Evaluation of selected South African plant species for antioxidant, antiplatelet, and cytotoxic activity

Abstract

The antioxidant, antiplatelet, and cytoxoxic effects of seven South African plant extracts, namely, Combretum vendae A.E. van Wyk (Combretaceae), Commiphora harveyi (Engl.) Engl. (Burseraceae), Khaya anthotheca (Welm.) C.DC (Meliaceae), Kirkia wilmsii Engl. (Kirkiaceae), Loxostylis alata A. Spreng. ex Rchb. (Anacardiaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae), and Protorhus longifolia (Bernh. Ex C. Krauss) Engl. (Anacardiaceae), were evaluated using established in vitro assays. All the extracts showed comparably low toxicity except for the extract of C. harveyi that showed high hemagluttination assay titer value, which indicates toxicity. The extracts of P. longifolia, K. wilmsii, O. natalitia, L. alata, C. harveyi, and C. vendae exhibited antioxidant properties in the qualitative assay using DPPH. In the quantification of antioxidation using ABTS, only the extracts of P. longifolia, L. alata, and C. vendae showed antioxidant activity with respective TEAC values of 1.39, 1.94, and 2.08. Similarly, in the quantitative DPPH assay, L. alata (EC₅₀, 3.58 ± 0.23 µg/mL) and K. wilmsii (EC₅₀, 3.57 ± 0.41 µg/mL) did not differ significantly (p ≤ 0.05) from the control. K. anthotheca showed a higher EC₅₀ (176.40 ± 26.56 µg/mL) value, and differed significantly (p ≤ 0.05) from all the other extracts and control. In addition, the extracts of C. vendae and C. harveyi showed significant (p ≤ 0.05) antiplatelet activity and did not differ from the control (aspirin) with EC₅₀ of 0.06 ± 0.01 µg/mL and 0.19 ± 0.00 µg/mL, respectively. Lower EC₅₀ values in the antioxidant and antiplatelet studies are indicative of superior activity of the plant extract against oxidation and platelet aggregation.

Keywords::

• South African plants
• antioxidant assay
• antiplatelet activity
• cytotoxicity effect

Introduction

Bioactive compounds commonly found in plants have been shown to have possible health benefits partly due to their antioxidative properties (Cao & Cao, 1999). It is well known that reactive oxygen species (ROS) are involved in a diversity of important processes in medicine, including among others: inflammation, atherosclerosis, cancer, and reperfusion injury (Kehrer, 1993). One of the major fundamental tissue-destructive mechanisms is oxidative stress through an excessive release of reactive oxygen metabolites (ROM) (McCord, 2000). Although their generation by phagocytes (and to a lesser extent by eosinophils, lymphocytes, and fibroblasts) is essential for an effective host defense against bacterial infection, continuous overproduction during inflammatory processes may also cause extensive tissue destruction (Weiss, 1989). One way by which a substance can interfere with these processes is by acting as an antioxidant or free radical scavenger. Antioxidants abate inflammation and protect tissues from oxidative damage caused by free radicals. Inflammation has been described as the release of chemicals from tissues and migrating cells. Agents such as prostaglandins, leukotrienes, histamine, bradykinin, platelet-activating factor (PAF) 1, and interleukin 1 (IL 1) play some part in the inflammatory processes. In addition, prostaglandins and other inflammatory mediators produce oxidant products that play an important role in tissue oxidation (Pekoe et al., 1982). Many anti-inflammatory drugs are able to react with oxidants in vitro, so considerable interest has been expressed in the possibility that oxidant scavenging contributes to the action of these drugs in vivo (Halliwell et al., 1988). Despite the presence of wide arrays of anti-inflammatory drugs (steroidal and nonsteroidal), they still present a wide range of side effects for which the major reason is nonselective inhibition of cyclooxygenase I (COX I) and cyclooxygenase II (COX II) (Vane & Botling, 1995). Prostaglandins, thromboxanes, and platelet-activating factor (PAF) contribute to platelet aggregation, and, moreover, platelets have been shown to play an important role in acute inflammation by releasing arachidonic acid (AA) metabolites and PAF (Holmsen et al., 1977; Vincent et al., 1977). Furthermore, thromboxane A₂ (TXA₂), an AA product formed via the COX pathway in platelets, has been reported to be a potent vasoconstrictor and pro-aggregatory agent (Page et al., 1984). COX inhibitors, such as aspirin, are known to inhibit platelet aggregation (Saeedu et al., 1997).

A large number of naturally occurring compounds, such as flavonoids, catechins, lignans, and phenolic acids contained in plants and herbal remedies, have been shown to have antioxidant properties (Dall’Acqua et al., 2008). These reasons have recently prompted research into natural antioxidants. The aim of this study was to test the antioxidant, antiplatelet, and cytotoxicity effects of acetone extracts of seven South African tree leaves that were previously screened and shown to have antifungal, antibacterial, and other pharmacological effects.

Materials and methods

Plant collection

The leaves of Commiphora harveyi (Engl.) Engl. (Burseraceae), Combretum vendae A.E. van Wyk (Combretaceae), Khaya anthotheca (Welm.) C.DC (Meliaceae), Loxostylis alata A. Spreng. ex Rchb. (Anacardiaceae), and Protorhus longifolia (Bernh. Ex C. Krauss) Engl. (Anacardiaceae) were collected at the University of Pretoria Botanical Garden, South Africa. Kirkia wilmsii Engl. (Kirkiaceae) and Ochna natalitia (Meisn.) Walp. (Ochnaceae) were collected at the Lowveld National Botanical Garden in Nelspruit, South Africa. All plants were collected in November 2006 and were identified and authenticated by Lorraine Middleton, the herbarium curator, and Magda Nel at the Botanical Garden of the University of Pretoria. Voucher specimens of the plants were deposited at the herbarium of the Department of Plant Sciences, University of Pretoria, South Africa.

Plant storage

Immediately after collection and transportation to our laboratory, leaves were separated from stems and dried at room temperature with good ventilation. The dried plants were milled to a fine powder in a Macsalab mill (model 200 LAB; Eriez®, Bramley, South Africa) and stored at room temperature in closed containers in the dark until used.

Plant extraction

Plant samples from each species were individually extracted by weighing four aliquots of 1 g of finely ground plant material and extracting with 10 mL of acetone, hexane, dichloromethane (DCM), or methanol (technical grade; Merck) in polyester centrifuge tubes. Tubes were vigorously shaken for 1 h using a Labotec model 20.2 shaking machine at a moderate speed. Extracting at lower speed for a longer period allows the solvent to penetrate more into the plant tissues, allowing the extraction of more of the compounds contained in the plant species (Silva et al., 1998). After centrifuging at 3500 × g for 10 min, the supernatant was decanted into pre-weighed, labeled containers. The whole process was repeated three times to exhaustively extract the plant material and the extracts were combined. The solvent was removed under a stream of air in a fume cupboard at room temperature to quantify the extraction.

Evaluation of antioxidant activity

Qualitative antioxidant screening was employed using 2,2-diphenyl-1-picryl hydrazyl (DPPH) (Takao et al., 1994). Thin layer chromatography (TLC) plates loaded with 100 µg of each extract were developed in a chloroform/ethyl acetate/formic acid (5:4:2; CEF) solvent system and sprayed with 0.2% DPPH in methanol. Compounds with antioxidant activity were visualized as yellow bands against a purple background (Bors et al., 1992). Similarly, quantification of antioxidant activity was done by spectrophotometric means using two radicals, ABTS [2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] and DPPH.

In the ABTS method, the Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) equivalent antioxidant capacity (TEAC) assay (Re et al., 1999) was determined. This was based on scavenging of the ABTS radical into a colorless product by antioxidant substances. The blue/green chromophore ABTS⁺ was produced through the reaction between ABTS and potassium persulfate. The absorbance was read at 734 nm using a Versamax microplate reader (Molecular Devices). Trolox is a vitamin E analog and was used as a standard in this assay. The percentage change in absorbency was calculated as shown below:

Initial absorbency of ABTS⁺ – New absorbency of ABTS⁺/Initial absorbency of ABTS⁺ × 100

Curves were plotted with the dependent variable being the percentage change in absorbency and the independent variable being the different concentrations at which test substances were analyzed. Mathematical comparison of the antioxidant activities of different plant extracts was done by dividing the slope obtained for extract by that of Trolox, to get the Trolox equivalent antioxidant capacity (TEAC). An extract with a TEAC value of 1 indicates an antioxidant value equivalent to that of Trolox. A decrease or increase of antioxidant activity is depicted by a lower or higher value of TEAC, respectively.

The DPPH free radical assay was conducted as described by Mensor et al. (2001). Briefly, 10 µL of 0.3 mM DPPH in ethanol was added to 25 µL of each concentration of extract tested and allowed to react at room temperature in the dark for 30 min. Appropriate blank and negative control solutions were prepared for each test. L-ascorbic acid (vitamin C) was used as positive control. The decrease in absorbance was measured at 518 nm. Values obtained were converted to percentage antioxidant activity (AA%) using the formula:

AA% = 100 – {[(Abs_sample – Abs_blank) × 100]/Abs_control}

where Abs_sample is the absorbance of the sample, Abs_blank is the absorbance of the blank, and Abs_control is the absorbance of the control. The EC₅₀ value, defined as the concentration of sample leading to a 50% reduction of the initial DPPH concentration, was calculated from the linear regression of plots of concentration of the test extracts (µg/mL) against the mean percentage of antioxidant activity obtained from three replicate assays, using Microsoft Office Excel. EC₅₀ values obtained from the regression lines showed a coefficient of determination r² ≥ 95%. A lower EC₅₀ value indicates high antioxidant activity.

In vitro platelet aggregation assay

The modified method of Fratantoni and Poindexter (1990) was used to determine the inhibitory effect of the plant extracts on platelet aggregation. Briefly, fresh equine (Equus caballus) blood was collected from healthy representative breeds in the Equine Research Centre, Faculty of Veterinary Sciences, University of Pretoria, by Mrs, Stellest de Villiers into sterile 5 mL glass tubes containing 3.8% trisodium citrate solution as anticoagulant at a ratio of 1:9 volume of anticoagulant to blood.

The platelet rich plasma (PRP) and platelet poor plasma (PPP) were separated by centrifugation at 160 × g for 10 min and 1600 × g for 15–30 min at 20–25°C, respectively, using a Beckman® GS-15R centrifuge. PRP was later centrifuged at 1000 × g for 15 min at 20–25°C to sediment the platelets. The platelet count of the PRP was adjusted to 300,000/pL by adding PPP. Both PRP and PPP were then stored at room temperature. The cell suspension was adjusted to approximately 3.0 × 10⁸ platelets per mL using phosphate buffered saline (PBS). Different concentrations of the extracts and aspirin as the reference drug were added and incubated at 37°C for 3 min. After incubation, platelet aggregation was induced by the addition of 50 µL of adrenaline.

The degree of platelet aggregation was determined spectrophotometrically at 600 nm after 30 min. Percentage platelet aggregation inhibition was calculated using the following equation:

X (%) = A – B/A × 100

where A = maximal aggregation of the control and B = maximal aggregation of sample-treated PRP.

The EC₅₀ values were calculated by linear regression of plots using Microsoft Office Excel. The abscissa represented the concentration of tested plant extracts and the ordinate the average percent of antioxidant activity from three separate tests.

Cytotoxicity assay

MTT assay

The plant extracts were tested for cytotoxicity against the Vero monkey kidney cell line. The cells were maintained in minimal essential medium (MEM; Highveld Biological, Johannesburg, South Africa) supplemented with 0.1% gentamicin (Virbac) and 5% fetal calf serum (Adcock-Ingram). To prepare the cells for the assay, cell suspensions were prepared from confluent monolayer cultures and plated at a density of 0.5 × 10³ cells into each well of a 96-well microtiter plate. After overnight incubation at 37°C in a 5% CO₂ incubator, the subconfluent cells in the microtiter plate were used in the cytotoxicity assay. Stock solutions of the plant extracts were prepared by reconstitution to a concentration of 100 mg/mL in dimethylsulfoxide (DMSO). Serial 10-fold dilutions of each extract were prepared in growth medium (1–1000 µg/mL). The method described by Mosmann (1983) was used to determine the viability of cell growth after 120 h incubation with plant extracts. 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was used as an indicator of cell growth. The absorbance was measured at 570 nm. Berberine chloride (Sigma) was used as a positive control. Tests were carried out in quadruplicate and each experiment was done in triplicate.

Hemagglutination assay

Fresh equine (Equus caballus) blood was collected from a representative breed in the Equine Research Centre, Faculty of Veterinary Sciences, University of Pretoria into sterile 5 mL glass tubes containing 3.8% trisodium citrate solution. Erythrocytes were fixed with formalin and prepared according to the method of Sadique et al. (1989) as modified by Iwalewa et al. (2005). Briefly, 20 mL of the mixed blood was centrifuged at 4000 rpm for 10 min, using a Beckman® GS-15R centrifuge. The packed red blood cells (RBCs) were washed with 10 mM PBS, pH 7.2, until a clear supernatant was obtained. The washed, packed RBCs were suspended in 5% (v/v) formaldehyde–PBS (1:12.3, v/v) solution. The mixture was left at room temperature for 24 h. The final, fixed RBCs were washed and centrifuged with PBS three times, and preserved with 1 mL (50 mg/mL) gentamicin containing 0.1% methyl paraben to prevent microbial growth, and stored at 4°C.

PBS (100 µL) was added to wells of 96-well microtiter plates. The first row was used as a control without extracts. The extracts (100 μL) were added into the first well of the second row, and a two-fold serial dilution was made until the last well (well 12). Then, 50 μL of equine RBCs was added to all the wells. They were incubated at room temperature for 1 h. The presence of buttons in the center of the well indicated no agglutination, and the hemagglutination titer values of the extracts were read as the reciprocal of the last dilution showing agglutination

Statistical analysis

Antioxidant and antiplatelet experiments were done in triplicate. The results are presented as mean ± standard error of the mean (SEM). A one-way analysis of variance (ANOVA) was used for comparison of means using Microsoft Office Excel. A difference was considered statistically significant when p ≤ 0.05.

Results

The extraction yields of acetone extracts of plants used in this study are presented in Table 1. C. vendae gave the highest yield of 15.7%, while K. wilmsii possessed the lowest yield (6.9%). The TLC DPPH method of qualitative antioxidant detection showed that the acetone extracts of P. longifolia, K. wilmsii, O. natalitia, L. alata, C. harveyi, and C. vendae displayed antioxidant compounds due to their DPPH free radical scavenging activity. Antioxidant compounds were seen as yellow bands against a purple background. The acetone extract of K. anthotheca, however, did not show any antioxidant compound on the TLC plate (Figure 1).

Figure 1.  Chromatogram of 100 μg acetone extracts of the leaves of P. longifolia (PL), K. wilmsii (KW), O. natalitia (ON), K. anthotheca (KA), L. alata (LA), C. harveyi (CH), and C. vendae (CV), separated with CEF mobile phase and sprayed with 0.2% DPPH. Antioxidant compounds are indicated by yellow areas.

Table 1.  Antioxidant and antiplatelet activity of acetone extracts of seven South African plants.

Plant	Voucher specimen number	Extract yield (%)	Antioxidant value		Antiplatelet activity (EC50 ± SEM, µg/mL)
			TEAC	DPPH (EC50 ± SEM, µg/mL)
C. vendae	PRU96507	15.7	2.08	4.41 ± 0.14^b	0.06 ± 0.01ª
C. harveyi	PRU96506	14.2	0.15	10.47 ± 1.96^b	0.19 ± 0.00ª
K. anthotheca	PRU96509	8.9	0.10	176.40 ± 26.56^c	0.97 ± 0.03
K. wilmsii	PRU96503	6.9	0.67	3.57 ± 0.41ª	0.22 ± 0.02
L. alata	PRU96508	13.7	1.94	3.58 ± 0.23^a	0.35 ± 0.03
O. natalitia	PRU96504	12.6	0.79	7.50 ± 0.13^b	0.39 ± 0.03
P. longifolia	PRU96505	15.3	1.39	6.57 ± 0.23^b	0.23 ± 0.02
l-ascorbic acid	–	N/A	N/A	1.59 ± 0.80ª	N/A
Aspirin	–	N/A	N/A	N/A	0.04 ± 0.00ª

Note. N/A, not available. Means within the same column and with different superscript letters (a, b, or c) differ significantly (p ≤ 0.05).

In the TEAC antioxidant assay, extracts of P. longifolia, L. alata, and C. vendae showed superior free radical scavenging activity when compared with other extracts and the standard antioxidant (Trolox) used in this study, with respective TEAC values of 1.39, 1.94, and 2.08 (Table 1). The results corroborate those of the qualitative assay where the extracts showed the presence of antioxidant compounds. Extracts of O. natalitia, K. wilmsii, C. harveyi, and K. anthotheca showed lower TEAC values of 0.79, 0.67, 0.15, and 0.10, respectively.

In the quantitative DPPH assay (Table 1), L-ascorbic acid had an EC₅₀ (1.59 ± 0.80 µg/mL) value lower than all the extracts; however, L-ascorbic acid showed no significant (p ≤ 0.05) difference in its free radical scavenging effect when compared with L. alata (EC₅₀, 3.58 ± 0.23 µg/mL) and K. wilmsii (EC₅₀, 3.57 ± 0.41 µg/mL). K. anthotheca showed a higher EC₅₀ (176.40 ± 26.56 µg/mL) value, and differed significantly (p ≤ 0.05) from all the other extracts and l-ascorbic acid. Extracts of P. longifolia, C. vendae, C. harveyi, and O. natalitia had EC₅₀ values of 6.57 ± 0.23, 4.41 ± 0.14, 10.47 ± 1.96, and 7.50 ± 0.13 µg/mL, respectively. The lower the EC₅₀ of a substance, the more effective is its free radical scavenging effect.

The antiplatelet actions of C. vendae and C. harveyi did not differ significantly (p ≤ 0.05) from that of aspirin (standard agent) (Table 1). The low EC₅₀ shown by C. vendae (0.06 ± 0.01 µg/mL) and C. harveyi (0.19 ± 0.00 µg/mL) depicts good antiplatelet activity when compared with that of aspirin (0.04 ± 0.00 µg/mL).

The cytotoxic activities of the extracts in the Vero monkey kidney cell line assay are provided in Figure 2. In the assay, the extracts of O. natalitia, K. anthotheca, L. alata, C. harveyi, and C. vendae at the highest concentration used were relatively toxic; they caused complete death of the Vero cells. However, at lower concentrations, all the extracts showed relatively lower toxicity when compared with the reference agent berberine (cytotoxic agent). Over 90% of Vero cells treated with berberine at the concentration of 100 µg/mL were not viable. The effects of the extracts on fixed equine erythrocytes are shown in Table 2. All the plant extracts exhibited very low hemagglutination assay (HA) titer values with a wide range of concentrations at which agglutination occurred. However, the acetone extract of C. harveyi showed a very high HA titer value and a very low concentration at which agglutination occurred.

Figure 2.  Viability of Vero monkey kidney cell line treated with different concentrations of acetone extracts of seven South African plants. Values are mean ± SEM.

Table 2.  Cytotoxic effect of acetone extracts of seven South African plants on formaldehyde-fixed equine erythrocytes.

Plant extract	Concentration value where agglutination occurred (mg/mL)	Hemagglutination assay titer value
C. vendae	1.25	0.80
C. harveyi	0.31	3.23
K. anthotheca	1.25	0.80
K. wilmsii	1.25	0.80
L. alata	1.25	0.80
O. natalitia	2.5	0.40
P. longifolia	1.25	0.80

Discussion

In this study, we applied established in vitro assays in order to evaluate the antioxidant, antiplatelet, and cytotoxic actions of seven South African tree leaves. These plants had shown promising antifungal and bacterial activities in previous studies. This study is the first to report the antioxidant, antiplatelet, and cytotoxicity activities of these plants. These natural products were able to scavenge free radicals in a concentration-dependent fashion in two separate antioxidant assays. DPPH is a free radical, stable at room temperature, which produces a violet solution in ethanol. It is reduced in the presence of an antioxidant molecule, giving rise to uncolored solutions. The use of DPPH provides an easy and rapid way to evaluate antioxidants (Mensor et al., 2001). The extracts that showed antioxidant activity separated poorly from the origin when applied and eluted on TLC. This could have been the result of overloading the TLC plates or due to the presence of polyphenolic compounds (Naidoo et al., 2006). Polyphenols do not move well from the origin due to their high polarity, leading to tight binding with normal phase silica (Davidson, 1964).

The ABTS⁺ assay described here involves direct production of the blue/green ABTS^.+ chromophore through the reaction between ABTS and potassium sulfate, which has absorption maxima at wavelengths 645 nm, 734 nm, and 815 nm (Miller et al., 1993; Re et al., 1999).

Both Trolox and l-ascorbic acid have been used as standards in the quantification of antioxidant activity (Fukomoto & Mazza, 2000). The EC₅₀ values of P. longifolia, K. wilmsii, O. natalitia, L. alata, C. harveyi, and C. vendae were lower than that of Ginkgo biloba extract (EGb 761), whose EC₅₀ is 40.72 μg/mL (Aderogba et al., 2004; Bridi et al., 2001; Mensor et al., 2001). The extract of Ginkgo biloba (EGb 761) has been widely employed for its significant benefit as an antioxidant for the prevention of neurodegenerative disorders (Bridi et al., 2001). Similarly, these extracts showed free radical scavenging in the DPPH TLC assay.

It has been suggested that more than one method of antioxidant testing should be used to obtain detailed knowledge of the antioxidant activity of test substances, and in addition, the extrapolation of in vitro data to in vivo situations is often difficult (Aruoma, 2003). The TEAC assay is used commonly for screening compounds, food products, and extracts for antioxidant activity, and is particularly useful in providing a ranking order of antioxidants (van den Berg et al., 1999), despite the limitations it carries.

The agonist adrenaline (epinephrine) induced platelet aggregation in equine platelets. The extracts of P. longifolia, K. wilmsii, O. natalitia, K. anthotheca, L. alata, C. harveyi, and C. vendae inhibited platelet aggregation induced by this agonist with different potencies. However, only the extract of C. vendae showed a statistically (p < 0.05) significant platelet inhibitory effect.

Platelet activation is usually accompanied by a rise in cytosolic Ca²⁺ levels and this occurs through stimulation of the enzymes that are not fully functional at the low Ca²⁺ concentration present in the resting platelets (Berridge, 1993; Heemskerk & Sage, 1994). In platelets, either the stimulation of phospholipase C (PLC) or the activation of inhibitory G-protein (Gi)-linked receptors elevates the cytosolic Ca²⁺ levels (Puri et al., 1995). This takes place through the release of Ca²⁺ from internal stores or through the entry of Ca²⁺ across the plasma membrane from an external medium (Berven et al., 1995; Obberghen-Schilling & Pouyssegur, 1993).

An alternative pathway of increasing the Ca²⁺ influx is through activation of the Gi-linked pathway. Agonists such as adrenaline are known to inhibit adenylyl cyclase activity in platelets, leading to a decrease in intracellular cyclic adenosine monophosphate (cAMP) levels (Puri et al., 1995). Multiple studies have shown that agents that decrease cAMP levels stimulate platelet aggregation (Nieuwland et al., 1994; Siess et al., 1993). This occurs through activation of α₂-adrenergic receptors (Siess & Lapetina, 1989). α₂-Adrenoceptors in platelets are known to be coupled to the guanine nucleotide-binding protein G, which mediates inhibition of adenylate cyclase. This mainly takes place either through an increase in Ca²⁺ influx (Shah et al., 1996) or as a result of activation of some other proteins (Musgrave & Seifert, 1995). It is possible that the extracts in this study may contain components which block these Ca²⁺ channels or act via an unknown mechanism(s) to cause increased levels of intracellular cAMP in platelets, as demonstrated through the inhibition of platelet aggregation.

Anti-inflammatory drugs could affect oxidant damage in several ways. First, they might directly scavenge such reactive oxidants as ^•OH and HOCl. Most, if not all, anti-inflammatory drugs are capable of reacting quickly with .OH. Hence, drugs with good anti-inflammatory activity could be of use as free radical scavengers (Halliwell et al., 1988).

Platelet aggregation plays a pathophysiological role in a variety of thromboembolic disorders. Therefore, prevention of platelet aggregation by drugs should provide effective prophylactic and/or therapeutic treatments for such diseases (Hsiao et al., 2003).

Studies have demonstrated that botany and medicine are related. Free radicals and lipid peroxidation have been suggested as potentially important causative agents of several diseases in animals and humans (Nair et al., 2003). It would be worthwhile to explore and find safer and efficacious remedies from natural sources, particularly from the relatively undiscovered and unexplored rich flora of South Africa.

The antioxidant and antiplatelet effects of these extracts, and the additional benefit of their low cytotoxicity, provide strong motivation for the development of these plants as possible drugs for the control of diseases in animals and humans. Most considerably, these properties substantiate the use of plant screening exercises for detecting disease control agents.

In conclusion, these plant species appear to be potential sources of antioxidant and anti-inflammatory agents. From this study, we can rightly infer that the South African flora offers great potential in the search for natural compounds with disease controlling agents. The bioassay-guided fractionation procedure to isolate and characterize active compounds from those plant extracts that show good activity is currently being undertaken in our laboratory.

Acknowledgements

The permission granted to us by the University of Pretoria Botanical Garden and the Lowveld National Botanical Garden, Nelspruit to collect the plant leaves is also greatly acknowledged.

Declaration of interest

We have acknowledged funds received from the SA-NRF for the study. The funds were provided to one of the authors (MM Suleiman) as PhD bursary at the University of Pretoria.