Table 1.
Different media used for the optimization of fungal VCR and VBL production.
Fig 1.
Isolation of endophytic fungi from C. roseus plant.
Arrowheads indicate the emergence of endophytic fungi from the plant cuttings. A-leaf, B-stem, C-pedicel and D-flower petal.
Fig 2.
PCR products of approximately 550 to 600 bp of ITS region in endophytic fungi of C. roseus.
Lane M: 100-bp DNA marker. Lanes 1 to 22: PCR products of CrP1 to CrP22.
Table 2.
Details of the endophytic fungi isolated from C. roseus and the antiproliferative effects (IC50 values) of their crude extracts against human HeLa cells.
Fig 3.
Effects of the ethyl acetate extracts of mycelium on the antiproliferative activity of HeLa cells.
Fig 4.
Effects of the ethyl acetate extracts of filtrate on the antiproliferative activity of HeLa cells.
Fig 5.
Genomic PCR analyses to determine the presence of the TDC gene.
Lane M: 100-bp ladder; Lanes 1–22: the PCR amplification products of the TDC gene in 22 endophytic fungal isolates. Arrowheads show the amplified products in T. radicus—CrP20 and C. roseus (which served as the positive control).
Fig 6.
Macromorphology of Ten-day-old TIA-producing fungus T. radicus—CrP20 on PDA medium, showing mycelial growth and green-colored spores.
Fig 7.
Micromorphology of T. radicus—CrP20, showing phialides with chains of conidia under light microscope (40X magnification).
Fig 8.
Thin-layer chromatography analysis of T. radicus—CrP20 crude extract on a silica gel aluminum sheet.
Fig 9.
LC-ESI-MS analysis. of fungal VCR.
The mass spectrum of the fungal extract showed a (M+H+) peak at a molecular mass of 825.46, which was identical to that observed in the mass spectrum of the VCR standard.
Fig 10.
LC-ESI-MS analysis of fungal VBL.
The mass spectrum of the fungal extract showed a (M+H+) peak at a molecular mass of 811.51, which was identical to that observed in the mass spectrum of the VBL standard.
Fig 11.
HPLC profile of T. radicus—CrP20 culture extract showing VCR (RT-11.43) and VBL (RT-12.83).
Fig 12.
Production of VBL and VCR by T. radicus—CrP20 grown in different media.
Fig 13.
Cytotoxic activity of ‘fungal VCR’ in different cancer cell lines.
Fig 14.
Cell cycle distribution of HeLa cells treated with different concentrations of ‘fungal VCR’.
The sub-G0/G1, G1, S and G2/M phases are represented on the histogram as P5, P2, P4 and P3, respectively. A—control, B—fungal VCR (5 μg/ml), C—fungal VCR (10 μg/ml), D—fungal VCR (25 μg/ml) and E—percent apoptosis.
Fig 15.
Induction of apoptosis in HeLa cells treated with different concentrations of ‘fungal VCR’, as determined by annexin V-FITC/PI dual staining.
A- untreated cells, B—cells + FITC, C—cells + PI, D—cells + FITC + PI, E—cells + FITC + PI + fungal VCR (5 μg/ml), F—cells + FITC + PI + fungal VCR (10 μg/ml), G—cells + FITC + PI + fungal VCR (25 μg/ml) and H—percentage of cells undergoing early apoptosis.
Fig 16.
Induction of mitochondrial membrane depolarization in HeLa cells treated with various concentrations of ‘fungal VCR’.
A—cells alone, B—cells + JC1 stain, C—cells + JC1 + 25 μM valinomycin (+ ve control), D—cells + JC1 + fungal VCR (5 μg/ml), E—cells + JC1 + fungal VCR (10 μg/ml), F—cells + JC1 + fungal VCR (25 μg/ml), G—standard VCR (2 nM) and H—percent loss of mitochondrial membrane depolarization.
Fig 17.
DNA fragmentation in HeLa cells treated with ‘fungal VCR’.
A—control, B—cells treated with fungal VCR (10 μg/ml), C—cells treated with fungal VCR (25 μg/ml) and D—1-kb DNA ladder.