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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 12  |  Issue : 2  |  Page : 112-116

Cytotoxic activity of endophytes isolated from Allamanda blanchetii A. DC


1 Department of Ethanomedicine, National Institute of Traditional Medicine (Indian Council of Medical Research), KLE University; Department of Cell Culture and Microbiology, MMDS, Belagavi, Karnataka, India
2 Department of Ethanomedicine, National Institute of Traditional Medicine (Indian Council of Medical Research), KLE University, Belagavi, Karnataka, India
3 Department of Microbiology, Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, Belagavi, Karnataka, India

Date of Web Publication4-Jun-2019

Correspondence Address:
Dr. Harsha V Hegde
National Institute of Traditional Medicine (Indian Council of Medical Research), Nehru Nagar, Opposite to KLE University, Belagavi - 590 010, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/kleuhsj.kleuhsj_7_19

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  Abstract 


AIM: To evaluate the cytotoxic potential of endophytic fungi isolated from A. blanchetii.
MATERIALS AND METHODS: Endophytic fungi were isolated from A. blanchetii using surface sterilization methods, grown in different types of media to ascertain their suitability for growth. Mass culture was prepared using suitable media and extracts using several solvents were prepared and dried. These extracts were later evaluated using MTT assay for their cytotoxic potential.
RESULTS: Hydroalcoholic extract of Phomopsis species exhibited better cytotoxicity (80 μg/ml) when compared ethyl acetate extract of the same 160 μg/ml. Similarly, ethyl acetate extract of Phomopsis species, ethyl acetate extract of Alternaria aff. raphani J. W. Groves and Skolko also demonstrated cytotoxicity of 160 μg/ml in MTT assay against MCF 7 cell line.
CONCLUSION: Both the species of Phomopsis and Alternaria have shown significant cytotoxicity in MTT assay.

Keywords: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, alternaria, anticancer, endophytes, MCF-7, phomopsis


How to cite this article:
Pednekar HD, Hegde HV, Hurakadale PJ, Wagawade JD, Bhat KG, Bogar C. Cytotoxic activity of endophytes isolated from Allamanda blanchetii A. DC. Indian J Health Sci Biomed Res 2019;12:112-6

How to cite this URL:
Pednekar HD, Hegde HV, Hurakadale PJ, Wagawade JD, Bhat KG, Bogar C. Cytotoxic activity of endophytes isolated from Allamanda blanchetii A. DC. Indian J Health Sci Biomed Res [serial online] 2019 [cited 2019 Jun 20];12:112-6. Available from: http://www.ijournalhs.org/text.asp?2019/12/2/112/259645




  Introduction Top


Endophytes are group of microbes, which reside in living tissues, intra and/or inter cellularly, without causing physical manifestations of diseases.[1],[2] They gained widespread publicity due to their ability to produce an array of compounds, both known and unknown, having a variety of uses in medical, agriculture and industry.[3] This has led to special interest in these organisms, especially as an alternative source of biomedically important secondary metabolites.

Interest in the generation of secondary metabolites from source other than plants began initially with the discovery of Taxol. Taxol was isolated from Taxus brevifolia followed by Taxus wallinchiana, which harbor endophytic fungi, namely Taxomyces andreanae and Pestalotiopsis microspora, respectively.[4] From the discovery of taxol, it became evident that microbial sources could be used as secondary source of compounds apart from plants.

Breast cancer is one of the most common female cancers. It is estimated that worldwide, nearly one-fifth of all cancers in women are breast cancer.[5] With the increasing reports on the side effects associated with chemotherapy, there is a need to find newer therapeutic molecules, and hence, efforts are on global scale to tap the unconventional resources, such as endophytic fungi. Further, from discovering novel substances to finding its way to clinical research, analysis of substances demonstrating cytotoxic property became very important.[6] Cell-based cytotoxicity assays such as 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) mainly deal with screening to determine whether the compound/extract has cell proliferation or direct cytotoxic effects which will eventually lead to cell death. These types of assay are widely used for high throughput screening.[7]

Fractions from Allamanda blanchetii A. DC demonstrated varied biological properties such as free radical scavenging activity 2,2- diphenyl-1-picryl-hydrazyl-hydrate (DPPH), brine shrimp lethality bioassay, thrombolytic activity, whereas methanolic crude extract, hexane soluble fraction, carbon tetrachloride soluble fraction, and chloroform (CHL) soluble fraction revealed zone of inhibition against bacterial strains ranging from 7.0 to 13.0 mm in disc diffusion assay.[8] Antihyperglycemic activity was demonstrated by CHL, chloroform: methanol (4:1), and chloroform: methanol (3:2) fractions of A. blanchetii (Family: Apocynaceae), whereas the floral ether and CHL extract showed anti-oxidant and hypolipidemic properties in rats.[9]

However, no work has been reported on either endophytes or the activity of endophytes isolated from A. blanchettii, and hence, the present work is an effort isolate the endophytes from the plant to screen their cytotoxic activity to find the better alternatives for further downstream applications.


  Materials and Methods Top


Collection of plant source and sterilization

Leaves were collected from the healthy plants of A. blanchettii from RMRC campus in Belagavi and processed immediately for isolation.

With minor modification, the standard sterilization procedures were followed.[10] The collected leaves were surface sterilized with 0.05% tween 20 solution, dipped in 5% sodium hypochlorite for a minute followed by dipping in ethanol for 1 min. This procedure was repeated twice. In the final step, the leaf materials are rinsed twice, with double distilled water (DDW) and dried in laminar air flow hood.

Leaf imprinting on potato dextrose agar (PDA) was done to ascertain the effectiveness of sterilization procedure and eliminating epiphytic fungi if any.[11]

Isolation and identification of endophytic fungi

With slight modification, 1 g leaves were carefully macerated in 10 ml of DDW in sterile mortar pestle. One ml each of it was transferred to different media, namely PDA, sabouraud dextrose agar (SDA), yeast extract sucrose agar (YESA), czapek dox agar (CDA), and sabouraud dextrose malt agar (SDMA) with streptomycin (20 mg/1000 ml media) incorporated in it to obtain diversity among fungi and to eliminate bacterial isolates. These plates were incubated at 28°C for 15 days. Each fungal isolates were further subcultured in PDA, SDA, YESA, CDA, and SDMA separately without antibiotic to assess its media suitability. The entire procedure was repeated thrice in aseptic conditions. Later, the axenic cultures obtained were maintained in FA (fungal agar) slants, sealed, and stored at 4°C.[12]

The cultures were tentatively identified using lactophenol cotton blue stain under light microscope (Olympus, USA). The axenic cultures obtained were sent for confirming to National Fungal Culture Collection of India, Agharkar Research Institute, Pune. The morphological identity of these strains was confirmed as Phompsis species, Alternaria aff. raphani J. W. Groves and Skolko and Alternaria tenuissima (Kunze.) Wiltshire.

Preparation of fungal extract

0.5 cm of cultured agar blocks were used as inoculum from CDA to czapek dox broth (CDB) with slight modification and incubated at 28°C for 30 days in 12 h light/dark cycle. After incubation period, mycelia were carefully separated, washed, and soaked in 70% ethyl alcohol, followed by maceration to obtain hydroalcoholic (HA) extract.

The filtered culture broth was used to prepare ethyl acetate (EA), CHL and HD (hexane: dichloromethane, 1:1) extract. The filtrate broth was soaked in solvent and extracted with 50 ml of solvent. Extraction was repeated thrice for each solvent. The solvent was pooled together and dried with rotatory evaporator (Heidolph-Heivep Advantage, Germany) under vacuum at 40°C till dry extract was obtained. The dried extract was weighed and stored at-20°C till further use.[13]

Cytotoxicity activity was performed quantitatively using the MTT assay with slight modification.[14],[15] MCF 7 breast cancer cell lines were added in 96 well plate-containing Dulbecco's modified eagle medium with 10% heat-inactivated fetal calf serum, containing 5% of mixture of gentamicin (10 μg), penicillin (100 units/ml), and streptomycin (100 μg/ml) in the presence of 5% CO2 at 37°C for 48 to 72 h. The cells were seeded in 96 well plate capacity at a density of 2.0 × 104 in 100 μl per well of the medium. Concentrations of drug ranging from 10 to 160 μg/ml were added to seeded wells and were incubated at 37°C in 5% CO2 with humidity not >94% for 24 h. Phosphate-buffered saline-containing 20 μl of MTT (stock 5 mg/ml) was added and incubated for 4 h. Dimethyl sulfoxide (DMSO) (100 μl) was added for solubilization of formazan crystals after supernatant was aspirated. Viability of cells was calculated by comparing the absorbance of treated versus untreated cells. Cells treated with equivalent concentration of DMSO were used as negative control. Optical density (OD) was calculated at 492 nm by using ELISA reader (Thermo Scientific, USA) and inhibitory concentration by 50% (IC50) value was calculated by using the following formula.[16]



IC50 value was obtained by plotting % cytotoxicity/growth inhibition against drug concentration (in μg/ml) using MS-excel worksheet.[17]

% cytotoxicity was derived using formula: (1 – [test/control]) ×100


  Results Top


A total of four endophytes were isolated from A. blanchetii A. DC (Apocynaceae), of which three have been identified morphologically. Mycelia of endophytes were grown in different media (PDA, SDA, YESA, SDMA, and CDA) to assess their colony morphology and media suitability [Table 1].
Table 1: Media comparison and suitability of fungal isolates from Allamanda blanchetii

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Accordingly, CDA was selected as media of choice and CDB was used for further mass culture of these selected fungal strains. HD, EA, CHL, and HA extracts were prepared from all the four axenic cultures of endophytic fungal isolates. Among these, three extracts exhibited cytotoxicity in the MTT assay [Figure 1]. Standard doxorubicin was used for comparison [Figure 2].
Figure 1: Cytotoxicity of extracts from fungal isolates of Allamanda blanchetii

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Figure 2: Cytotoxicity of control (doxorubicin)

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Doxorubicin at different concentrations (1–6 μg/mL) was administered to MCF 7 cell lines, which induced significant inhibition as compared to control. The Dox was highly cytotoxic, and the reduction in cell viability observed at 3.7 μg/mL in Dox-treated MCF 7 cell lines.[12]

HA extract of Phomopsis species exhibited better cytotoxicity (80 μg/ml) when compared to EA extract of the same 160 μg/ml. Similar to EA extract of Phomopsis species, EA extract of Alternaria aff. raphani J. W. Groves and Skolko also demonstrated cytotoxicity of 160 μg/ml in MTT assay against MCF 7 cell line.


  Discussion and Conclusion Top


Earlier, it was reported that various species of Phomopsis are producing a vast array of compounds and the extracts showed potent biological activities. Apart from several reports of their antimicrobial activities[17],[18],[19] and free radical scavenging activities,[20] there are few reports on their cytotoxic activities. Phompsis liquidambari QH4, an isolate of Artemisia annua showed antitumor activity against MCF 7 breast with growth inhibition of 53.66% in EA extract (20 μg/ml).[21] A new xanthone 1,5-dihydroxy-3-hydroxyethyl-6-methoxycarbonyl, isolated from Phomopsis, was reported to be cytotoxic to MCF7 cells with IC50 value of 2.7 μM.[22]

Alternaria species also led to the isolation of various compounds, and as a species, it also exhibited a wide range of activities. A. alternata, isolated from grapes, exhibited antimicrobial effect on plant pathogen Plasmopara viticola.[23] In a study conducted,[24] ZJ9-6B (Alternaria species) isolated from the fruit of Aegiceras corniculatum two compounds, compound 1 and 2 have been isolated, which showed IC50 value of 29.11 μM and 20.04 μM, respectively, in MCF 7 cells.

Further study and implications

In the present study, both the species of Phomopsis and Alternaria have shown significant cytotoxicity. Further research work is essential to validate the anti-cancer potential of these endophytes, which may provide the real application of the present work.

Financial support and sponsorship

This study was financially supported by RMRC, Belagavi for intramural research.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Strobel G, Yang X, Sears J, Kramer R, Sidhu RS, Hess WM, et al. Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiology 1996;142(Pt 2):435-40.  Back to cited text no. 4
    
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Pednekar HD, Hegde HV, Hurakadale PJ, Bhat K, Bogar C, Wagawade J.In vitro anticancer activity of endophytic fungal extracts from cladosporium family isolated from Cyathocline purpurea (D. Don.) O. Ktze. EJBPS 2017;4:412-6.  Back to cited text no. 12
    
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Prema R, Sekar DS, Sekhar KB, Jeevanandham S.In vitro cytotoxicity study on combined plants extracts (Cissus quadrangularis and Aegle marmelos). Eur J Exp Biol 2012;2:882-8.  Back to cited text no. 16
    
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Desale MG, Bodhankar MG. Antimicrobial activity of endophytic fungi isolated from Vitex negundo linn. Int J Curr Microbiol Appl Sci 2013;2:389-95.  Back to cited text no. 17
    
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Rakshith D, Santosh P, Sreedharamurthy S. Isolation and characterization of antimicrobial metabolite producing endophytic Phomopsis sp. from Ficus pumila Linn. (Moraceae). Int J Chem Anal Sci 2013;4:156-60.  Back to cited text no. 18
    
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Redko F, Clavin M, Weber D, Anke T, Martino V. Search for active metabolites of Erythrina crista-galli and its endophyte Phomopsis sp. Mol Med Chem 2006;10:24-6.  Back to cited text no. 19
    
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Qian Y, Kang J, Lu W, Lei B, Hyde KD.In vitro antioxidant and antitumor activities of an endophytic fungus Phomopsis liquidambari QH4 from Artemisia annua. Chiang Mai J Sci 2014;41:992-1006.  Back to cited text no. 21
    
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Yang HY, Gao YH, Niu DY, Yang LY, Gao XM, Du G, et al. Xanthone derivatives from the fermentation products of an endophytic fungus Phomopsis sp. Fitoterapia 2013;91:189-93.  Back to cited text no. 22
    
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Musetti R, Polizzotto R, Vecchione A, Borselli S, Zulini L, D'Ambrosio M, et al. Antifungal activity of diketopiperazines extracted from Alternaria alternate against Plasmopara viticola: An ultrastructural study. Micron 2007;38:643-50.  Back to cited text no. 23
    
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Huang CH, Pan JH, Chen B, Yu M, Huang HB, Zhu X, et al. Three bianthraquinone derivatives from the mangrove endophytic fungus Alternaria sp. ZJ9-6B from the South China Sea. Mar Drugs 2011;9:832-43.  Back to cited text no. 24
    


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