Silymarin, a polyphenolic flavonoid impede Plasmodium falciparum growth through interaction with heme

A polyphenolic flavonoid, Silymarin isolated from Silybum marianum is widely known for its hepatoprotective action. In the present study anti-plasmodial activity of Silymarin has been demonstrated for the first time having IC 50 of 14±0.33 μM against the NF-54 strain of P. falciparum with high selectivity index (>100). The parasitostatic action is exerted through inhibition of β-hematin/hemozoin formation which is due to the interaction ( Kd =3.63±0.9µM) of silymarin with free heme in a Stoichiometry of 1:1 silymarin: heme complex resulting into heme-induced membrane damage in the parasite. Silymarin could hinder the glutathione and hydrogen peroxide-induced heme detoxification. Silymarin also induces apoptosis in the parasite through the elevation of caspase-3 level in a dose-dependent manner. Results from the docking studies suggest that silymarin interacts with heme.

described previously by Trager and Jensen 1976. Culture was maintained in a standard gas mixture consisting of 5% CO2. The medium was changed after every 24 h and monitored through Geimsa staining. The culture was synchronized using 5% D-sorbitol treatment to obtain ring-stage parasites (Lambros and Vanderberg 1979).

In vitro antiplasmodial assay
Silymarin was dissolved in DMSO and further diluted with culture medium to achieve the required concentrations. Precautions was taken that the final concentration of DMSO does not exceed 1%. Parasite growth was determined spectrophotometrically in control and treated cultures using a parasite lactate dehydrogenase assay (pLDH) as described previously with minor modifications (Makler et al. 1993). A synchronous ring stage culture with 1% parasitemia and 1% hematocrit was incubated in 96-well tissue culture plate with different concentrations of silymarin (1, 10, 50 and 100 μg/ml) at 37°C for 72 h. Chloroquine and artemisinin were used as positive control. After incubation, plates were subjected to three 20 minute freeze-thaw cycles to release cell content. Parasite culture was carefully mixed and aliquots of 20 μl were added to another 96-well plate containing 25 μl of NBT-PES (1.9 μM NBT and 0.24 μM PES) and 100 μl of Malstat reagent (0.125% Triton X-100, 130 mM Llactic acid, 30 mM Tris buffer and 0.62 μM APAD) solution per well. The plate was incubated in dark for 30 minute and absorbance was recorded at 650 nm using a microplate reader (FLUOStar Omega, BMG Labtech). The antiplasmodial activity of silymarin was expressed as IC 50 (mean ± SEM), calculated from dose-response curve data by nonlinear regression analysis. The percent growth inhibition at different concentration was calculated using the following formula: Growth inhibition (%) = [absorbance of control -absorbance of test] / [absorbance of control] × 100.

Drug-haematin-induced red blood cell lysis
Experiments that evaluated the lysis of human red blood cells by haematin and drug-haematin complexes were conducted by incubating 0.03% (vol/vol) cell suspensions in phosphatebuffered saline (pH 7.4) at 37°C for 1 h with various concentrations of haematin in the absence or presence of silymarin and measuring the decrease in absorbance at 700 nm.

Determination of selectivity ratio
Selectivity index (SI) is used as a parameter of clinical significance. Generally, selectivity index > 2.0 is considered as safe for natural products. SI of silymarin was calculated from the following expression as described previously (Omoregie and Sisodia 2012): SI = IC 50 of against Vero cells ⁄ IC 50 against P. falciparum

In vitro β-haematin formation inhibition assay and Measurement of hemozoin content in parasite lysate
In order to determine the mode of action of silymarin, its ability to inhibit β-hematin (or hemozoin) formation by the ferriprotoporphyrin IX (FP) biomineralization was studied (Ncokazi and Egan 2005). Briefly, the assay was performed in a 96-well plate, each well containing a mixture of 50 μl silymarin solution of different concentrations, 50 μl of 0.5 mg/ml hemin chloride freshly dissolved in DMSO, and 100 μl of 0.5 M sodium acetate buffer (pH 4.4). After incubation at 37 °C for 12 h, the plate was centrifuged at 550 X g for 8 minute . The supernatant was discarded by vigorously flipping the plate upside down and pellet was washed twice with 200 μl DMSO. Then the pellet was dissolved in 200 μl of 0.1 M NaOH solution, and absorbance was measured at 405 nm using a micro plate reader (FLUOStar Omega, BMG Labtech). The results were expressed as percent of inhibition compared to the negative control (DMSO). The positive control was chloroquine (in water).
All the experiments were performed in triplicate.
P. falciparum culture of 10 % parasitemia was treated with different concentrations of silymarin, after incubation at 37°C for 36 h parasite was pellet down by centrifugation at 2,000 rpm and lysed by using 0.08% saponin (1X PBS). After centrifugation at 10,000 rpm, supernatant was discarded and pellet was washed thrice with an equal volume of 2.5% SDS buffered with 25 mM Tris-HCl (pH 7.4). The pellet was hydrolyzed to monomeric heme in 1N NaOH and measured spectrophotometrically using methods described earlier (Huy et al. 2002). β-haematin /hemozoin formation inhibition calculated as : Heme content in control -heme content in sample Heme content in control

Inhibition of hydrogen peroxide-mediated hemin degradation
The monitoring of peroxidative decomposition of hemin was adapted from (Loria et al. 1999

Inhibition of glutathione-mediated hemin degradation
Inhibition of glutathione-mediated hemin degradation was performed according to method

Silymarin-haematin induced red blood cell lysis
The effect of silymarin-haematin induced hemolysis was evaluated by following the method described earlier (Huy et al. 2007). Briefly, 100 µl of heme (0 to 20 µM) was incubated with or without silymarin (10 µM) for 10 Then 50 µl of erythrocyte suspension in PBS was added and incubated 2 h at 37°C and centrifuged at 2000 rpm for 2 minute, 150 μl of supernatant transferred to 96-well plate and absorbance was measured at 630 nm. The absorbance of the control well, which contained only erythrocytes, was considered to be 0% hemolysis and was used to convert the remaining absorbance values to the percentage hemolysis.

Cytotoxicity and Hemocompatibility
Cytotoxicity was performed using Vero cell line (VERO C1008; ATCC CRL-1586) which was cultured in RPMI-1640 medium supplemented with 0.2% NaHCO3, 10% FBS, and 1× antibiotic-antimycotic solution at 37°C and 5% CO2. The cells were seeded in flat bottom 96-well cell culture plate and allowed to grow for 24 h. Cells were treated with different concentrations (1 to 100 µg/ml) of silymarin and incubated for 48 h. Cytotoxicity was assessed in terms of cell viability using MTT assay as described earlier (Woerdenbag et al. 1993).Experimentally After incubation 10 μl of MTT (5 mg/ml in PBS) solution was added to each well, gently mixed and incubated for another 4 h at 37 o C. After incubation culture medium was removed and 100 μl of DMSO was added to each well and mixed gently. The absorbance of control and treated wells was recorded using a microplate reader (FLUOStar Omega, BMG Labteck) at 570 nm. Cytotoxicity of test compounds was expressed as IC 50s (mean ± SEM), calculated from dose-response curve data by nonlinear regression analysis.
Hemolysis was performed as described (Huy et al. 2007). Briefly Fresh blood from healthy donors was collected in heparinized tube and 250 µl of 10% (v/v) RBCs suspension was incubated under agitation at room temperature for 2 h containing 1 to 25 µg/ml concentration of Silymarin . Triton X-100 1% (v/v) was used as a positive control (100% lysis) and PBS (pH 7.4) as a negative control (0% lysis). The mixtures were then centrifuged at room temperature for 5 min at 8,000 × g and the absorbance of the supernatants was measured at 550 nm with a microplate reader (FLUOStar Omega, BMG LabTech). The red blood cell lysis percentage was determined as follows: Percent RBCs lysis = (OD 550nm sample − OD 550nm PBS ) / (OD 550nm Triton X-100 1% − OD 550nm PBS ).

Statistical analysis
One-way analysis of variance (ANOVA) was used. Dunnett's test was used to compare the treatment and control. P < 0.05 is considered statistically significant.