Diversity and biological activities of endophytic fungi associated with Catharanthus roseus

Abstract

Background: The present study involves diversity and bioactivity of the endophytic fungal community from Catharanthus roseus inhabiting the coastal region. This study has been conducted hypothesizing that the microbial communities in the coastal regions would tolerate a range of abiotic stress such as salinity, humidity, temperature and soil composition, and it may produce new metabolites, which may possess bioactive property. Therefore in the current study, the cytotoxicity and free radical scavenging potential of the fungal organic extracts have been investigated. Moreover, the apoptotic and the antioxidant potential of the fungus that exhibited the best activity in preliminary screening has also been demonstrated.

Results: Twenty endophytic fungal isolates were obtained from different parts of the plant, and identified using internal transcribed spacer region analysis. Based on the colonization frequency, the dominant genera were found to be Colletotrichum, Alternaria and Chaetomium with colonization frequency % of 8.66, 7.00 and 6.33, respectively. It was observed that the species diversity and richness was the highest in bark followed by leaf and stem regions of the plant. On screening the fungal ethyl acetate extracts for cytotoxicity against the HeLa cells, the Chaetomium nigricolor extract exhibited potent cytotoxic activity of 92.20% at 100 μg mL^{- 1} concentration. Comparison between the different organic extracts (ethyl acetate, chloroform, dichloromethane and hexane) of Chaetomium nigricolor mycelial and culture filtrate, it was observed that the mycelial as well the culture filtrate ethyl acetate extracts and the culture filtrate hexane extract showed significant cytotoxic potential against the HeLa and MCF-7 cells, respectively. The apoptotic- and mitochondrial membrane depolarisation-induction potential of the Chaetomium nigricolor ethyl acetate extract has also been demonstrated in this study. Further the screening of antioxidant potential of the ethyl acetate fungal extracts using DPPH scavenging assay showed that Chaetomium nigricolor extract exhibited potential activity with a significant EC₅₀ value of 22 μg mL^{- 1}. The ethyl acetate extract of Chaetomium nigricolor also exhibited superoxide radical scavenging potential.

Conclusion: These results indicated that diverse endophytic fungal population inhabits Catharanthus roseus. One of the fungal isolate Chaetomium nigricolor exhibited significant cytotoxic, apoptotic and antioxidant potential.

Keywords: Antioxidant potential; Apoptotic activity; Cancer; Catharanthus roseus; Chaetomium nigricolor; Cytotoxic activity; Endophytic fungus.

Fig. 1

Phylogenetic tree of endophytic fungi isolated from C. roseus plant based on ITS region. The phylogenetic tree was constructed using maximum parsimony method. 100% bootstrap value showed each genera were distinguished by monophyletic group in different subclades from outgroup. Nomenclature of the endophytic fungi and the isolate number are indicated

Fig. 2

Morphological observation of the fungus C. nigricolor. a) The colonies after 7 days at 30 °C on PDA plate b) Light microscopy image of the C. nigricolor spore (40 X magnification)

Fig. 3

Effect of EA extract of C. nigricolor on loss of mitochondrial membrane potential (MMP) in HeLa cells. HeLa cells were treated with the indicated concentrations of the EA extract, stained with JC-1 and subsequently analyzed by flow cytometry, numbers in the bottom right gate represents percentage of cells with low MMP. B) Statistical analysis about loss of MMP obtained in flow cytometer.The experiments were repeated three times and the results are presented as mean ± SD

Fig. 4

The effect of C. nigricolor- EA extract treatment on HeLa cells apoptosis determined by annexin V/ PI double staining. HeLa cells were treated with or without the extract for 24 h. After incubation the cells were double stained with Annexin –V FITC and PI, and analyzed by flow cytometry. Dot-plot represents cell distribution as follows: Lower left (PI – ve/ FITC –ve) living cells, lower right (PI –ve/ FITC +ve) apoptotic cells, upper right (PI +ve /FITC +ve) late apoptotic cells, upper left (PI +ve /FITC -ve) dead necrotic cells. B) Bar diagram representing distribution of early and late apoptotic cells. The data are derived from atleast three independent experiments

Fig. 5

Antioxidant activity of C. nigricolor- EA extract a) DPPH scavenging activity b) Nitric oxide scavenging potential c) Hydroxyl radical scavenging activity d) Superoxide scavenging activity. Ascorbic acid at a concentration of 50 μg mL⁻¹ was used as a positive control for all the antioxidant assays. The data are derived from atleast two independent experiments each performed in triplicates (n = 6). P value was calculated by comparing the means ± SD of the % scavening activity observed with adding and without adding the extract to the reaction mixture, using Student’s T test and the statistical significance are presented as follows in the bar graph: ***,P ≤ 0.001; **P ≤ 0.01; ns, P > 0.05