Cytotoxic Potency of Bioactive Metabolites from an Endophytic Fungi Pestaloptiopsis breviseta against MCF-7 and A 549 Cancer cell lines

Vardhana Janakiraman*, Kathiravan Govindarajan²

¹Department of Biotechnology, Alpha Arts and Science College, University of Madras, Chennai- 600116.

²Department of Botany, RKM Vivekananda College, Chennai.

*Corresponding Author E-mail: vardhana88@ymail.com

ABSTRACT:

Fungi are organisms which lack chlorophyll and play a major role in plants. Some cause the disease to the plant, some live inside it as parasites and some as endophytes. Endophytic fungi play a very important role such that some endophytes are host specific and others just reside in it. In this study, one of the frequently recurring endophytic fungi isolated were identified as Pesatloptiopsis breviseta. The maximum absorbance was recorded at 302 nm in the UV spectra. The peaks above 3000 - 3400 cm^-1 in the FT-IR analysis, confirms the existence of free OH, NH, and NO₂ groups. The presence of peaks at 1900 - 2900 cm^-1confirms the presence of alkyl groups attached to an aromatic moiety and confirms the presence of a double bond in the compound. The peak at 1965 cm ^-1 confirms the presence of unsaturation in the molecule. The NMR spectra confirm the presence of an aromatic compound. GC-MS analysis showed the maximum m/z valve at 15.5 retention time followed by 23.38 and 18.92 correspond to the compounds Neoisolongifolane, hydroxy-, 1, 2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester and 8-Octadecenoic acid, methyl ester respectively. From the MTT assay, the percentage of cell viability based on IC₅₀ value was observed. From the assay, it was proved that on using different concentrations, the viability of cells was minimum at 100μl. Thus this study shows that the endophytic fungi can be served as a source for the production of bioactive metabolites, with which the pharmaceutical products can be developed.

KEYWORDS: Endophytic fungi, Secondary metabolites, MTT assay, MCF-7, A549, Pestaloptiopsis breviseta.

INTRODUCTION:

Some Endophytes are widespread and others are highly specific to a single host in a single environment. These endophytes protect their hosts from infectious agents and adverse conditions by secreting bioactive secondary metabolites^[9-11]. The metabolic interactions of endophytes with its host may favor the synthesis of biologically active secondary metabolites. There has been a great interest in endophytic fungi as potential producers of the novel, biologically active products^[12,13]. In this study, one of the frequently recurring endophytes which were isolated from various plant sources was used for the analysis of secondary metabolites and the solvent extract was used to study the cytotoxic potency against MCF-7 and A549 cell lines.

MATERIALS AND METHODS:

Collection of plant sample:

Healthy leaf samples were collected from Chennai and it was rinsed in distilled water to remove the dust and the leaves were dried in tissue paper. After the leaves are dried, the leaves are cut over the midrib region. The alternative midrib pieces are taken to avoid colonization of the same organism.

Isolation of endophytic fungi:

The surface sterilized leaves were trimmed using a sterile blade and inoculated into plates containing sterile Potato Dextrose Agar medium. The plates were incubated at 28°C.

Extraction of Secondary Metabolites:

The test fungus used in the present study was grown in 2L Erlenmeyer flasks containing 500ml MID medium supplemented with 1g of soytone L- 1. Three mycelial agar plugs (0.5cm) were used as inoculum. The organism was grown at 24±2°C statically for 3-4 weeks. After incubating the culture for 3–4 weeks, the culture filtrate was passed through four-layered cheesecloth. The fungal mycelia were collected and it was fixed in Glutaraldehyde solution and stored for subsequent studies. The culture fluid was extracted with two equal volumes of Methylene chloride or DCM and the organic phase was evaporated to dryness under reduced pressure at 35°C.

Cytotoxic study:

The cytotoxic study was carried out using the MTT assay^[14]. MCF-7 (Breast cancer) and A549 (Lung cancer) were procured from National Centre for Cell Science, Pune, India. The cell lines were grown adherently and maintained in DMEM containing 10% fetal calf serum, and 1% antibiotic solution containing penicillin and streptomycin at 37°C in 5% CO2 at 37ºC until the monolayer was subconfluent^[15]. The cells were plated separately in 96 well plates at a concentration of 1 × 105 cells/well. After 24 h, cells were washed twice with 100μl of serum-free medium and starved for an hour at 37^oC. After starvation, cells were treated with a different test compound for 24 hrs. At the end of the treatment period, the medium was aspirated and serum-free medium containing MTT (0.5mg/ml) was added and incubated for 4 h at 37ºC in a CO₂ incubator. The MTT containing medium was then discarded and the cells were washed with PBS (200μl). The crystals were then dissolved by adding 100μl of DMSO and this was mixed properly by pipetting up and down. Spectrophotometrical absorbance of the purple-blue formazan dye was measured in a microplate reader at 570nm (Biorad 680). Cytotoxicity was determined using Graph pad prism5 software.

% Cell viability = A570 of treated cells / A570 of control cells × 100%.

RESULT AND DISCUSSION:

Identification of endophytic fungi:

The endophytic fungi isolated were identified as Pesatloptiopsis breviseta (Sacc, 1949) based on the morphological and microscopical observations (Fig 1). Spots definite, circular, gray, pustules small, punctiform, gregarious, subglobose, largely hypophyllous. Conidia 5- celled, elliptic fusiform, 18-24 x 6-8μm, hardly constricted at septa, intermediate colored cells olivaceous, concolorous, sometimes slightly contrasted, 12-16μm long, setulae 3, sometimes 2, 3-12μm long, exteriorhyaline cells short, apical cells, conic. Cylindric, the basal cells broad conic, Pedicles short.

Fig 1 Culture and Spores of Pestaloptiopsis breviseta

Extraction of secondary metabolites:

The test fungal colonies were inoculated in M1D medium. The inoculated flask cultures of the fungal species were harvested after 3-4 weeks when it has attained the maximum growth. The culture filtrate and the mycelia mat separated were extracted using dichloromethane solvent.

UV Spectroscopic Analysis:

The UV spectra 200–400nm using UV-VIS spectrophotometer was recorded. The maximum absorbance was recorded at 302nm (Fig 2)

Fig 2 UV Spectral analysis of the endophytic fungal extract

FT-IR spectral analysis:

The FT-IR spectra were observed both in the fingerprint region and functional group region (Fig 3). The peaks above 3000 - 3400 cm^-1 confirms the existence of free OH, NH and NO₂ groups. The presence of peaks at 1900 - 2900 cm^-1confirms the presence of alkyl groups attached to an aromatic moiety and confirms the presence of a double bond in the compound. These peaks reveal the SP³ hybridized stretching vibration of alkyl groups attached to an aromatic moiety. The peak at 1965 cm ^-1 confirms the presence of unsaturation in the molecule. The peaks at 1000 - 1500 cm^-1 in the fingerprint region is due to the out of plane (OOP) bending vibration, further confirms the presence of aromatic nuclei.

NMR Spectral Analysis:

NMR Analysis of the solvent extract was carried out using the Carbon and Hydrogen Spectra. From the carbon spectra, it was found that the presence of peaks at 150 – 160 δ indicates the aromatic nuclei in the compound. Peaks at 160- 165 δ indicates the presence of a carbonyl group as it is evident from C - 13 spectra. From the proton spectra, it was evident that the peaks at 1 δ – 3 δ- alkyl groups attached to an aromatic ring. 3.5- 4.5 δ- Benzylic CH and Omethoxy groups. 6- 6.5 δ- unsaturation in the molecule. 7 – 8 δ- aromatic group.

GC- MS SPECTRA ANALYSIS:

The maximum m/z valve at 15.5 retention time followed by the peak in 23.38 and 18.92 which supports the molecular weight of the substance which is not found in the GC- MS spectra of other retention time. The peaks correspond to the compounds Neoisolongifolane, hydroxy-, 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester and 8-Octadecenoic acid, methyl ester respectively. (Fig 4).

From the above results, it could be interpreted that the extracted compound from Pestalotiopsis breviseta should be an aromatic compound with the presence of one or more carbonyl group, benzylic group, O- methoxy group, and the aliphatic side chain should contain two or more alkyl group with one or more units of unsaturation.

CYTOTOXIC ASSAY:

The solvent extracted metabolite samples were treated on human breast cancer (MCF-7) and lung cancer (A549) cell lines. The samples were taken in different concentrations from 6μl to 100μl. The MTT assay was carried out for 24 hrs and 48 hrs interval time (Table 1) and the percentage of cell viability based on IC₅₀ value was observed using graph pad prism 5 software. From the assay, it was proved that on using different concentrations, the viability of cells was minimum at 100μl (Fig 5).

Fig 5: (A) Vero (B) MCF -7 and (C) A549 cell line treated with 100 μl of the solvent extract

Table 1 Percentage of cell viability at different concentrations of samples in MCF-7 and A549 cell line at 24 hr and 48 hrs.

The concentration of the sample	MCF 7		A549
Time	24 hrs	48 hrs	24 hrs	48 hrs
6 μg	89	90	93	94
12 μg	82	80	90	81
25 μg	69	68	73	70
50 μg	63	60	54	51
100 μg	43	47	50	46

DISCUSSION:

In the present investigation, the fungal secondary metabolites were extracted using dichloromethane solvent and the extract was subjected to spectral analysis to find out the group of compounds present in it. The crude solvent extract was treated on MCF-7 and A549 cancer cell lines. It was clearly evidenced that at the concentration of 100μg/ml of the sample, the cytotoxic potent was more effective. Thus this study shows that the endophytic fungi can be served as a source for the production of bioactive metabolites, with which the pharmaceutical products can be developed. Also, this study shows that the production of bioactive metabolites from microbes is a cost-effective and eco-friendly method.

ACKNOWLEDGMENT:

The authors express sincere thanks to the Department of Science and Technology (INSPIRE), New Delhi, India, for providing necessary funds to carry out the research work. The Authors also thank CNST Anna University for providing the instrumentation facility.

CONFLICT OF INTEREST:

The authors have no conflict of interest to declare.

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Received on 11.10.2019 Modified on 19.12.2019

Research J. Pharm. and Tech. 2020; 13(10):4683-4686

DOI: 10.5958/0974-360X.2020.00824.0