Fungal endophytes associated with Viola odorata Linn. as bioresource for pancreatic lipase inhibitors

The endophytic fungi were isolated from V. odorata as per the method described by Strobel and Daisy [1] with slight modifications. Different tissues (roots, leaf nodes and leaves) of the disease free plants were carefully excised with a sterile scalpel. In the first instance, these tissues were cleaned by thorough washing in running tap water, followed by deionized (DI) water. Clean tissue pieces were sterilized in a series of solution: 70% ethanol; 1.0% sodium hypochlorite (v/v); 70% ethanol for 1 min in each solution. Finally, they were rinsed twice with sterile distilled water. After surface sterilization, tissues were dried on blotting sheets and cut into 0.5 cm² pieces. These sterile small pieces were placed on water and potato dextrose agar (PDA) plates containing streptomycin (250 μg/mL) to inhibit the bacterial growth. The plates were wrapped with parafilm, incubated at 25 ± 2 °C and observed daily. The fringes of fungal mycelia growing from the tissues were subcultured on fresh PDA plates. The endophytic fungal isolates so obtained, were coded as per their tissue origin (VOR1, VOR3 from roots, VOLN1, VOLN2 from Leaf node and VOLF1, VOLF2, VOLF3 etc. from leaves). These endophytes were stored in paraffin oil at 4 °C and were deposited in RN Chopra, Microbial Repository, IIIM.

Fungal endophytic isolates were finally identified by ITS based rDNA sequencing. Genomic DNA of the endophytes was extracted from the in vitro grown biomass of endophytes using the protocol described by Reader and Broda [55]. Approximately 1 g of dried mycelia was kept in liquid nitrogen and crushed into a fine powder. It was transferred to 10 mL of extraction buffer and vortexed thoroughly. The samples were incubated in water bath at 65 °C for 30 min with intermittent mixing. The tubes were centrifuged at 10,000 g for 5–10 min followed by extraction of aqueous layer with chloroform: isoamyl alcohol (24:1). Aqueous layer was collected and DNA was precipitated with 2.5–3 V of absolute ethanol in presence of 1/10th volume of sodium acetate. Tubes were inverted slowly to mix the contents and centrifuged at 8000 g for 20 min at 4 °C. White/transparent pellets thus obtained were washed with ice cold 70% ethanol followed by air drying. Dried pellets were dissolved in 20 μL of water (molecular biology grade). ITS sequences containing ITS1–5.8S–ITS2 region spanning 500–600 bp were amplified with ITS1 and ITS4 universal primers [56]. PCR reaction was set up in 50 μL containing DNA (1–10 ng), 1× PCR buffer (with 15 mM MgCl₂), each dNTP (200 mM), each primer (10 pmol, IDT, Belgium) and 1 U Taq DNA polymerase (Promega, US). Cycling parameters were 5 min at 94 °C followed by 30 cycles of 94 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min and a final extension for 10 min at 72 °C. The PCR product (10 μL) was resolved using agarose gel electrophoresis at 80 V. The amplified product was purified using a Gel Extraction Kit (Qiagen, USA) and sequencing reaction was set up in a 10 μL: 40–60 ng of purified PCR product, 3.2 pmol forward/reserve primer, Big Dye Terminator sequencing mix 8 μL (v. 3.1, Applied Biosystems). Samples were sequenced on an automated sequencing system (Applied Biosystems). Resultant sequences (KX621956-KX621982) were submitted to a gene bank and were blasted against the nucleotide database using blastn Tool of the US National Centre for Biotechnology Information (NCBI) for final identification of endophytes [57].

For the extraction of biomolecules, the endophytic fungal isolates (twenty-seven) were cultured in 1 L Erlenmeyer flask containing 400 mL of potato dextrose broth (PDB) at 27 ± 2 °C for 10 days at 180 rpm (New Brunswick, USA). A 5 mm mycelial plug of 10-day old culture was used as inoculum. After 10 days, fermented culture of each endophyte was blended thoroughly in 20% methanol. Homogenate was extracted with one volume of methylene chloride (DCM) (HPLC grade). The extraction process was repeated four times. Solvent containing extract was striped off in a rotary evaporator leaving behind the solid powder, termed the crude extract. The stock solutions of extracts (10 mg/mL) were prepared in dimethyl sulfoxide (DMSO) and were used to evaluate the anti-obesity potential.

PL inhibition assay was performed using the reported protocol [58], which was previously optimized in our laboratory [59]. Briefly, 50 mg of porcine pancreatic lipase was suspended in 10 mL of Tris–HCl buffer (containing 2.5 mmol of Tris and 2.5 mmol of NaCl, adjusted to pH 7.4 with HCl). The solution was subjected to vigorous shaking for 15 min, followed by centrifugation (4000 rpm, 291 K for 10 min). The supernatant was collected and used afresh as the enzyme solution.

Stock solutions of the extracts and orlistat were prepared in DMSO at linear concentrations ranging from 1.56–2000 μg/mL and 0.78–1000 μg/mL, respectively. The final reaction mixture comprised of 875 μL of buffer, 100 μL of enzyme and 20 μL of the compounds of various stock concentrations, pre-incubated for 5 min at 37 °C, followed by addition of 10 μL of the substrate (4-nitrophenyl butyrate, 10 mM in acetonitrile). The amount of DMSO in the final concentration did not exceed 2%. The absorbance of the final mixture was taken in microplate reader (EPOCH, BioTek) after 5 min at absorbance maxima of 4-nitrophenol (405 nm). The assay was performed in triplicate and the percentage inhibition was calculated using the formula

Where, A_E is the absorbance of enzyme control (without inhibitor), and A_T is the difference between the absorbance of test sample with and without substrate. The IC₅₀ of the compounds was calculated by plotting linear regression curve, and was compared to that of orlistat (reference standard). Porcine pancreatic lipase exhibited a K_m value of 92.36 μM and V_max of 0.065 μM/min [59].

The inhibitory activity of endophytes against pancreatic lipase was examined with ANOVA and TUKEYS post hoc analysis using Graph Pad Prism software.

The endophytic fungi that were exhibiting inhibitory activity against pancreatic lipase were characterized on the basis of the morpho-cultural and molecular taxonomy and phylogenetic characteristics.

For phylogenetic evaluation, endophytic ITS DNA sequence and downloaded sequences of their nearest neighbors were aligned in Alignment Explorer of MEGA4 software [60] using Clustal W option. Trimming and verification of the sequence alignment were carried out using the MUSCLE (UPGMA) algorithm. The Maximum Composite Likelihood and Neighbor-Joining methods were used to compute the evolutionary distances and history respectively. The robustness of the tree was assessed by bootstrap analysis with 1000 replications.

Endophytic fungi were isolated from healthy and symptomless tissues of V. odorata to assess their anti-obesity potential and were identified on the basis of morpho-cultural and microscopic characteristics. Further confirmation was made on the basis of their molecular identification carried out by ITS based rDNA sequence analysis. Details of the fungal endophytes, their isolation source, GenBank accession numbers, and closest sequence homolog are given in Table 1.

The isolated endophytic fungi belonged to 11 different genera (Table 1). Most of the endophytic fungi belonged to Ascomycota phylum, except VOLF5 (Peniophora sp), which belonged to Basidiomycota phylum. Colletotrichum spp. (25.9%) showed the highest isolation frequency followed by Fusarium spp. (22.2%). Endophytes specifically isolated from the leaf tissues were Colletotrichum trifolii, Cladosporium tenuissimum, Aspergillus japonicus and Peniophora sp., whereas Daldinia eschscholtzii, Aspergillus niger and Colletotrichum siamense were found specific to leaf nodes and roots were found to harbor Nigrospora sp., Fusarium nematophilum, Fusarium solani, Colletotrichum truncatum, Nectria haematococca, Aspergillus awamori, Fusarium sp., Fusarium oxysporum, and Paecilomyces tenuis.

DCM extracts of the endophytes from V. odorata were subjected to PL inhibitory assay against porcine pancreatic lipase (Type II) using 4-nitrophenyl butyrate as substrate. Orlistat was used as reference standard and exhibited a very potent PL inhibition (IC₅₀ of 0.49 μg/mL). Approximately 61% extracts showed potent lipase inhibitory activity with IC₅₀ < 20 μg/mL, chosen as an acceptable limit (Table 2). Out of these 61% extracts, 7 endophytic extracts showed lipase inhibitory activity with IC₅₀ < 10 μg/mL. Rest of nine extracts displayed moderate activity with IC₅₀ 10–20 μg/mL. Further, VOLF4 (Aspergillus sp.) exhibited most potent PL inhibitory activity with an IC₅₀ of 3.80 μg/mL, followed by VOLF5 (Peniophora sp.) and VOR5 (Fusarium nematophilum) with IC₅₀ of 5.85 and 6.52 μg/mL, respectively. These results suggested that these extracts possess bioactive compounds with potential PL inhibitory activity. Furthermore, an analysis of the identified species against their IC₅₀ values indicated that Aspergillus spp. along with most of the species of Fusarium, Penicillium and Colletotrichum exhibited good PL inhibitory activity.

Morphological features of VOLF4 (Aspergillus sp.) VOLF5 (Peniophora sp.) and VOR5 (Fusarium nematophilum) were shown in Fig. 1. VOLF4 and VOLF5 were grown moderately over PDA medium while VOR5 was slow grower. Mycelium of VOLF4 was initially white later changed into light pink color, which is a rare color for this genus. Back was the pale in color. Rests of two endophytes were white in color. Phylogenetic positioning of these endophytes was shown in Fig. 2 suggesting unique identity of each of them.