Skip to main content

The anti-inflammatory and antioxidant activity of 25 plant species used traditionally to treat pain in southern African



Inflammation is a common risk factor in the pathogenesis of conditions such as infections, arthritis, type 2 diabetes mellitus, obesity and cancer. An ethnobotanical survey of medicinal plants used traditionally to treat inflammation and related disorders such as pain, arthritis and stomach aches in southern Africa led to the selection of 25 plant species used in this study.


The antioxidant activities of acetone extracts were determined by measuring the free radical scavenging activity and ferric reducing ability, respectively. The anti-inflammatory activities of the extracts were determined by measuring the inhibitory effect of the extracts on the activities of the pro-inflammatory enzyme, lipoxygenase and inducible nitric oxide synthase.


Extracts of Peltophorum africanum had good antioxidant activity with IC50 values of 4.67 ± 0.31 μg/mL and 7.71 ± 0.36 μg/mL compared to that of the positive control ascorbic acid (2.92 ± 0.14 μg/mL and 13.57 ± 0.44 μg/mL), using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and 2,2′-azinobis (3-ethylbenzthiazoline-6-sulphonic acid (ABTS) methods, respectively. The metabolism of linoleic acid to leukotriene derivatives by 15-lipoxygenase (15-LOX) was also inhibited by the crude acetone extracts of Peltophorum africanum (IC50 = 12.42 μg/mL), Zanthoxylum capense (IC50 = 14.92 μg/mL) compared to the positive control quercetin (IC50 = 8.75 μg/mL). There was a poor correlation between the flavonoid content and 15-LOX inhibition by the extracts (R2 = 0.05), indicating that flavonoids are not involved in LOX inhibition. Extracts of Clausena anisata, at a concentration of 6.25 μg/mL inhibited nitric oxide production by RAW 264.7 macrophage cell lines in vitro by 96 %. The extracts of Zanthoxylum capense were the least cytotoxic (IC50 > 1000 μg/mL) when the extract toxicity was determined against Vero (African green Monkey) kidney cell lines.


Some plant species used traditionally to treat pain have reasonable anti-inflammatory activity and flavonoids are probably not involved in this process.

Peer Review reports


Medicinal plants have long been recognised as important sources of therapeutically active compounds. Evidence-based research supports the medical and pharmacological benefits of plant-derived compounds, with increasing interest in the identification and characterization of bioactive compounds from natural sources [1].

One of the earliest recorded approaches for treating inflammation and pain was the application of extracts from willow leaves by Celsius in 30 AD [2]. This observation led to the discovery of acetyl salicylic acid, the active component of aspirin, a major anti-inflammatory drug widely used in clinical practice, along with many other non-steroidal anti-inflammatory drugs (NSAIDs) in current use [3].

Non-steroidal anti-inflammatory drugs are commonly prescribed for treatment of pain and inflammatory conditions such as rheumatoid arthritis, osteoporosis and Alzheimer’s disease. However, because many NSAIDs are associated with side effects such as gastrointestinal bleeding and suppressed function of the immune system [4], attention has shifted to alternative pharmacotherapies [5, 6]. Recent studies on Zingiber officinale, ginger, suggest that it might be as effective as some NSAIDs in the treatment of inflammation and related pain [7, 8].

In South Africa the use of plants to treat many diseases is widely practiced. According to Iwalewa et al. [9], more than 115 plant species of 60 families are used in South Africa to treat pain-related inflammatory disorders in humans and animals. The bioactive principles in these plant species have been linked to secondary metabolites such as phenolic compounds (curcumins, flavonoids and tannins), saponins, terpenoids and alkaloids [9, 10,]. Biological and therapeutic properties attributed to these plant metabolites include antioxidant, anti-inflammatory, antimicrobial and anticancer activities [10]. The mechanisms of action of many phenolic compounds such as flavonoids, tannins and curcumins are thought to be via their free radical scavenging activities or the inhibition of pro-inflammatory enzymes such as cyclo-oxygenases (COX) and lipoxygenases (LOX) in the inflammatory cascades [11, 12].

Flavonoids are a group of polyphenols thought to inhibit the biosynthesis of prostaglandins, end-products in the COX and LOX pathways of immunologic responses [13]. There are three known isomeric-forms of COX i.e. COX-1 and COX-2, with a recently described third isomeric-form, COX-3 that is selectively inhibited by acetaminophen and related compounds [14, 15]. The selective inhibition of COX-2 is more desirable because the inhibition of COX-1 in the gastric mucosa is associated with the undesirable effects of NSAIDs [16]. COX-2 is induced as an early response to pro-inflammatory mediators and stimuli such as endotoxins and cytokines [17]. Upon induction, COX-2 synthesizes prostaglandins that contribute to inflammation, swelling and pain [18]. Consequently, dual COX-2/LOX inhibitor compounds could potentially be developed into safer and more effective drugs for the treatment of inflammation since they could potentially inhibit biosynthesis of prostaglandins and leukotrienes respectively from arachidonic acid [16, 19], without the undesirable effects of NSAIDs.

Lipoxygenases are lipid-peroxidizing enzymes involved in the biosynthesis of leukotriene from arachidonic acid, mediators of inflammatory and allergic reactions. These enzymes catalyse the addition of molecular oxygen to unsaturated fatty acids such as linoleic and arachidonic acids [20]. There are four main iso-enzymes already described, namely, 5-LOX, 8-LOX, 12-LOX and 15-LOX, depending on the site of oxidation in the unsaturated fatty acids [20]. The common substrates for LOX are linoleic and arachidonic acids. For many in vitro studies, soy bean LOX is used due to difficulties in obtaining human LOX for bioassays [21].

During inflammation, arachidonic acid is metabolized via the COX pathway to produce prostaglandins and thromboxane A2, or via the LOX pathway to produce hydroperoxy-eicosatetraenoic acids and leukotrienes [22]. The LOX pathway is active in leucocytes and many immune-competent cells including mast cells, neutrophils, eosinophils, monocytes and basophils. Upon cell activation, arachidonic acid is cleaved from cell membrane phospholipids by phospholipase A2 and donated by LOX activating protein to LOX, which then metabolises arachidonic acids in a series of reactions to leukotrienes, a group of inflammatory mediators [23]. Leukotrienes act as phagocyte chemo-attractant, recruiting cells of the innate immune system to sites of inflammation. For instance in an asthmatic attack, it is the production of leukotrienes by LOX that causes the constriction of bronchioles leading to bronchospasm [8, 16]. Therefore, the selective inhibition of LOX is an important therapeutic strategy for asthma [8, 16, 24]. Inhibitors of the activities of LOX could provide potential therapies to manage many inflammatory and allergic responses. Medicinal plants may therefore be potential sources of inhibitors of COX-2/LOX that may have fewer side effects than NSAIDs [24].

Nitric oxide (NO) is a short-lived free radical that mediates many biological processes. One of the functions of NO is to enhance the bactericidal and tumoricidal activities of activated macrophages [25, 26]. Excessive production of NO could however potentially lead to tissue damage and activation of pro-inflammatory mediators [27, 28]. The potential of extracts from medicinal plants to scavenge these free radicals and modulate inflammatory reactions has been demonstrated [2931].

The objective of this study was to determine the anti-inflammatory activity of extracts in relevant bioassays in order to validate their use for pain relief and to identify plants that could be investigated in more detail.


Analytical grade chemicals were purchased from various suppliers in South Africa, and were used for the bioassays in the laboratory.

Preparation of plant materials

Fresh leaves of the selected plants species were collected from the Manie van der Schijff Botanical Garden, University of Pretoria in March 2012. The plant materials were dried at room temperature in a well-ventilated room for a week. After drying, the materials were ground to fine powder using a MacSalab Model 200 grinder and stored in closed honey jars in the dark. Herbarium specimens for each of the plant species were prepared and deposited at HGWJ Schweickerdt Herbarium, University of Pretoria. Herbarium voucher specimen numbers (PRU voucher numbers) are provided in Table 1.

Table 1 Percentage crude extract yield from the selected plant species

Preparation of crude extracts for biological assays

Ground leaf powders (3 g) were extracted in 30 mL of 70 % acetone in clean honey jars and vigorously shaken for 3 h (Labotec model 20.2 shaker). The crude acetone extracts were filtered through Whatman No. 1 filter papers into pre-weighed honey jars, and then left open overnight for solvent evaporation. The honey jars were weighed again to determine the percentage yield of the crude extracts. For the biological assays, the crude extracts were reconstituted in dimethyl sulphoxide (DMSO) at a concentration of 10 mg/mL.

Determination of total phenolics and flavonoids

Total phenolics were determined according to the method of Folin-Ciocalteu described by Makkar [32], with slight amendments. In brief, 25 μL of crude extract was treated with 250 μL Folin-Ciocalteu reagent for 5 min. The reaction was stopped by adding 750 μL 20 % anhydrous sodium carbonate. The volume was made up to 5 mL with distilled water and incubated in the dark at room temperature for 2 h. After incubation, the absorbance was read at 760 nm with a spectrophotometer (HELIOS βT60, Separation Scientific). The phenolic content was determined from a standard curve of different concentrations of gallic acid DMSO. The results were expressed as mg/g gallic acid equivalent (GAE).

Flavonoid content of the extracts was determined using the methods of Yadav and Agarwala, [33], also amended slightly. Crude extracts (100 μL) were dissolved in 300 μL methanol, to which 20 μL 10 % aluminium chloride was added. A further 20 μL of 1 M sodium acetate was added to the solution. The resultant solution was made up to 1 mL with distilled water. This was incubated at room temperature for 30 min in a microplate. After incubation, the absorbance was read at 450 nm in a microplate reader (SpectraMax 190, Molecular devices). Quercetin (10 mM) was used as a standard. The flavonoid content of each extract was expressed as mg/g quercetin equivalent (QE).

The 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay methods

The DPPH radical-scavenging activity was determined using the method of Brand-Williams et al. [34]. Ascorbic acid and Trolox were used as positive controls, methanol as negative control and extract without DPPH as blank. Results were expressed as percentage reduction of the initial DPPH absorption in relation to the control. The concentration of extract leading to 50 % reduction of DPPH (IC50) was also determined.

The 2, 2′-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging assay methods

The ABTS radical scavenging capacity of the samples was measured with modifications of the 96-well microtitre plate method described by Re et al. [35]. Trolox and ascorbic acid were used as positive controls, methanol as negative control and extract without ABTS as blank. The percentage of ABTS• + inhibition was calculated using the formula:

$$ \mathrm{Scavenging}\ \mathrm{capacity}\ \left(\%\right)=100-\left[\frac{{\mathrm{OD}}_{\mathrm{sample}}\hbox{-} {\mathrm{OD}}_{\mathrm{sample}\ \mathrm{blank}}}{{\mathrm{OD}}_{\mathrm{control}}\hbox{-} {\mathrm{OD}}_{\mathrm{control}\ \mathrm{blank}}}\right]\times 100\% $$

where OD represents the optical density or absorbance.

The IC50 values were calculated from the graph plotted as inhibition percentage against the concentration.

The ferric reducing ability of plasma (FRAP) assay methods

The FRAP assay was carried out according to the procedure of Benzie and Strain [36] with slight modification. The FRAP assay depends upon the reduction of ferric tripyridyltriazine (Fe (III)-TPTZ) reduction to ferrous tripyridyltriazine (Fe (II)-TPTZ) by a reductant at low pH. Ferrous (II)-TPTZ has an intensive blue colour and can be monitored at 593 nm. Briefly, the FRAP reagent was prepared using an acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM hydrochloric acid and 20 mM iron (III) chloride solution in proportions of 10:1:1 (v/v), respectively. Twenty five microliters of sample were added to 175 μL of the FRAP reagent. The absorbance of the reaction mixture was recorded at 593 nm (SpectraMax 190, Molecular devices) after 5 min. The standard curve was made using iron (II) sulphate solution (40–0.078 μg/mL), and the results were expressed as μg Fe (II)/g of extract. All the measurements were taken in triplicate and the mean values were calculated.

Inhibition of 15-lipoxygenase (15-LOX) enzyme

The 15-LOX (Sigma) was made up to a working solution of 200 units/mL and kept on ice. A volume of 12.5 μL of test sample or control (dissolved in DMSO) was added to 487.5 μL of 15-LOX in a 96-well microtitre plate and incubated at room temperature for 5 min. After incubation, 500 μL substrate solutions (10 μL linoleic acid dissolved in 30 μL ethanol, made up to 120 mL with 2 M borate buffer at pH 9.0) was added to the solution. After 5 min incubation at room temperature, the absorbance was measured with the microplate reader at 234 nm (SpectraMax 190, Molecular devices). Quercetin (1 mg/mL) was used as a positive control, while DMSO was used as the negative control (100 % enzyme activity or no enzyme inhibition). The percentage enzyme inhibition of each extract compared with negative control as 100 % enzyme activity was calculated using the equation;

$$ \%\ \mathrm{Inhibition}=\frac{\left({\mathrm{OD}}_{\mathrm{extract}}\hbox{-} {\mathrm{OD}}_{\mathrm{blank}}\right)}{\left({\mathrm{OD}}_{\mathrm{negative}\ \mathrm{control}}\hbox{-} {\mathrm{OD}}_{\mathrm{blank}}\right)}\times 100\% $$

The results were expressed as IC50, i.e. concentration of the extracts and controls that resulted in 50 % 15-LOX inhibition plotted on a graph.

Inhibition of nitric oxide (NO) production

Cell culture

The RAW 264.7 macrophage cell lines obtained from the American Type Culture Collection (Rockville, MD, USA) were cultured in plastic culture flasks in Dulbecco’s Modified Eagle’s Medium (DMEM) containing l-glutamine supplemented with 10 % foetal calf serum (FCS) and 1 % PSF (penicillin/streptomycin/fungizone) solution under 5 % CO2 at 37 °C, and were split twice a week. Cells were seeded in 96 well-microtitre plates and were activated by incubation in medium containing LPS (5 μg/mL) and various concentrations of extracts dissolved DMSO.

Measurement of nitrite

Nitric oxide released from macrophages was assessed by the determination of nitrite concentration in culture supernatant using the Griess reagent. After 24 h incubation, 100 μL of supernatant from each well of cell culture plates was transferred into 96-well microtitre plates and equal volume of Griess reagent was added. The absorbance of the resultant solutions in the wells of the microtitre plate was determined with a microtitre plate reader (SpectaMax 190 Molecular devices) after 10 min at 550 nm. The concentrations of nitrite were calculated from regression analysis using serial dilutions of sodium nitrite as a standard. Percentage inhibition was calculated based on the ability of extracts to inhibit nitric oxide formation by cells compared with the control (cells in media without extracts containing triggering agents and DMSO), which was considered as 0 % inhibition.

Cell viability

To ensure that the observed nitric oxide inhibition was not due to cytotoxic effects, the cytotoxicity was also determined against Vero Monkey kidney cells as previously described by Mosmann [37], with slight modifications. After removal of media, the cells were topped up with 200 μL DMEM. To each well, 30 μL of 15 mg/mL 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetra-zoliumbromide (MTT) was added. The cells were incubated at 37 °C with 5 % CO2. After 2 h, the medium was carefully discarded and the formed formazan salt was dissolved in DMSO. The absorbance was read at 570 nm (SpectraMax 190, Molecular devices). The percentage of cell viability was calculated with reference to the control (cells without extracts containing LPS taken as 100 % viability).

All the experiments to measure nitric oxide inhibition were conducted three times in triplicate.

Cytotoxicity assessments

The cytotoxicity of the extracts (dissolved in acetone) against Vero monkey kidney cells was assessed by the MTT reduction assay as previously described [37] with slight modifications. Cells were seeded at a density of 1 × 105 cells/mL (100 μL) in 96-well microtitre plates and incubated at 37 °C and 5 % CO2 in a humidified environment. After 24 h incubation, extracts (100 μL) at varying final concentrations were added to the wells containing cells. Doxorubicin (40–0.38 μM) was used as a reference compound. A suitable blank control with equivalent volume of acetone was also included and the plates were further incubated at 37 °C for 48 h in a CO2 incubator. The medium was removed by aspiration and cells were then washed twice with PBS, followed by suspension in fresh medium (200 μL). Then, 30 μL of MTT (5 mg/mL in PBS) was added to each well and the plates were incubated at 37 °C for 4 h. The medium was removed by aspiration and 100 % DMSO (100 μL) added to dissolve the formed formazan crystals. The absorbance was measured on SpectraMax 190 (Molecular devices) microtitre plate reader at 570 nm. The percentage of cell growth inhibition was calculated based on a comparison with untreated cell. The selectivity index (SI) values were calculated by dividing cytotoxicity LC50 values by the MIC values (SI = LC50/MIC).

Statistical analysis

All results are presented as the means of triplicate experiments. Differences between test extracts in these experiments was assessed for significance using analysis of variance (ANOVA) and student t-test, where probability (p ≤ 0.05) was considered significant.

Results and discussion

The results obtained in this study are presented below using Tables and Figures for ease of interpretation and data comparison.

Crude yield of extracts

Tulbaghia violacea yielded 22 % of crude acetone extract from 3 g plant material, the highest yield of all the plant species in this study. This plant grows as a bulbous rhizome, which had to be cut into pieces for proper drying. The presence of reserve materials might account for the high yield of extract from the plant unlike the other plant species in the study, whose leaves could be easily dried when left open in the drying room for three days (Table 1).

Total phenolics and flavonoid contents

The high extract yield from T. violacea did not correlate well with its total phenolics and flavonoid content. This may be due to high concentrations of carbohydrates as reserve material in the rhizome. Terminalia phanerophlebia and Terminalia prunioides with lower crude extract yield of 7 % and 5.7 % respectively contained more total phenolics than T. violacea (Table 1). The highest amounts of total phenolic compounds were obtained from T. phanerophlebia (86 mg/g GAE) followed by T. prunioides (79 mg/g GAE) and M. comosus (64.7 mg/g GAE).

In terms of flavonoid content, the three highest yields were obtained from D. cinerea (0.54 mg/g QE), T. phanerophlebia (0.53 mg/g QE) and S. birrea (0.52 mg/g QE), respectively (Fig. 1). The overall results suggest that generally, there was poor correlation between total phenolics and flavonoid contents in the selected plant species (R2 = 0.05); however, T. phanerophlebia seems to be an exception. Not much study has been done on phyto-chemical screening of the leaves of T. phanerophlebia, but available literature data indicates the presence of triterpenoids and tannins [10]. The dried leaves of the plant are generally used as decoction in water to treat rheumatism, stomach pains and diarrhoea [38]. The high content of total phenolics and flavonoids, possibly tannins, triterpenoids and other secondary metabolites may be responsible for its therapeutic uses.

Fig. 1
figure 1

Relationship between total phenolic and flavonoid contents. Results indicated that there was no correlation between the total phenolic content and flavonoid content of the extracts tested (R2 = 0.05)

Data from literature sources on the secondary metabolites present in the leaves of T. prunioides is scarce. Its antibacterial [39], Thin Layer Chromatography profile and antifungal activity [40], and antioxidant activity [41] has been reported. However, the dried leaves are used as decoction traditionally for the relief of stomach pains. Our study indicated that it contained relatively high amounts of phenolic compounds, possibly flavonoids, tannins and terpenoids, this may be responsible for the antimicrobial and antioxidant activity. The third plant species with a high phenolic content among the selected plants was M. comosus. Potential anti-fungal and lipoxygenase inhibitory properties of this plant species have already been reported. This may be associated with its flavonoid and cardiac glycoside content [42]. Phenolic compounds, especially flavonoids are well known for their anti-oxidant activitiy and lipoxygenase enzyme inhibitory activity [43].

Anti-inflammatory activities

The main objective of the study was to evaluate the anti-inflammatory activity of the selected extracts using the anti-15 LOX model of inhibition. Therefore the three plants extracts with promising inhibitory activity of 15-LOX were selected for further investigation. As illustrated in Fig. 2, crude extracts harvested from two of the plant species tested, P. africanum (IC50 = 12.42 μg/mL) and Z. capense (IC50 = 14.92 μg/mL), had promising 15-LOX inhibitory activities compared with quercetin (IC50 = 8.75 μg/mL) used as a positive control. These complex crude extracts may contain compounds with higher activity than quercetin. These results suggest that the bioactive constituent(s) of P. africanum had both antioxidant and anti-inflammatory activities. Antioxidants act by scavenging free radicals such as reactive oxygen species, hydroxyl radicals and nitric oxide while anti-inflammatory mediators act by modulating the activities of pro-inflammatory enzymes and cytokines.

Fig. 2
figure 2

Inhibitory activities of crude plant extracts on 15-LOX. The extract with the highest inhibitory activity on 15-LOX was obtained from P. africanum (IC50 = 12.42 μg/mL) compared with quercetin controls (IC50 = 8.75 μg/mL)

The lipoxygenase group of enzymes (5, 8, 12 and 15-LOX) plays a role in many inflammatory disorders. The isomeric enzyme, 15-LOX is an important enzyme involved in the synthesis of leukotrienes from arachidonic acids. Biologically active leukotrienes are mediators of many pro-inflammatory and allergic reactions, therefore the inhibition of the synthesis of leukotrienes by 15-LOX is considered as one of the therapeutic strategies in the management of inflammatory conditions [17, 24]. Assessment of extracts derived from more than 180 different plant species indicated their potential dual COX/LOX inhibitory capacity [24]. Extracts or compounds from plants inhibiting the pro-inflammatory activities of these enzymes may contain potential leads or templates for the development of potent anti-inflammatory drugs [44]. Further work is required to properly characterize the compound(s) responsible for the anti-inflammatory principles in these plant species, and also understand their mechanisms of action. The three plants extracts with promising inhibitory activity on 15-LOX were selected for further investigation.

Inhibition of nitric oxide (NO) production by the three extracts with promising 15-LOX inhibitory activity

The inhibitory activity of the extracts on NO production by induced RAW 264.7 macrophage cell lines is presented in Table 2. Clausena anisata crude acetone extracts had the best inhibitory activity on NO production (96.9 % inhibition/ 71.3 % cell viability) at 6.25 μg/mL compared with P. africanum (91.3 %/ 65.7 %), Z. capense (62.2 %/ 82.5 %) and quercetin (91.1 %/ 73.8 %) respectively (Table 2). Extracts with good inhibitory activity on NO production and a low cytotoxicity are more useful. Release of NO promotes inflammation, therefore extracts that could act as scavengers of NO, or inhibitors of its production, especially with corresponding low cytoxicity could be used to mitigate the propagation of inflammation by NO. The inhibition of NO production by extracts derived from medicinal plants may be due to the inhibition of inducible nitric oxide synthase activity and/or its expression [30, 31].

Table 2 Inhibitory activities of Peltophorum africanum (PA),Clausena anisata (CA) andZanthoxylum capense (ZC) on the LPS-activated NO production in RAW 264.7 macrophages

Antioxidant activities

Phytochemical evaluation of extracts derived from P. africanum has yielded bergenin [45] and betulinic acid [46]. Secondary metabolites are stored in various parts of the plant; however coumarins constitute the major compounds in the leaves [47]. The plant is widely used traditionally for treating wounds, back and joint pains and dysentery, among others [48], but reported biological activities of the extracts are limited to antimicrobial activity [49]. The bioactive compounds responsible for the observed effects have not been properly characterized and the mechanism of activity has not been explored. In the case of Z. capense, biological activities such as anti-mycobacterial [50] and anti-proliferative effects [51] have been reported, subsequent to a bio-assay guided isolation of six alkaloids from the roots of the plant [52].

Extracts of P. africanum had the best antioxidant activity among the three extracts that were tested (Table 3). With IC50 of 4.67 ± 0.31 and 7.71 ± 0.36 μg/mL using the DPPH and ABTS assays respectively, the results were comparable to that of Trolox positive controls (2.74 ± 0.08 and 7.21 ± 0.42 μg/mL). This is consistent with the findings of Bizimenyera, 2007 [53]. An extensive review of extracts of African medicinal plants with potent anti-oxidant activities by Atawodi [54] indicated that the mechanism(s) of action extracts was by free radical scavenging. In addition, the synergistic effects of natural products also enhance their antioxidant activities [53]. These results suggest that the bioactive constituent(s) of P. africanum had both antioxidant and anti-inflammatory activities. Antioxidants acts by scavenging free radicals such as reactive oxygen species, hydroxyl radicals and nitric oxide while anti-inflammatory mediators act by modulating the activities of pro-inflammatory enzymes and cytokines. Accumulation of free radicals result in cellular injury, and may be the cause of many diseases.

Table 3 Cytotoxicity, antioxidant activity, total phenolics and total flavonoids content of acetone extracts of Clausena anisata, Peltophorum africannum and Zanthoxylum capense


Our results indicated that extracts of Z. capense had the lowest cytotoxicity on Vero Monkey kidney cell lines (Table 3) among those tested (IC50 > 1000 μg/mL). Peltophorum africanum extracts also had a relatively low toxicity of with an IC50 of 103 μg/mL that was comparable to values in an earlier report [53]. The safety of herbal remedies remains a concern because few reports exist on the safe use of these products. Many extracts have been shown to contain potentially harmful substances that could impact adversely on human health when consumed [55]. Although, our study suggests that extracts of Z. capense had low toxicity on Vero cell lines (≥1000 μg/mL) (Table 3), this observation has not yet been confirmed using in animal studies.


Our results provide further scientific evidence supporting the use of P. africanum, Z. capense and C. anisata as anti-inflammatory and pain relief remedies in traditional medicine. To be used as herbal products the safety in animal experiments have to be confirmed. The good inhibitory activity of crude extracts containing many other compounds on 15-LOX inhibition in these plant species means that it probably contains compounds with excellent activities. Further work is required to isolate, identify and characterize the bioactive compounds that are responsible for the activities. Once the active compounds have been isolated the mechanism of activity can be examined.


  1. Sandoval M, Okuhama NN, Zhang X-J, Condezo LA, Lao J, Angeles FM, et al. Anti-inflammation and antioxidant activities of cat’s claw (Uncaria tomentos and Uncaria guianensis) are independent of their alkaloid content. Phytomedicine. 2002;9:325–37.

    Article  CAS  PubMed  Google Scholar 

  2. Vane J, Botting R. Inflammation and the mechanism of action of anti-inflammatory drugs. FASEB J. 1987;1:89–96.

    CAS  PubMed  Google Scholar 

  3. Yuan G, Wahlqvist ML, He G, Yang M, Li D. Natural products and anti-inflammatory activity. Asia Pac J Clin Nutr. 2006;15(2):143–52.

    CAS  PubMed  Google Scholar 

  4. Hougee S. Plant-derived modulators of inflammation and cartilage metabolism. In: PhD Thesis. The Netherlands: Utrecht University; 2008.

    Google Scholar 

  5. Conforti F, Sosa S, Marreli M, Menichini M, Statti GA, Uzunov D, et al. In vivo anti-inflammatory and in vitro antioxidant activities of Mediterraean dietary plants. J Ethnopharmacol. 2008;116:144–51.

    Article  CAS  PubMed  Google Scholar 

  6. Buhrmann C, Mobasheri A, Busch F, Aldeinger C, Stahlmann R, Montaseri A, et al. Curcumin modulates nuclear factor kappa β (NF-Кβ)-mediated inflammation in human tenocytes in vitro; Role of the phosphatidylinositol 3-kinase/Akt pathway. J Biol Chem. 2011;286(32):28556–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Black CD, Herring MP, Hurley DJ, O’Connor PJ. Ginger (Zingiber officinale) reduces pain caused by eccentric exercise. J Pain. 2010;11(9):894–903.

    Article  PubMed  Google Scholar 

  8. Shah BN, Seth AK, Maheshwari KM. A review on medicinal plants as a source of anti-inflammatory agent. Res J Med Plant. 2011;5(2):101–15.

    Article  CAS  Google Scholar 

  9. Iwalewa EO, McGaw LJ, Naidoo V, Eloff JN. Inflammation: the foundation of diseases and disorders. A review of phytomedicines of South African origin used to treat pain and inflammatory conditions. Afr J Biotech. 2007;6(25):2868–85.

    Article  CAS  Google Scholar 

  10. Hodzic Z, Pasalic H, Memisevic A, Srabovic M, Saletovic M, Poljakovic M. The influence of total phenols content on antioxidant capacity in the whole grain extract. Eur J Sci Res. 2009;28(3):471–7.

    Google Scholar 

  11. Sadik CHD, Sies H, Schewe T. Inhibition of 15-lipoxygenase by flavonoids: structure activity relation and mode of action. Biochem Pharm. 2003;65:773–81.

    Article  CAS  PubMed  Google Scholar 

  12. Lee S, Lee I, Mar W. Inhibition of inducible nitric oxide synthase and cycloxygenase-2 activity by 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose in murine macrophage cells. Arch Pharm Res. 2003;26:832–9.

    Article  CAS  PubMed  Google Scholar 

  13. Nilvetjdis R, van Nood E, van Hoorn DEC, Boelens EG, van Norren K, van Leeuwen PAM. Flavonoids: a review of probable mechanisms of action and potential application. Am J Clin Nutr. 2001;74:418–25.

    Google Scholar 

  14. Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsik J, Elton TS, et al. “COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression”. Proc Natl Acad Sci USA. 2002;99(21):13926–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Botting R. COX-1 and COX-3 inhibitors. Thromb Res. 2003;110(5–6):269–72.

    Article  CAS  PubMed  Google Scholar 

  16. Bezáková L, Grancăi D, Obložinsky M, Vanko M, Holková I, Pauliková I, Garaj V and Gáplovskŷ M: Effects of flavonoids and cynarine from Cynara cardunculus L., on lipoxygenase activity. Acta Facul Pharm Univ Com. Tomus Liv. 2007, Tomus Liv, 48-52

  17. Chedea VS, Jisaka M. Inhibition of soybean lipoxygenases-structural and activity models for the lipoxygenase iso-enzymes family. Recent trends for enhancing the diversity and quality of soybean products. InTech. 2005;6:109–30.

    Google Scholar 

  18. Brand-Williams W, Cuveleir ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT-Food SciTechnol. 1995;28(1):25–30.

    Article  CAS  Google Scholar 

  19. De Wet B. Medicinal plants and human health. S Afr Pharm J. 2011;78(6):38–40.

    Google Scholar 

  20. Porta H, Rocha-Sosa M. Plant lipoxygenases, physiological and molecular features. Plant Physiol. 2002;130:15–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Skrzypczak-Jankun E, Zhou K, Jankun J. Inhibition of lipoxygenase by (−)-epigallocatechin gallate: X-ray analysis at 21 A reveals degradation of ECGC and shows soybean LOX-3 complex with EGC instead. Int J Mol Med. 2003;12:415–20.

    CAS  PubMed  Google Scholar 

  22. Akula US, Odhav B. In vitro 5-lipoxygenase inhibition of polyphenolic antioxidants from undomesticated plants of South Africa. J Med Plant Res. 2008;2(9):207–12.

    Google Scholar 

  23. Radmark O, Samuelsson B. 5-lipoxygenase: mechanism of regulation. J Lipid Res. 2009;50(suppl):S40–5.

    PubMed  PubMed Central  Google Scholar 

  24. Schneider I, Bucar F. Lipoxygenase inhibitors from natural plant sources. Part 1: Medicinal plants with inhibitory activity on arachidonate 5-lipoxygenase and 5-lipoxygenase/cycloxygenase. Phytother Res. 2005;19:81–102.

    Article  CAS  PubMed  Google Scholar 

  25. Stuehr DJ, Nathan CF. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med. 1989;169:1543.

    Article  CAS  PubMed  Google Scholar 

  26. Nathan CF, Hibbs Jr JB. Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr Opin Immunol. 1991;3:65.

    Article  CAS  PubMed  Google Scholar 

  27. MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol. 1997;15:323.

    Article  CAS  PubMed  Google Scholar 

  28. Guzik TJ, Korbut R, Adamek-Guzik T. Nitric oxide and superoxide in inflammation and immune regulation. J Physiol Pharmacol. 2003;54:469.

    CAS  PubMed  Google Scholar 

  29. S-i K, Shouji A, Tomizawa A, Hiura T, Osanai Y, Ujibe M, et al. Inhibitory effect of naringin on lipopolysaccharide (LPS)-induced endotoxin shock in mice and nitric oxide production in RAW 264.7 macrophages. Life Sci. 2006;78:673–81.

    Article  Google Scholar 

  30. Lee C-J, Chen L-G, Liang W-L, Wanga C-C. Anti-inflammatory effects of Punica granatum Linne in vitro and in vivo. Food Chem. 2010;118:315–22.

    Article  CAS  Google Scholar 

  31. Lee MH, Lee JM, Jun SH, Lee SH, Kim NW, Lee JH, et al. The anti-inflammatory effects of Pyrolae herba extract through the inhibition of the expression of inducible nitric oxide synthase (iNOS) and NO production. J Ethnopharmacol. 2007;112:49–54.

    Article  PubMed  Google Scholar 

  32. Makkar HPS, Quantification of tannin in tree foliage. A laboratory manual for the FAO/IAEA co-ordinated research project on use of nuclear and related techniques to develop simple tannin assay for predicting and improving the safety and efficiency of feeding ruminants on the Tanniferous Tree Foliage. Vienna, Austria: Joint FAO/IAEA division of nuclear techniques in food and agriculture; 1999.

    Google Scholar 

  33. Yadav RNS, Agarwala M. Phytochemical analysis of some medicinal plants. J Phytol. 2011;3(12):10–4.

    CAS  Google Scholar 

  34. Brand-Williams W, Cuveleir ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT-Food SciTechnol. 1995;28(1):25–30.

    Article  CAS  Google Scholar 

  35. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation de-colourization assay. Free Radic Biol Med. 1999;26(9–10):1231–7.

    Article  CAS  PubMed  Google Scholar 

  36. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem. 1996;239(1):70–6.

    Article  CAS  PubMed  Google Scholar 

  37. Mosmann T. Rapid colorimetric assay for cellular growth and survival - application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1–2):55–63.

    Article  CAS  PubMed  Google Scholar 

  38. Olorunnisola OS, Bradley G, Afolayan AJ. Antioxidant properties and cytotoxic evaluation of methanolic extract of dried and fresh rhizomes of Tulbaghia violaceae. Afr J Pharm Pharmacol. 2011;5(22):2490–7.

    Article  Google Scholar 

  39. Eloff JN. The antibacterial activity of 27 southern African members of the Combretaceae. S Afr J Sci. 1999;95:148–52.

    Google Scholar 

  40. Masoko P, Picard J, Eloff JN. Antifungal activities of six South African Terminalia species (Combretaceae). J Ethnopharmacol. 2005;99:301–8.

    Article  CAS  PubMed  Google Scholar 

  41. Masoko P, Eloff JN. Screening of 24 South African Combretum and 6 Terminalia (Combretaceae) species for antioxidant activities. Afr J Tradit Complement Altern Med. 2007;4:231–9.

    Google Scholar 

  42. Thring TSA, Wietz FM. Medicinal plant use in the Bredadoro/Elim region of the Southern overberg in the Western Cape Province of South Africa. J Ethnopharmacol. 2006;103:261–75.

    Article  CAS  PubMed  Google Scholar 

  43. Albano SM, Lima AS, Miguel MG, Pedro LG, Barroso JG, Figueiredo AC. Antioxidant, anti-5-lipoxygenase and anti-acetylcholine esterase activities of essential oils and decoction water of some aromatic plants. Rec Nat Prod. 2012;6(1):35–48.

    CAS  Google Scholar 

  44. Adebayo SA, Shai LJ, Eloff JN. The anti-inflammatory and antioxidant activities of extracts derived from three South African medicinal plant species. SAfr J Bot. 2013;86:148.

    Article  Google Scholar 

  45. Mebe PP, Makhunga P. 11-(E)-p-Coumaric acid ester of bergenin from Peltophorum africanum. Phytochemistry. 1992;31(9):3286–7.

    Article  CAS  Google Scholar 

  46. Theo A, Masebe T, Suzuki Y, Kikuchi H. Peltophorum africanum a traditional South African medicinal plant contains an anti-HIV-1 constituent, betulinic acid. Tohoku J Exp Med. 2009;217(2):93–9.

    Article  CAS  PubMed  Google Scholar 

  47. Mazimba O: Pharmacology and phytochemistry studies in Peltophorum africanum. Bulletin Faculty of Pharm, Cairo University 2014; 52 DOI:10.1016j.bfopcu.2014.01.001.

  48. Bizimenyera SE, Swan GE, Chikoto H, Eloff JN. Rationale for using Peltophorum africanum (Fabaceae) extracts in veterinary medicine. J South Afr Vet Ass. 2005;76(2):54–8.

    CAS  Google Scholar 

  49. Bizimenyera ES, Githiori JB, Eloff JN, Swan GE. In vitro activity of Peltophorum africanum Sond. (Fabacea) extracts on egg hatching and laval development of the parasitic nematode Trichostrongylus colubriformis. Vet Parasitol. 2006;142(3–4):336–43.

    Article  CAS  PubMed  Google Scholar 

  50. Luo X, Pires D, Aínsa JA, Gracia B, Duarte N, Mulhovo S, et al. Zanthoxylum capense constituents with antimycobacterial activity against Mycobacterium tuberculosis in vitro and ex vivo within human macrophages. J Ethnopharmacol. 2013;146(1):417–22.

    Article  CAS  PubMed  Google Scholar 

  51. Mansoor TA, Borralho PM, Luo X, Mulhovo S, Rodrigues CMP, Ferreira MJ. Apoptosis inducing activity of benzophenanthridine-type alkaloids and 2-arylbenzofuran neolignans in HCT116 colon carcinoma cells. Phytomedicine. 2013;20(10):923–9.

    Article  CAS  PubMed  Google Scholar 

  52. Luo X, Pedro L, Milic V, Mulhovo S, Duarte A, Duarte N, et al. Antibacterial benzofuran neolignans and benzophenanthridine alkaloids from the roots of Zanthoxylum capense. Planta Med. 2012;78(2):148–53.

    Article  CAS  PubMed  Google Scholar 

  53. Bizimenyera E. The potential role of antibacterial, antioxidant and antiparasitic activity of Peltophorum africanum Sond (Fabaceae) extracts in the ethnoveterinary medicine. In: PhD thesis. South Africa: University of Pretoria; 2007, page 83.

  54. Atawodi SE. Antioxidant potential of African medicinal plants. Afr J Biotechnol. 2005;4(2):128–33.

    Google Scholar 

  55. Watt JM, Breyer-Brandwijk MG. The medicinal and poisonous plants of Southern and Eastearn Africa. 2nd ed. London: Livingstone; 1962.

    Google Scholar 

  56. Hutchings A, van Staden J. Plants used for stress-related ailments in traditional Zulu, Xhosa and Sotho medicine. Part 1: Plants used for headaches. J Ethnopharmacol. 1994;43(2):89–124.

    Article  CAS  PubMed  Google Scholar 

  57. Ngueyem TA, Brusotti G, Caccialanza, Finzi PV. The genus bridelia: a phytochemical and ethnopharmacological review. J Ethnopharmacol. 2009;128(3):339–49.

    Article  Google Scholar 

  58. Hutchings A, Scott AH, Lewis G, Cunningham AB. Zulu Medicinal Plants: An Inventory. Pietermaritzburg: University of Natal Press; 1996.

    Google Scholar 

  59. McGaw LJ, Jäger AK, van Staden J. Prostaglandin synthesis inhibitory activity in Zulu, Xhosa and Sotho medicinal plants. Phytotherapy Res. 1997;11:113–7.

    Article  Google Scholar 

  60. Jäger AK, Hutchings A, van Staden J. Screening of Zulu medicinal plants for prostaglandin-synthesis inhibitors. J Ethnopharmacol. 1996;52:95–100.

    Article  PubMed  Google Scholar 

  61. Owolabi OJ, Omogbai EKI. Analgesic and anti- inflammatory activities of the ethanolic stem bark extract of Kigelia africana (Bignoniaceae). Afri J Biotechnol. 2007;6(5):582–5.

    Google Scholar 

  62. Ojewole JAO. Evaluation of the analgesic, anti-inflammatory and anti-diabetic properties of Sclerocarya birrea (A. Rich.) Hochst. Stem-bark aqueous extract in mice and rats. Phytptherapy Res. 2004;18(8):601–8.

    Article  Google Scholar 

  63. Nair JJ, Aremu AO, van Staden J. Anti-inflammatory effects of Terminalia phanerophlebia (Combretaceae) and identification of the active constituent principles. South Afr J Botany. 2012;81:79–80.

    Article  CAS  Google Scholar 

  64. Kubec R, Velisck J, Musa RA. The amino acid precursors and odour formation in society garlic (T. violacea Harv.). Phytochemistry. 2002;60:21–5.

    Article  CAS  PubMed  Google Scholar 

  65. van Wyk B-E, van Oudtshoorn B, Gericke N. Medicinal plants of south africa. Pretoria: Briza Publications; 1997. p. 262.

    Google Scholar 

Download references


The Tshwane University of Technology, Pretoria, South Africa, and the National Research Foundation (South-Africa) provided funding. This study was carried out at in the Phytomedicine Programme of the University of Pretoria. Mr. Jason Sampson, the curator of the Manie van der Schijff Botanical Gardens, University of Pretoria provided support in plant collection and identification.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Jacobus N. Eloff.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

The biological assays analyses and writing the draft manuscript were done by SAA and JPD. LJS critically reviewed the manuscript and participated in the study design and choice of assay methods. JNE conceived the idea, reviewed the draft and final manuscripts and interpretation of results. All authors read and approved of the final manuscript for submission.

Rights and permissions

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit

The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Adebayo, S.A., Dzoyem, J.P., Shai, L.J. et al. The anti-inflammatory and antioxidant activity of 25 plant species used traditionally to treat pain in southern African. BMC Complement Altern Med 15, 159 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: