVMO activity) at 340 nm for 180 s in 1 ml cuvettes by using a UV–Vis spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The assays were performed in Tris-HCl buffer (50 mM, pH 8.5) containing 0.8 mM NADPH, 10 mM cyclohexanone, by adjusting absorbance between 0.9 and 1.0 at 340 nm and followed by addition of appropriate amount of the crude enzyme extracts. One unit (U) of the enzyme activity was defined as the amount of enzyme to oxidize 1 μmol of NADPH for 1 min under the reaction conditions. Protein content was determined by following Bradford’s method, using bovine serum albumin (BSA, Sigma-Aldrich) as standard (Bradford 1976).
