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ORIGINAL ARTICLE
Year : 2018  |  Volume : 10  |  Issue : 2  |  Page : 188-194  

Antioxidant and cytotoxic potential of endophytic fungi isolated from medicinal plant Tragia involucrata L.


1 Department of Studies and Research in Biochemistry, PG Centre, Mangalore University, Kodagu, India
2 Department of Biochemistry, Davanagere University, Davanagere, India
3 Department of Biotechnology, A Constituent College of University of Agriculture, Sciences, Bangalore, India

Date of Web Publication20-Apr-2018

Correspondence Address:
Dr. Chandrashekhar G Joshi
Department of Studies in Biochemistry, PG Centre, Mangalore University, Chikka Aluvara, Thorenur, Kushalnagar Hobli, Somwarpet, Kodagu - 571 232, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pr.pr_137_17

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   Abstract 


Objective: The aim of the present study was to test the antioxidant and cytotoxic and DNA protection activities of ethyl acetate extracts of endophytic fungi isolated from Tragia involucrata Linn. (Euphorbiaceae). Materials and Methods: The 1, 1-diphenyl-2-picrylhydrazyl scavenging, reducing power, and total antioxidant assay were used to evaluate the antioxidant activity. Cytotoxic activity of endophytic fungal extracts against MCF-7 and MOLT-4 cell lines was carried out using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide method. Results: We have successfully isolated four endophytic fungi from T. involucrata, namely Penicillium citrinum CGJ-C1 (GenBank accession No. KT780618), P. citrinum CGJ-C2 (GenBank accession No. KP739821), Cladosporium sp. CGJ-D1 (GenBank accession No. KP739822), and Cryptendoxyla hypophloia CGJ-D2 (GenBank accession No. KT780619). The ethyl acetate extract of P. citrinum CGJ-C2 showed the highest antioxidant as well as cytotoxic activity among the four fungal extracts taken for the study. All the extracts showed moderate DNA protection activity. Conclusions: Further studies on the isolation and purification of the lead molecule will help in designing the novel therapy for different ailments associated with free radical generation.
Abbreviations used: MCF-7: Breast Cancer Cell Line; MOLT-4: Human leukemia cell line; DPPH: 1, 1-diphenyl-2-picrylhydrazyl; MTT:3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; DMSO: Dimethyl sulfoxide; H2O2: Hydrogen peroxide; NCI: National Cancer Institute.

Keywords: Antioxidants, cytotoxicity, endophytes, Tragia involucrata


How to cite this article:
Danagoudar A, Joshi CG, Ravi SK, Rohit Kumar HG, Ramesh BN. Antioxidant and cytotoxic potential of endophytic fungi isolated from medicinal plant Tragia involucrata L. Phcog Res 2018;10:188-94

How to cite this URL:
Danagoudar A, Joshi CG, Ravi SK, Rohit Kumar HG, Ramesh BN. Antioxidant and cytotoxic potential of endophytic fungi isolated from medicinal plant Tragia involucrata L. Phcog Res [serial online] 2018 [cited 2020 Feb 25];10:188-94. Available from: http://www.phcogres.com/text.asp?2018/10/2/188/230751

Summary

Totally four endophytic fungi (Penicillium citrinum CGJ-C1, P. citrinum CGJ-C2, Cladosporium sp. CGJ-D1, and Cryptendoxyla hypophloia CGJ-D2) were isolated from the medicinal plant T. involucrata. High amount of phenolic (112.00 ± 4.12 mg/g GAE) and flavonoid content (40.00 ± 1.14 mg/g quercetin equivalent/g) was found in ethyl acetate extract of P. citrinum CGJ-C2 and C. hypophloia CGJ-D2 respectively. Free radical scavenging ability was measured using DPPH method and a significant anti-radical activity was observed in C. hypophloia CGJ-D2 (260.00 ± 1.45 mg/g) extract. The endophytic fungal extracts were subjected to cytotoxic activity against MCF-7 as well as MOLT-4 cell lines using MTT assay. P. citrinum CGJ-C2 showed a potential cytotoxicity against cancer cells used in this study. The fungal extracts also exhibited DNA protection capability at higher concentration. We are for the first time reporting on the isolation and biological study of endophytic fungi isolated from T. involucrata. Further studies are going on in our laboratory to isolate the novel anticancer lead/s from these fungi.




   Introduction Top


Endophytic fungi are microscopic organisms that grow within plant tissue without causing immediate symptoms of disease.[1] Natural products from fungal endophytes have vast applications in medicine as they exhibit diverse therapeutic properties.[2],[3] Many antitumor agents such as taxol and beauvericin are extracted from the endophytic fungi such as Pestalotiopsis microspore and Fusarium oxysporum on Taxus wallachiana and Ephedra fasciculate plants, respectively.[4],[5] Even though the endophytes are studied for various applications, still many are unexplored with respect to their therapeutic potential. According to Strobel and Daisy, each plant identified on earth hosts an endophyte.[6] Endophytes acquire the medicinal properties of the host plant in which they exist.[7] Hence, they are the reservoirs for various novel molecules with therapeutic efficacy.

Tragia involucrata belongs to Euphorbiaceae family and widely distributed in Asian subcontinent. This plant has been reported to contain several medicinal properties such as cytotoxic, antitumor, anti-fertility,[8],[9],[10] antimicrobial,[11] antidiabetic,[12] wound healing,[11] and anti-inflammatory activities.[13] Even though T. involucrata has been extensively studied for its medicinal activity, there are no reports on the therapeutic potential of endophytes of T. involucrata. To the best of our knowledge, no one has isolated and studied the medicinal properties of endophytes of T. involucrata. Hence, the aim of the present study was to isolate endophytic fungi from T. involucrata and to study their antioxidant, cytotoxic, and DNA protection potential.


   Materials and Methods Top


Collection of plant material

Healthy stems of T. involucrata were collected in the month of May 2013 in and around Sullia (T), Dakshina Kannada (D), Karnataka, India. The Global Positioning System locations were 12.5581° N and 75.3892° E. The plant was shifted to laboratory using sterile polyethylene bags at 8°C. The plant was identified by Dr. Jagath Timmaiah, Department of Botany, Karnataka, India. The plant was authenticated by CSIR-National Institute of Science Communication and Information Resources, New Delhi. Herbarium was peevishly deposited by one of the authors to Gandhi Krishi Vignana Kendra, Bengaluru, which has been taken as a reference (Herbarium No. 3687).

Isolation and identification of endophytic fungi from Tragia involucrata

T. involucrata was washed with running tap water to remove excess debris followed by washing with distilled water. The samples were cut into small pieces (0.6–0.8 cm) and rinsed with 70% alcohol. The alcohol-washed pieces were again rinsed with 0.5% sodium hypochlorite followed by washing thrice with sterile distilled water. The sterilized samples were transferred aseptically to potato dextrose agar media containing chloramphenicol (150 mg/L) and incubated at (28.00°C ± 1°C) for 7 days.[14]

The genomic DNA was isolated and purified using fungal genomic DNA mini kit using the manufacturer's protocol (xcelgenCat No: XG2416-01 from Xceleries genomics Pvt. Ltd, India), and molecular identification was achieved by internal transcribed spacer (ITS) region and 18S rDNA analysis. The sequences were used as query sequences to search for similar sequences from GenBank using the basic local alignment search tool program. The most similar reference sequences with query sequences were obtained and used for subsequent phylogenetic analyses [Figure 1]. These sequences were aligned using the CLUSTALX program.[15]
Figure 1: Neighbor-joining phylogenetic tree analysis of fungal endophyte sp. (a) Penicillium citrinum CGJ-C1, (b) Penicillium citrinum CGJ-C2, (c) Cladosporium sp. CGJ-D1, and (d) Cryptendoxyla hypophloia CGJ-D2. Confidence values above 50% obtained from a 1000-replicate bootstrap analysis are shown at the branch nodes. Bootstrap values from neighbor-joining method were determined

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Preparation of ethyl acetate extracts of endophytic fungi

Extraction of secondary metabolites was carried according to the procedure explained by Higginbotham et al. with slight modification. Each liquid culture with evident growth (incubation for 18 days in static condition at 25°C–28°C) on potato dextrose broth was mixed with an equal volume of ethyl acetate (100%). The mixture was blended using pestle and mortar. The resulting homogenate was filtered with Whatman ® No. 1 filter paper and extracted twice with an equal volume of ethyl acetate. The aqueous and organic layers were dried and stored at −20°C until use.[14]

Total phenolic content

The total phenolic content of the fungal extract was estimated using Folin–Ciocalteu (FC) reagent. Different aliquots (0–1 mL) of gallic acid (Sigma-Aldrich, USA) and extracts were taken in tubes and the volume was made up to 1 mL with distilled water. One mL of (1:1 diluted) FC reagent and 1.5 mL of 20% sodium carbonate were added to each tube. After the addition of 6 mL of distilled water, the reaction mixture was incubated at room temperature for 30 min. The absorbance of the sample and tests was measured at 760 nm against blank in ultraviolet-visible (UV-Vis) spectrophotometer (Optima Tokyo, Japan). The total phenolics in fungal extract was calculated using standard curve and expressed as gallic acid equivalents.[16]

Total flavonoid content

Total flavonoid content of the fungal extract was estimated by the protocol published by Zhishen et al.[17] Different aliquots of endophytic extracts and quercetin (100 μg/ml) were taken in different tubes, and the volume in each tube was made up to 1 mL with distilled water. 1.5 mL of ethanol, 0.1 mL of 10% aluminum chloride, and 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water were added to each tube and the absorbance of the reaction mixture was measured at 450 nm in UV-Vis spectrophotometer (Optima Tokyo, Japan).

Total antioxidant assay

The total antioxidant assay was carried out using molybdenum reagent. Phosphomolybdate (3 mL) was added into tubes containing either ascorbic acid or extract and incubated at 95°C in boiling water bath for 90 min after shaking vigorously for 15 min at room temperature. The optical density was measured at 695 nm using UV-Vis spectrophotometer (Optima Tokyo, Japan). Total antioxidant potential of endophytic fungal extracts was expressed as ascorbic acid equivalents.[18]

1, 1-diphenyl-2-picrylhydrazyl radical scavenging activity

The free radical scavenging activity of the fungal extracts was studied by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) method based on the procedure of Brand-Williams et al.[19] with modifications. Standard ascorbic acid and extracts (100 μg/mL) were taken in different aliquots (0–1 mL) and volume was made up to 1 mL using methanol. Three mL of 0.1 M DPPH (Sigma, USA) was added and tubes were incubated in dark for 30 min. The absorbance was measured at 517 nm using UV-Vis spectrophotometer (Optima Tokyo, Japan). DPPH scavenging activity was calculated by using the following equation,

DPPH scavenging (%) = (AControl-AExtract/AControl) × 100 where A denotes absorbance.

Cytotoxic activity

Assessment of cytotoxicity by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide assay

Cytotoxic effect of the endophytic fungal extracts was determined by the procedure explained by Kuriakose et al.[20] Cells were grown on 96-well flat-bottomed microtiter plates at a final volume of 100 μL culture medium per well and incubated at 37°C and 5% CO2 for 24 h, 48 h, and 72 h. Supernatant was removed after incubation and the cells were washed. Fungal extracts (25–200 μg/mL for MCF-7 and 10–100 μ g/mL for MOLT-4) were added into the respective labeled wells. After 24 h, 48 h, and 72 h incubation, 10 μL (5 mg/mL) 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) solution in phosphate-buffered saline was added to each well and incubated for 2 h in dark. Thereafter, medium was removed and 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. Plates were read at 570 nm using a microplate reader (Bio-Rad, USA) and results were expressed as viability percentage against the untreated control cells (100% of viability).

DNA protection ability

Oxidative λ-DNA damage was prevented by endophytic fungal extract and it was assayed.[21] λ-DNA (0.5 μg) with and without endophytic fungal extract (200 μg) was incubated with Fenton's reagent (1 mM FeSO4, 25 mM H2O2 in Tris buffer 10 mM, pH 7.4) at a final reaction volume of 30 μL for 1 h at 37°C. Relative difference between oxidized and native DNA was analyzed on 1% agarose gel prepared in tris-acetate-ethylenediaminetetraacetic acid buffer (pH 8.5) at 50 V for 3 h at room temperature. The gel was documented (Uvitec Company, software platinum 1D, UK) and the band intensity was determined.

High-pressure liquid chromatography analysis of endophytic fungal extracts

Extract samples were filtered with 0.45 μ membrane filter prior to high-pressure liquid chromatography (HPLC) analysis. The system was equipped with an auto-injector and photodiode array detector (Waters Milford USA). Analysis was performed on a Grace Smart reverse-phase chromatography − 18 column (5 μm, 250 mm × 4.6 mm), at a flow rate of 1 mL/min, using an injection volume of 20 μL, detection at 280 nm. The mobile phases employed were A (methanol 100%) and B (0.5% acetic acid). The gradient program followed was 0–28.6 min, 90%–40% B and 28.6–30 min, 40%–90% B, equilibrated till 35 min.

Statistical analysis

All the experiments were performed in triplicates and the data were compared with one-way analysis of variance using Graphpad prism software version 5.0 (GraphPad Software 7825 Fay Avenue, Suite 230 La Jolla, CA 92037 USA). A significant difference (P< 0.05) among the equation was evaluated by Turkey's test.


   Results Top


Isolation and identification of endophytic fungi from Tragia involucrata

Four endophytic fungi were isolated from T. involucrata. The identity of the endophytes was established by comparing the 18S rDNA sequence. These endophytic fungi were identified as Penicillium citrinum CGJ-C1 (GenBank accession number- KT780618), P. citrinum CGJ-C2 (GenBank accession number KP739821), Cladosporium sp. CGJ-D1 (GenBank accession number KP739822), and Cryptendoxyla hypophloia CGJ-D2 (GenBank accession numberKT780619) as shown in [Figure 1].

Determination of total phenolics

Total phenolic content in the fungal extracts was measured by Folin–Ciocalteu's method. The total polyphenol and flavonoid content of each extract is shown in [Figure 2]a and [Figure 2]b. P. citrinum CGJ-C2 extracts showed the highest amount of phenolic content (112.00 ± 4.12 mg/g GAE) followed by C. hypophloia CGJ-D2, P. citrinum CGJ-C1, and Cladosporium sp. CGJ-D1 extracts [Figure 3]a.
Figure 2: Cytotoxic activity of fungal extracts on MCF-7 cell line. (a) Penicillium citrinum CGJ-C1, (b) Penicillium citrinum CGJ-C2, (c) Cladosporium sp. CGJ-D1, and (d) Cryptendoxyla hypophloia CGJ-D2 in 48 h and 72 h. (*P = 0.05; **P = 0.01; ***P = 0.001)

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Figure 3: (a) Total phenolic content, (b) Total flavonoid content, (c) Total antioxidant of endophytic fungal extract, (d) 1, 1-diphenyl-2-picrylhydrazyl radical scavenging activity of the fungal extracts. All values are reported as means ± standard deviation (n = 3). * EF1 (Penicillium citrinum CGJ-C1 extract), EF2 (Penicillium citrinum CGJ-C2), EF3 (Cladosporium sp. CGJ-D1), and EF4 (Cryptendoxyla hypophloia CGJ-D2). All the experiments were performed in triplicates and the data were compared with one way analysis of variance using GraphPad prism software version 5.0. Different alphabets above the bars have mean values that are significantly different (P < 0.05).

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Flavonoid content

The total flavonoid content of four endophytic extracts was depicted in [Figure 3]b. Flavonoid content varied in different extracts. Among the four extracts analyzed, the highest amount of flavonoids was found in C. hypophloia CGJ-D2 extract (40.00 ± 1.14 mg/g quercetin equivalent/g) compared to the other three extracts.

Total antioxidant activity

The total antioxidant activity of the endophytic extracts was measured spectrophotometrically by phospho-molybdenum method. The principle involved in this method is the reduction of molybdenum (Mo) (IV) to Mo (V) by the extracts, and the final green-colored Mo (V) end product is measured as absorption at 695 nm.[22] In our study, significant antioxidant activity was observed in C. hypophloia CGJ-D2 (260.00 ± 1.45 mg/g) extract. The three other extracts also showed considerable total antioxidant activity [Figure 3]c.

1, 1-diphenyl-2-picrylhydrazyl radical scavenging activity

The free radical scavenging ability of ethyl acetate extracts of endophytic fungi was evaluated using DPPH substrate. DPPH is a stable free radical with characteristic absorption at 517 nm, and antioxidants react with DPPH and convert it to 2,2-diphenyl-1-picrylhydrazine. DPPH-scavenging activities of all the extracts were comparable to standard ascorbic acid. C. hypophloia CGJ-D2 extract showed 50% radical scavenging activity at 4.01 μg/mL [Figure 3]d and [Supplementary Table 1 [Additional file 1]].

Cytotoxic activity

Cytotoxic effect of the endophytic extract on MCF-7 and MOLT-4 cell line was assessed using MTT assay. The cytotoxic activity of the endophytic extracts is depicted in [Figure 2] and [Figure 4]. P. citrinum CGJ-C2 extract exhibited a significant cytotoxic effect on MCF-7 cell line in 48 h and 72 h compared to other extracts. The cytotoxic activities of all the four extracts were found to be dose dependent. In our study, P. citrinum CGJ-C2 extract showed significant cytotoxicity against MCF-7 as well as MOLT-4 cell lines. The IC50 value of the P. citrinum CGJ-C1, P. citrinum CGJ-C2, Cladosporium sp. CGJ-D1, and C. hypophloia CGJ-D2 on MCF-7 cells was 1.50, 1.09, 3.87, and 2.90 μg/mL, respectively, at 72 h of incubation after the treatment of extract. The IC50 value of the P. citrinum CGJ-C1, P. citrinum CGJ-C2, Cladosporium sp. CGJ-D1, and C. hypophloia CGJ-D2 on MOLT-4 cells was 2.97, 2.38, 8866, and 5.41 μg/mL, respectively [Supplementary Table 2 [Additional file 2]].
Figure 4: Cytotoxic activity of fungal extracts on MOLT-4 cell line. (a) Penicillium citrinum CGJ-C1, (b) Penicillium citrinum CGJ-C2, (c) Cladosporium sp. CGJ-D1, and (d) Cryptendoxyla hypophloia CGJ-D2 in 24 h, 48 h, and 72 h.(*P = 0.05; **P = 0.01)

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DNA protection assay

Incubation of radicals with λ-DNA for 1 h resulted in the total disappearance of DNA bands on 1% agarose gel compared to DNA control and DMSO vehicle (Lanes 1, 2, and 7). However, the addition of 200 μg of endophytic extracts to the mixture of λ-DNA and Fenton's reagent prevented the radical-induced DNA damages (Lanes 3–6) as shown in [Figure 5].
Figure 5: DNA protection by four isolated endophytic fungal extracts. Lane 1: λ DNA (0.5 μg), Lane 2: λ DNA (0.5 μg) + Fenton's reagent (10 μl), Lane 3: λ DNA (0.5 μg) + EF1 (200 μg) + Fenton's reagent (10 μl), Lane 4: λ DNA (0.5 μg) + EF2 (200 μg) + Fenton's reagent (10 μl), Lane 5: λ DNA (0.5 μg) + EF3 (200 μg) + Fenton's reagent (10 μl), Lane 6: λ DNA (0.5 μg) + EF4 (200 μg) + Fenton's reagent (10 μl), Lane 7: λ DNA (0.5 μg) + dimethyl sulfoxide. Fenton's reagent (30 mM H2O2, 50 mM ascorbic acid, and 80 mM FeCl3). * EF1 (Penicillium citrinum CGJ-C1 extract), EF2 (Penicillium citrinum CGJ-C2), EF3 (Cladosporium sp. CGJ-D1), EF4 (Cryptendoxyla hypophloia CGJ-D2)

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High-pressure liquid chromatography analysis

The polyphenols present in the fungal extracts were identified by using reverse-phase C-18 column in HPLC. Different peaks with respect to retention time confirmed the presence of polyphenols in endophytic fungal extracts [Figure 6]. P. citrinum CGJ-C1 showed the presence of vanillin, caffeic acid, ferulic acid, quercetin, and coumaric acid. Each extract was quantitated for the presence of phytochemicals, and the quantity of individual chemical was varying with the species. Caffeic acid was found to be a major constituent in P. citrinum CGJ-C1 (15.22 ± 0.12 μg/mg), while quercetin (6.904 ± 0.44 μg/mg) and vanillin (1.70 ± 0.84 μg/mg) were present in abundant quantity in P. citrinum CGJ-C2 and Cladosporium sp. CGJ-D1 extracts, respectively, when compared to other extracts. The chemicals present in the C. hypophloia CGJ-D2 did not match any standards that were used for this study [Table 1].
Figure 6: High-pressure liquid chromatography chromatograms of ethyl acetate extract of (a) Penicillium citrinum CGJ-C1, (b) Penicillium citrinum CGJ-C2, (c) Cladosporium sp. CGJ-D1, (d) Cryptendoxyla hypophloia CGJ-D2, detected at 280 nm

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Table 1: Polyphenolic profiles of endophytic fungal extracts

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   Discussion Top


The endophytes present in the plant produce similar secondary metabolites as that of the host plant. These secondary metabolites even exhibit the medicinal properties of the plant in which they exist. Endophytes isolated from medicinal plants have many advantages over the plant as they can be easily grown and harvested in laboratories. Even the process involved in the isolation of secondary metabolite is less complicated than medicinal plants.[7] This information prompted us to isolate the endophytes from T. involucrate and to test the antioxidant and cytotoxic activities as well as DNA protection potential

We have successfully isolated identified four endophytic fungi. Out of the four endophytic fungi, two were different strains of the same species P. citrinum CGJ-C1 (KT780618) and P. citrinum CGJ-C2 (KP739821). Organisms with 99% or more sequence similarity to be considered from the same species. If the sequence similarity between the organisms ranges from 93% and 98% ; the organisms to be considered from the same genus. However, the organism is considered to be previously unidentified if the ITS Sequence similarity is below 93%.[23] The endophyte had 99% similarity to P. citrinum CGJ-C1, P. citrinum CGJ-C2, Cladosporium sp. CGJ-D1, and C. hypophloia CGJ-D2.

According to Cui et al. (2011), medicinal properties of the endophytic fungi vary even though they belong to the same genus and isolated from the single host species.[24] Our study supported the observation of Cui et al. (2011) regarding the different bioactivity in different strains of the same species of endophytes (P. citrinum CGJ-C1 [KT780618] and P. citrinum CGJ-C2 [KP739821] showed different bioactivity).[24]

Reactive oxygen species (ROS) such as O2, H2O2, and OH− are generated as a result of metabolic process in living organisms. These ROS are involved in various pathophysiologies such as aging, cancer, and neurodegenerative processes. However, they are essential for certain vital functions in the body such as stress-response signaling and programmed cell death. ROS and antioxidants are the key mediators of symbiotic interactions between the host and microorganisms as they are ubiquitous and evolutionarily conserved.[25],[26] The microorganism may produce the antioxidant molecules to protect the host plant from different harmful ROS.[27],[28]

DPPH free radical scavenging activity is considered to be one of the basic, accurate, and widely used methods to find out the antioxidant potential of various natural products.[19] Electron donation capacity and free radical scavenging potential of a substance determine its reducing power. These properties of antioxidant compounds help them to prevent lipid peroxidation of the membrane as a consequence of a series of reactions of free radicals.[29],[30] Some of the previous studies have reported about the linear relationship between total phenolic content and antioxidant activity.[31] However, some researchers have shown that there is no correlation between the phenolic content and antioxidant activity.[32],[33] Our study supports the view of a nonlinear relationship between the phenolic content and antioxidant activity. This trend may be attributed to the presence of other phytochemicals along with the phenolics that could act as radical scavengers. Similar results were reported by Wang et al.[32] and Devi et al.[33]

Assessment of cytotoxicity of natural products is the first step in the development of anticancer compounds.[33] In our study, P. citrinum CGJ-C2 showed significant cytotoxicity against MCF-7 as well as MOLT-4 cell lines. All the endophytic extracts except Cladosporium sp. CGJ-D1 showed IC50 value much lesser than the National Cancer Institute (NCI) guideline values against P. citrinum CGJ-C2 in MCF-7 cell lines. According to the NCI guidelines, any crude extract showing IC50 value below 30 μg/mL can be considered as a promising anticancer substance.[34] Interestingly, the earlier work of this group have demonstrated the cytotoxic activity of the methanol extract of T. involucrata against KB (subline of the ubiquitous KERATIN-forming HeLa tumor cell line) and MCF-7 cell line, which was well below the NCI guidelines.[8] This finding supports the hypothesis of Zhao et al.[7] who proposed that the endophytes share the medicinal properties of a host plant. Our present study will be helpful in finding a new source for anticancer compounds from endophytes. Many studies on endophytes have shown their cytotoxic potential against various cell lines.[5],[35] However the IC50 value of those endophytic extracts are not within the NCI-guided limits.

Ruma et al. reported the PBR-322 plasmid protection by the organic extracts of endophytes (isolated from Garcinia sp.).[36] 50 μg/mL was able to protect the plasmid DNA damage by free radicals. However, in the present study, much higher amount of the extract [Figure 5] was needed to protect the λ DNA from Fenton-induced OH− radicals. Polyphenolic compounds are known to have free radical scavenging activity.[35],[37] Hence, the radical scavenging activity of different fungal extracts in this study may be contributed by the phenolic compounds. Even though all the extracts showed significant cytotoxic activity against MCF-7 and MOLT-4 cell lines, extracts also prevented the λ DNA damages at higher concentrations. This contradictory trend can be attributed to the presence of various phytochemicals (as we have taken the crude extract) present in the endophytic extracts (as confirmed by HPLC analysis). As these extracts contain several phytochemicals that are responsible for radical scavenging activity; the prevention of Fenton radical generation might have prevented the DNA damage in fungal extract-treated group. Our observation is supported by the finding of Siddaraju and Dharmesh.[37]

Financial support and sponsorship

This work is funded by the University Grant Commission (UGC), New Delhi, Government of India.

Conflicts of interest

There are no conflicts of interest.



 
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