Bioactive sesquiterpenes from an endophytic fungus Bipolaris sorokiniana isolated from a popular medicinal plant Costus speciosus

ABSTRACT Bipolaris sorokiniana, a fungal endophyte, was isolated from the leaves of Costus speciosus, a popular medicinal plant used to control diabetes. Fermentation of the fungus in potato dextrose broth and chromatographic purification of the ethyl acetate (EtOAc) extracts of the culture broth and mycelium yielded two rare sesquiterpenes helminthosporal acid (1) and helminthosporol (2), together with ergosterol. Compounds 1 and 2 showed a strong phytotoxic effect on lettuce seed germination and toxicity against brine shrimps. Compound 1 also showed antifungal activity. Complete assignments of 1H and 13C NMR data of compound 1 are described for the first time.


Introduction
Endophytes are microorganisms that live in the intercellular spaces of stems, petioles, roots and leaves of plants causing no discernible manifestation of their presence and have typically gone unnoticed (Strobel and Long 1997). It is well documented that endophytic fungi are capable of producing bioactive secondary metabolites. The first discovery of endophytic fungi was recorded as far back as 1904 (Freemann 1904). However, endophytes did not receive much attention until the recent realisation of their ecological relevance and potential of yielding metabolites with diverse structures and biological functions (Gunatilaka 2006). In a continuation of our studies on the search for bioactive compounds from Sri Lankan flora, secondary metabolites produced by an endophytic fungus Bipolaris sorokiniana (Class: Ascomycetes & Order: Pleosporales) isolated from the leaves of Costus speciosus of the family Costaceae have been investigated. C. speciosus is a widely used medicinal plant in Sri Lanka and India especially due to its antidiabetic properties and for its ability to cure various diseases (Pawar and Pawar 2014).

General
Extractions were taken using a sonicator (VWR Ultrasound cleaner, model-USC 1700 D). Thin layer chromatography (TLC) analysis was conducted on silica gel plates (Merck 1.05554, 60F 254 , 0.20 mm thickness). TLC spots were located using a UV lamp and by heating after spraying with acidic anisaldehyde. Silica gel (Merck Art. 7734 & 9385) and Sephadex LH-20 were used for column chromatography. 1 H NMR and 13 C NMR were recorded on a Bruker DRX-500 or JEOL JNM-ECP500 (both are 500 MHz for 1 H and 125 MHz for 13 C) spectrometer in CDCl 3 solution. FABMS (+) were obtained on a JEOL JMS-700 spectrometer with NBA as matrix. UV absorptions were measured on a Thermo Scientific Multiskan GO Microplate spectrophotometer.

Isolation and identification of the endophytic fungus
Fresh leaves of C. speciosus were collected from the Central Province of Sri Lanka in January 2014. Endophytic fungus was isolated from the leaves as CONTACT Lalith Jayasinghe ulbj2003@yahoo.com This paper is dedicated to the late Prof. G.P. Wannigama, Sri Lanka, an inspirational scientist and teacher, on the occasion of his second death anniversary.
Supplemental data for this article can be accessed here.
described in our previous publication (Qader et al. 2015;Thanabalasingam et al. 2015). The fungus was identified as Bipolaris sorokiniana by sequence analysis of the rDNA gene. DNA was extracted using Promega, Wizard Genomic DNA purification kit (A1120), and amplification of the ITS region was carried out using the universal eukaryotic primers of ITS1 and ITS4. These experiments were performed by the GeneTech Institute, Sri Lanka. BLAST search indicated that the sequence of the ITS region had 99% similarity to that of Bipolaris sorokiniana isolate A1S6-2 (Gene Bank Accession No. KJ767094.1). Photographic evidence of the leaves of C. speciosus and B. sorokiniana strain (IFS/MQ/TFP-2014) are deposited at the National Institute of Fundamental Studies.

Results and discussion
An endophytic fungus isolated from the leaves of C. speciosus was identified as B. sorokiniana (syn. Helminthosporum sativum and Cochliobolus sativus (Duczek 1982) by molecular means. This is the first report of the isolation of endophytic fungus B. sorokiniana from a popular medicinal plant C. speciosus. Large-scale fermentation of B. sorokiniana in potato dextrose broth media for 4 weeks and extraction of the culture broth and mycelium with EtOAc yielded an extract that displayed antifungal activity against C. cladosporioides (minimum inhibitory concentration [MIC] <250 ppm), antioxidant activity against DPPH (IC 50 346 ppm), lettuce (Lactuca sativa) seed germination assay (IC 50 400 ppm), brine shrimp (Artemia salina) lethality (LD 50 407 ppm) and α-amylase inhibitory activity (IC 50 627 ppm). Chromatographic separation of the EtOAc extract furnished compounds 1 and 2 and ergosterol. Compound 1 was characterised as helminthosporal acid by comparison of the 1 H NMR data with the reported values (Rodriguez et al. 1988). First complete assignments of 1 H and 13 C signals for 1, which were established by 2D-NMR including COSY, HMQC and HMBC spectra, are reported. Compound 2 was identified by comparison of the 1 H and 13 C NMR data with literature values (Osterhage et al. 2002). The absolute configurations of 1 and 2, shown in Figure 1, have been determined previously through the synthesis of helminthosporal (3) (Corey and Nozoe 1963) and chemical oxidative conversion of 2 and 3 to 1 (de Mayo et al. 1962;Tamura et al. 1965).
Compounds 1 and 2, and related sesquiterpenes such as helminthosporal and helminthosporic acid, are rare sesquiterpenes, which were previously isolated from B. sorokiniana (Mayo et al. 1961;Tamura et al. 1965;Rodriguez et al. 1988). Isolation of 2 is also reported from the fungus Drechslera dematioidea, which was isolated from the marine red alga Liagora viscida (Osterhage et al. 2002). B. sorokiniana is known to be a plant pathogenic fungus that produces root rot, leaf spot disease, seedling blight and head blight of barley and wheat, causing significant yield loss (Mayo et al. 1961). It was reported that helminthosporol (2) is a gibberellin-like plant growth regulator, which exhibited elongation effect to the shoots of rice seedlings at concentrations ranging from 10 to 300 ppm, whereas rice root growth was inhibited above 50 ppm of 2 and emergence of roots was suppressed at 300 ppm. Moreover, compound 2 also inhibits shoot growth of wheat seedlings above 30 ppm (Tamura et al. 1963).
In the present phytotoxic assay, both compounds 1 and 2 inhibited (100%) the germination of lettuce seeds at 62.5 ppm (the positive control (+)-abscisic acid inhibited the germination (100%) at 10 ppm). Compounds 1 and 2 inhibited hypocotyl growth with IC 50 values of 45.5 and 28.6 ppm, respectively, and percentage radicle growth with IC 50 values of 31.6 and 20.6 ppm, respectively. Our results indicated that compounds 1 and 2 showed consistently deleterious effects on lettuce seed germination, hypocotyl and radicle growth at several 10 ppm, among which the effect on radicle growth of lettuce seeds was opposite to that reported for the shoots of rice seedlings. In addition, compounds 1 and 2 showed toxicity against brine shrimp (LD 50 27.1 and 28.3 ppm, respectively). Furthermore, compound 1 displayed antifungal activity against C. cladosporioides (MIC 31.3 ppm).
In addition to these activities, compounds 1 and 2 were reported to have significant neuraminidase inhibitory activity (Zhang et al. 2011). Furthermore, compound 1 was shown to be an inhibitor of Acyl-CoA: cholesterolacyltransferase in rat liver microsome (Park et al. 1993).
It would be interesting to study about endophytic fungi diversity in other parts of C. speciosus, because the secondary metabolites produced by endophytic fungi of C. speciosus are less studied and the possibility of production of similar bioactive compounds in a large scale will be beneficial for agriculture, industry and medicine.