Comparison of the Antioxidant and Cytotoxic Activities of Phyllanthus virgatus and Phyllanthus amarus ExtractsPoompachee K. · Chudapongse N.
School of Biology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
Objective: To determine the antioxidant activity and cytotoxicity of Phyllanthus virgatus crude extract compared to Phyllanthus amarus.Methods: Phenolic contents of the hydromethanolic extracts were measured using Folin-Ciocalteu reagent. Antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl hydrate free radical scavenging and antilipid peroxidation assays. Cytotoxicity on human hepatoma HepG2 cells was assessed by trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assays. A stereomicroscope was used to observe and photograph the morphology of the cells. Oxygen consumption of the HepG2 cells was measured using a Clark oxygen electrode. Results: The extract of P. virgatus, which contained more phenolic compounds than P. amarus extract, had higher cytotoxicity and showed higher free radical scavenging activity and more inhibition of peroxidation in a linoleic acid system. P. virgatus extract conspicuously increased the oxygen consumption of HepG2 cells, while P. amarus extract had little stimulatory effect. Conclusion: The hydromethanolic extract of P. virgatus had stronger antioxidant and cytotoxic action than P. amarus extract. The stimulation of HepG2 cell respiration by P. virgatus extract suggests the extract alters mitochondrial function; this action could play a role in the cytotoxicity of this plant.
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Oxidative stress induced by oxygen radicals, causing damage to lipids, proteins and nucleic acid, has been shown to be involved in the initiation and progression of various diseases such as inflammation, liver injury, renal failure, cardiovascular diseases and cancer . Several studies have revealed the potential of medicinal plants for serving as antioxidants against various diseases induced by oxidative radicals [2,3,4]. It is widely known that almost all chemotherapeutic agents currently used produce serious toxicities to several organs. Naturally occurring phenolic compounds of plants have been shown to have antioxidant properties [5,6,7] and to exhibit cytotoxic activity on a variety of cancer cell types, such as Jurkat and human hepatoma HepG2 cells, without toxicity to noncancer cells . Moreover, there is increasing evidence showing the chemopreventive potential of many plant compounds such as green and black tea polyphenols, lycopene, soy isoflavones, curcumin, phenethyl isothiocyanate, indole-3-carbinol and perillyl alcohol [9,10,11].
The genus Phyllanthus consists of several species in the family Euphorbiaceae. Phyllanthus virgatus and another two species, P. amarus and P. urinaria, are closely related in appearance and phytochemical structure . In Thailand, they have the same local name (Look Tai Bai). All of them traditionally have been used for the treatment of many ailments such as gonorrhea, jaundice, diabetes and liver diseases . The anticancer activity of Phyllanthus species has also been documented [14,15,16,17,18,19]. For example, P. amarus inhibits the growth of human adenocarcinoma cell line Caco-2 , hepatoma induced by N-nitrosodiethylamine in rats  and sarcoma induced by 20-methylcholanthrene in mice . Similarly, P. urinaria has also been shown to possess cytotoxic activity against several cancer cell lines including human promyelocytic leukemia , Lewis lung carcinoma cells  and HepG2 cells . However, the pharmacological studies on P. virgatus mostly involved its antimicrobial [20,21] and antihyperglycemic effects . Cytotoxicity of P. virgatus to cancer cells, particularly HepG2 cells, has never been reported. In this survey, we studied and compared the antioxidant and cytotoxic activities of the hydromethanolic extracts of P. virgatus and P. amarus. In addition, the effects of both extracts on HepG2 cell oxygen consumption were also investigated.
Materials and Methods
Plants were collected from June to September 2005 in the Pak Tong Chai and Mueang districts of Nakhon Ratchasima province, Thailand, and were identified by a botanist, Dr. Paul J. Grote, School of Biology, Suranaree University of Technology. Specimens of the plants are archived at the same School of Biology. Whole plants were thoroughly washed with distilled water to remove dirt and contaminants, and then oven-dried at 40°C. Each dried plant (10 g) was extracted twice with 100 ml of 50% methanol in distilled water. The pooled extracts were filtered and concentrated at 40°C using a rotary evaporator under low pressure. The residue was freeze-dried in a lyophilizer and stored at –20°C until used.
The concentration of phenolic compounds in an extract was measured according to the method described previously , using gallic acid as a standard. The reaction mixture consisted of 250 µl of the extract (1 mg/ml) or standard (0.025–0.5 mg/ml gallic acid), and 2.5 ml of 2% Na2CO3 was added with 100 µl of 50% Folin-Ciocalteu reagent. After 30 min of incubation, the absorbance was measured at 750 nm using a spectrophotometer. Results were expressed as milligrams per gram of gallic acid equivalents.
To measure antioxidant activity, the 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) radical scavenging assay was carried out according to the procedure previously described . Briefly, each sample (500 µl; final concentration range: 0–300 µg/ml) was added to 4.0 ml of 50 µM DPPH in methanolic solution, and the final volume was adjusted to 5.0 ml with distilled water. After vortexing, the mixture was incubated for 30 min in the dark at room temperature. The decrease in absorbance at 517 nm was then measured using a spectrophotometer. Antioxidant activity was expressed as half maximal inhibitory concentration (IC50), which was defined as the concentrations of the extracts required to inhibit the formation of DPPH radicals by 50%.
The antilipid peroxidation activity of the extracts was determined using the ferric thiocyanate method, as previously described . Each sample (500 µl; final concentration range: 0–2,000 µg/ml) was mixed with 2.5 ml of linoleic acid emulsion (0.2 M, pH 7.0) and 2.0 ml of phosphate buffer (0.2 M, pH 7.0). The linoleic acid emulsion was prepared by mixing 0.2804 g of linoleic acid with 0.2804 g of Tween 20 and 50 ml of phosphate buffer. The reaction mixture was incubated in the dark at 60°C for 8 h. An aliquot (0.1 ml) of the mixture was diluted with 4.5 ml of 75% ethanol, followed by the addition of 0.2 ml of 30% ammonium thiocyanate and 0.2 ml of 20 mM ferrous chloride in 3.5% HCl. Absorbance of the solution was measured at 500 nm using a spectrophotometer. The percentage of inhibition of lipid peroxidation in linoleic acid emulsion was calculated as follows:
inhibition of lipid peroxidation (%) = (1 – A1/A0) × 100%,
where A0 is the absorbance of the control, while A1 is the absorbance in the presence of the extracts.
HepG2 cells (ATCC HB-8065; Manassas, Va., USA) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 U/ml antibiotic-antimycotic (Gibco, Langley, Okla., USA). The cells were grown at 37°C in a fully humidified atmosphere of air with 5% CO2. The cells were used to determine the cytotoxicity of the extracts by trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays .
Cells (5 × 105 cells/well) were plated in triplicate in a 96-well culture plate overnight. They were treated with various concentrations (ranging from 0 to 1,000 µg/ml) of the extracts for 24 h. Cells were harvested after digestion with 0.25% trypsin-EDTA solution at 37°C for 5 min. The cell suspension was mixed with an equal volume of 0.4% (w/v) trypan blue. The numbers of viable (unstained) and dead (stained) cells were counted by hemacytometer under a light microscope and the results calculated and expressed as the percentage of live cells compared to control.
HepG2 cells (5 × 105 cells/well) were prepared as described above for the trypan blue exclusion assay and treated with each extract for 24 h. MTT solution (0.5 mg/ml) was added to each well and incubated for a further 4 h. The medium was removed and 100 µl of DMSO (99.9%) added to each well to dissolve purple crystals of formazan. Absorbance was measured with a spectrophotometer at a wavelength of 540 nm using a microplate reader (Biorad, USA). Data are expressed as percent cell viability compared to control.
Rates of oxygen consumption by HepG2 cells were determined with a Clark oxygen electrode (Hansatech Oxygraph, King’s Lynn, UK). HepG2 cells (2 × 107 cells/ml) were incubated in an oxygraph chamber. Glucose (2 mg/ml) was used as a substrate. After a stable rate of oxygen consumption, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which is a well-known and potent mitochondrial uncoupler (as a positive control), or the extracts were added to the chamber. Respiratory rates were expressed as nanomoles of oxygen per milliliter per minute.
Data are expressed as means ± SD, and the unpaired t test was used to compare phenolic content and antioxidant activity between P. virgatus and P. amarus groups. However, the data from cytotoxicity and oxygen consumption studies were not normally distributed. The data were expressed as medians with interquartile ranges. The Mann-Whitney U test was used to compare cytotoxicity (IC50) between P. virgatus and P. amarus groups. Statistical differences between treated and control groups in the oxygen consumption study were determined using the Friedman repeated measures analysis of variance by ranks followed by the Student-Newman-Keuls method for multiple comparisons. Differences between groups were considered significant when p < 0.05 (n = 5 in each group for all experiments).
The amounts of phenolic compounds in extracts as well as antioxidant activity are shown in table 1. P. virgatus extract contained more phenolic compounds than P. amarus extract. The former also showed significantly greater antioxidant capacity both in DPPH scavenging (p < 0.05) and inhibition of linoleic acid oxidation (p < 0.01). Significant differences in the inhibition of linoleic acid oxidation were found at concentrations of ≧250 µg/ml of the extracts, and the maximal inhibition rates of the P. virgatus and P. amarus extracts were about 84.0 and 65.5% (p < 0.001), respectively.
|Table 1. Comparison of total phenolic contents and antioxidant activity of Phyllanthus extracts|
The cytotoxic activity of the extracts of P. virgatus and P. amarus, expressed as IC50 values, from the two assays was similar (fig. 1). The results showed that P. virgatus extract was more toxic to HepG2 cells than P. amarus extract. The IC50 of P. amarus and P. virgatus extracts calculated from trypan blue exclusion were 500 (424–604) and 380 (305–401) µg/ml, respectively (fig. 1a), whereas those calculated from MTT assays were 372 (336–593) and 258 (208–296) µg/ml, respectively (fig. 1b).
|Fig. 1. Cytotoxic effects of Phyllanthus extracts. Cell viability was measured by two different methods. Values are expressed as medians with interquartile ranges in parentheses. The IC50 of P. amarus and P. virgatus measured by the trypan blue method (a) were 500 (424–604) and 380 (305–401) µg/ml, respectively (p < 0.05; n = 5). The IC50 of P. amarus and P. virgatus measured by MTT viability assays (b) were 372 (336–593) and 258 (208–296) µg/ml, respectively (p < 0.05; n = 5).|
Morphological features of cells treated with the extracts at 500 µg/ml were studied after 24 h of incubation (fig. 2). Normal cell morphology characterized by epithelial-like features forming a monolayer on the surface of the culture flask was seen in controls and cells treated with 1% DMSO. In the presence of the plant extracts, morphological changes were observed, such as rounding up, loss of contact with cells and detachment from the plate. P. virgatus extract caused more extensive morphological changes in HepG2 cells than P. amarus.
|Fig. 2. Effects of Phyllanthus extracts on HepG2 morphology. Morphological features of cells treated with the extracts (500 µg/ml each) were observed under a microscope after 24 h of incubation. a Control. b 0.5% DMSO. cP. amarus.dP. virgatus.|
The rate of oxygen consumption by HepG2 cells was measured in the presence of the extracts (fig. 3). It was found that P. amarus extract caused a small and statistically insignificant respiratory stimulation (p > 0.05), while P. virgatus extract clearly enhanced the respiration of HepG2 cells. This effect of P. virgatus extract was comparable to that produced by CCCP.
|Fig. 3. Effects of Phyllanthus extracts on oxygen consumption of HepG2 cells. Tracings a and b show the effects of vehicle (0.5% DMSO) and CCCP (1 µM) as negative and positive controls, respectively. Tracings c and d show the effects of P. amarus and P. virgatus (500 µg/ml each), respectively. Arrows: time at which the vehicle, CCCP and the extracts were added. Numbers in parentheses: rates of oxygen consumption in nanomoles of oxygen per milliliter per minute. The tracings are representative of 5 experiments (n = 5). The respiration rates of HepG2 cells in the presence of P. virgatus extract [16.9 (13.3–20.7) nmol O2/ml/min] and CCCP [9.8 (8.1–14.2) nmol O2/ml/min], but not of P. amarus extract [7.5 (5.7–11.0) nmol O2/ml/min], were significantly higher (p < 0.05) than that of the negative control [5.5 (4.2–8.2) nmol O2/ml/min]. Data are presented as medians with interquartile ranges.|
The findings of a higher total phenolic content of P. virgatus extract from five separate plant collections and the stronger antioxidant activity of P. virgatus extract are comparable to those presented by Kumaran and Karunakaran , who have previously reported that the total phenolic content and antioxidant activity of five Phyllanthus species from India can be placed in the following order: P. debilis > P. urinaria > P. virgatus > P. maderaspatensis > P. amarus.P. virgatus extract also showed a stronger cytotoxic effect than P. amarus.
Experiments on the effect of Phyllanthus extracts on HepG2 cell respiration showed that at a concentration of 500 µg/ml, P. virgatus extract clearly stimulated oxygen consumption, whereas P. amarus extract produced but a small and insignificant increase in respiration. Since oxygen consumption by cells is mostly due to mitochondrial oxidative phosphorylation, changes in cellular respiration rate as a result of experimental treatment may indicate alterations in mitochondrial activity. This finding therefore suggests that P. virgatus extract affects mitochondrial function. The greater cytotoxic activity of P. virgatus extract compared to that of P. amarus may be related to a higher phenolic content since it has been reported that phenolic compounds could be cytotoxic .
The ability of the extracts to stimulate HepG2 cell oxygen consumption may be involved in cell death. Apoptosis signaling transduction can be induced through the death receptor pathway, such as the Fas ligand and Bcl-2 family, as well as through a mitochondrial pathway . Mitochondrial dysfunction can cause a necrosis-type cell death via ATP depletion and Ca2+ dysregulation . Further experiments are needed to determine whether dysregulation of mitochondrial respiration and function is caused by phenolic compounds and is thereby involved in the cytotoxicity of Phyllanthus extracts; these might include, for example, measurement of oxygen consumption and oxidative phosphorylation by isolated mitochondria treated with known phenolic compounds of these extracts.
The results of the present study demonstrate that the hydromethanolic extract of P. virgatus exerted greater antioxidant and cytotoxic effects on human hepatoma HepG2 cells than P. amarus extract. It also produced stronger stimulation of HepG2 cell respiration, which indicates the possibility of mitochondrial involvement in the cytotoxic action.
This study was supported by the National Research Council of Thailand. We thank Dr. Paul J. Grote for plant verification, and Ms. Matcha Kamkhunthod for her technical assistance.
Dr. Nuannoi Chudapongse
School of Biology, Institute of Science
Suranaree University of Technology
Nakhon Ratchasima 30000 (Thailand)
Tel. +66 4422 4620, E-Mail firstname.lastname@example.org
Received: November 10, 2010
Accepted: May 9, 2011
Published online: October 20, 2011
Number of Print Pages : 6
Number of Figures : 3, Number of Tables : 1, Number of References : 27
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