A new antifungal eudesmanolide glycoside isolated from Sphaeranthus indicus Linn. (Family Compositae)

Abstract A new antifungal eudesmanolide glycoside 11,13-dihydro-3-O-(β-digitoxopyranose)-7α-hydroxy eudasman-6,12-olide (2) in addition to known compounds 1 and 3, has been isolated from Sphaeranthus indicus Linn. Its structure was determined by spectral analysis (UV, IR, 1D and 2D NMR and mass spectrum).


Introduction
India has been endowed with a very rich flora owing to the extreme variations in climate and geographical conditions prevalent in the country. With the advent in science, many of the crude drugs used in traditional system have been investigated scientifically (Galani et al. 2010). The therapeutic areas of infectious diseases and oncology have benefited from these numerous drug classes, able to interact with many specific targets within the cell, and indeed for many years have been central in the drug discovery and development (Mishra & Tiwari 2011;Singh et al. 2014). The plant Sphaeranthus indicus Linn., a herb of about 30 cm in height, with spreading branches and round purple flowers, is found all over the Indian plains as weed in the rice fields (Shekhani et al. 1990;Chatterjee & Pakrashi 2003). It is used ABSTRACT as folk medicine to treat wide range of diseases like wound healing, anthelmintic, insanity, tuberculosis, bronchitis, spleen diseases, elephantiasis, etc. (Farzana et al. 2006;Nisha et al. 2007;Mathew et al. 2012).

Results and discussion
The IR spectrum (KBr) of compound 2 showed characteristic 5-membered γ-lactone absorption band at 1780 cm -1 and also revealed the presence of oh and non-conjugated olefin function. elemental analysis (Found: h,8.17%;C,63.64%;o,28.32%/requires: h,8.14%;C,63.60%;o,28.26%) in combination with a molecular ion peak at m/z 396 (45%) and 21 carbon resonance signals observed in 13 C NMR spectrum (75 Mhz, CD 3 oD) of compound 2 confirmed the molecular formula to be C 21 h 32 o 7 . In eI-MS spectrum, the prominent peaks at m/z 299, 214, 200, 165 and 158 showed a related eudesmanolide skeleton (Irwin & Geissman 1969, 1971. A base peak observed at m/z 265 (100%), indicated for the compound 2 to be a eudesmanolide monoglycoside with a dideoxyhexose as a sugar unit.
In 1 h NMR spectrum (300 Mhz, CD 3 oD) of compound 2, a three-proton singlet appeared at δ 1.94 was evidenced for angular methyl resonance while another singlet integrated to three protons was identified for the presence of a methyl group attached to olefinic carbon.
The hydrolysis of compound 2 with 2N h 2 So 4 in 50% ethanol furnished an aglycone. The proton and 13 C NMR shift pattern were compared and found similar to frullanolides (Bohlmann et al. 1980;Segal et al. 1984). The sugar residue was evidenced as β-digitoxopyranose by paper chromatographic comparison with different known sugars.
The antifungal activity of compound 2 was evaluated against pathogenic fungi by disc diffusion assay (DDA), broth microdilution assay (BMA) and percent spore germination inhibition assay (PSGIA). The results were compared using standard antifungal drug Clotrimazole

General experimental procedures
The solvents were of pure analytical grade. Melting points were measured in open capillary tubes on a Buchi 530 apparatus and are uncorrected. Thin-layer chromatography was performed on 60 F254 silica gel, precoated on aluminium plates and revealed with either a UV lamp (λ max = 254 nm) or by spraying with methanolic h 2 So 4 solution and subsequent charring by heating at 100 °C. 1 h and 13 C NMR were recorded at 300 and 75 Mhz, respectively. Chemical shifts given in ppm downfield from internal TMS; J values in hz. Mass spectra recorded using electrospray ionisation mass spectrometry (eSI-MS). Infrared spectra recorded as Nujol mulls in KBr plates. elemental analysis was done using a C, h, N analyser and results were found to be within ±0.4% of the calculated values.

Plant material
The

Micro-organism and media
Clinical isolates of A. fumigatus, A. flavus, A. niger, Candida parapsilosis, Candida tropicalis, Candida albicans, Candida neoformans, Trichophyton mentagrophytes, Sporothrix schenckii, Trichoderma viride, Microsporum gypseum, Absidia ramosa, Pseudallescheria boydii and S. cerevisiae obtained from the Microbiology Department, Vallabhbhai Patel Chest Institute, Delhi, India were used for antifungal assay. The pathogenic strains of micro-organism were cultured on Sabouraud dextrose agar plates. Spores/conidia from the fungal colonies of 72-96 h cultures were incubated at 28 °C, the conidia were counted using hemocytometer and the number was adjusted to 1 × 10 6 spores/mL.

Disc diffusion assay
The radiation sterilised Petri dishes (10 cm diameter) were poured with adequate amount of Sabouraud dextrose agar . one ml of conidial suspension equivalent to 1 × 106 conidia was prepared in agar plate. The disc diffusion test was performed with different concentrations of compound 2 ranging from 50 to 1.56 μg. Medium was impregnated in a sterilised disc Whatman filter paper No. l, 5.0 mm in diameter and the discs were placed on the surface of agar plates already impregnated with fungi. The plates were incubated for 48 h at 37 °C and examined for around the disc, and the lowest concentration that develops zone of inhibition of 6 mm diameter or more, was considered as MIC, Clotrimazole was used as positive control in the assay.

Broth microdilution assay
For broth microdilution assay , autoclaved Sabouraud dextrose broth was placed in each well of 96 well culture plates (Nunc, Nunclon). Various concentrations of compound 2 in the range of 1000.0-7.86 μg/mL were prepared in the wells by twofold dilution method. The wells were inoculated with 1 × 106 spores in 10 μL of spore suspension. Appropriate control wells treated with Clotrimazole or without any treatment were included in the study. The plates were incubated at 28 °C and examined visually after 48-72 h of cultures for the growth of fungal mycelia. The activity was represented as −ve if visible growth was there and +ve if medium appeared clear without any growth of fungal mycelia.
The lowest concentration, which inhibits the growth of fungi, was considered as the MIC of the compound.

Percent spore germination inhibition assay
For the spore germination assay (Rajesh & Sharma 2002), various concentrations ranging from 1000.0 to 7.86 μg/mL of compound 2 in 90.0 μL of culture medium were prepared in 96 well flat bottom micro-culture plates (Nune, Nunclon) by double dilution method. The wells were prepared in triplicates for each concentration. The concentration ranged from 1000.0 to 7.86 μg/mL. each well was then inoculated with 10 μL of spore's suspension containing 100 ± 5 spores. The plates were incubated at 28 °C for 24 h and then examined for spore germination under inverted microscope. The numbers of germinated and non-germinated spores were counted. The PSGI was calculated using following formula.
The tests were repeated at least three times. The lowest concentration that gave 97-100% inhibition of germination of conidia in the wells was considered as MIC.

Conclusion
In conclusion, we have identified a new compound, 11,13-dihydro-3-O-(β-digitoxopyranose)-7α-hydroxyeudesman-6,12-olide (2) from ethanolic extract of aerial parts of S. indicus exhibiting significant in vitro antifungal activity against pathogenic strains of fungi. Synthesis of this naturally occurring compound 2 and its analogues may provide a lead for the development of a potent antifungal drug.