Fig 1.
Flow diagram of Extraction and Isolation of chemical constituents from P. fulgens roots.
Fig 2.
Cells were treated for 24 h with various concentrations of crude extract of P. fulgens root (PRE) and its ethyl acetate, hexane and nbutanol soluble fractions.
After the indicated time of treatment, cells were maintained without test materials for 10 days. At day 10, the grown colonies were counted and percentage of survival fraction was then calculated and shown in (A and B) in MCF-7 cells and (C and D) in U87 cells. Results were expressed as means ± SD for three independent determinations.
Table 1.
Trypan Blue Exclusion Assay in human lymphocytes and in cancer cell lines after treatment with PRE or EA-fraction.
Fig 3.
Analysis of cell cycle (A) The flow cytometry analysis on cell cycle distribution of PI-labeled MCF-7 cells and (B) in U87 cells with and without treatment with PRE, ethyl acetate (EA) and hexane (Hex) soluble fractions of P. fulgens root extract.
Right panel-The percentage of cells at different stages in both MCF7 and U87 cells with and without treatment. The values are the mean±SEM of three independent experiments. *p<0.05, Students’t-test as compared with untreated control.
Fig 4.
Analysis of apoptosis in (A) MCF7 and (B) U87 cells by measuring the mitochondrial membrane potential after JC1 staining in untreated and treated with PRE, EA, Hex fractions and mixture of EC+GA+UA of EA fraction of P. fulgens root.
The upper part indicates the percentage of cells show polarization of mitochondrial membrane and the lower part shows the percentage of cells having mitochondrial membrane depolarization. All these experiments were repeated twice. Right panel shows percentage of depolarized cells after each treatment. *p<0.05, Students’ t-test as compared with untreated control. (C) Effect of PRE, Hexane and ethyl acetate soluble fraction treatment on the DNA degradation and fragmentation in MCF7 and U87 cells. DNA samples are labeled as: (M) molecular weight marker, (1) untreated cells, (2) cells treated with hexane-fraction, (3) cells treated with PRE and (4) cells treated with ethyl acetate fraction. Results are representative of two independent experiments. The concentration was used 100 μg/ml and the treatment was given for 24 h.
Fig 5.
Protein expression analysis of MCF-7 and U87 cells by Western blotting after treatment with PRE, EA, Hex fractions and EC+GA+UA of EA fraction of P. fulgens root extracts in comparison with the untreated samples.
(A) Determination of PARP cleavage. Lower panel shows quantitative densitometric analysis of the level of PARP with and without cleavage in the treated and untreated MCF-7 and U87 cells. The values are the mean±SEM of three independent experiments. Two independent untreated samples were used. The values are normalized to respective β-actin values. Expression pattern of Bcl2, survivin, XIAP and cIAP using semiquantitative RT- PCR in (B) MCF-7 and (C) U87 cells treated with or without PRE, EA, Hex fractions and EC+GA+UA (two independent samples). Right-side panel shows quantitative densitometric analysis of the expression profile of genes mRNA level. The values are the mean±SEM of two independent experiments and are normalized to respective GAPDH values.
Fig 6.
Expression pattern of GCLC in (A) MCF-7 cells and in (C) U87 cells treated with or without PRE, EA, Hex fractions and mixture of EC+GA+UA.
Two independent untreated samples were used in this study. Lower panel shows quantitative densitometric analysis of the expression profile of GCLC mRNA level. The values are the mean±SEM of two independent experiments and are normalized to respective GAPDH values. Levels of glutathione in (B) MCF-7 cells and (D) U87 cells treated with or without PRE, EA, Hex fractions and EC+GA+UA. Two independent untreated samples were used in this study. Values are mean±SEM of three different experiments. *p<0.05, Students’ t-test as compared with untreated control.