Background
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 A
2, 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 A
2 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 [
29‐
31].
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.
Methods
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
Acacia burkei
| Fabaceae | PRU/120581/1/Adebayo SA | Black monkey thorn | Painful back and eye [ 10] | 110 | 3.7 |
Acacia sieberiana
| Fabaceae | PRU/120582/1/Adebayo SA | Paperback thorn | Fever, back aches and pain [ 10] | 210 | 7.0 |
Acokanthera oppositifolia
| Apocynaceae | PRU/120583/1/Adebayo SA | Bushman’s poison | | 520 | 17.3 |
Bridelia micrantha
| Phyllanthaceae | PRU/120584/1/Adebayo SA | Coast gold leaf | Anti-inflammatory, abdominal pain [ 57] | 340 | 11.3 |
Clausena anisata
| Rutaceae | PRU/120585/1/Adebayo SA | Maggot-killer | Abdominal pain, fever, rheumatism [ 58] | 190 | 6.3 |
Dichrostachys cinerea
| Fabaceae | PRU/120586/1/Adebayo SA | Sickle bush | | 50 | 1.7 |
Ekebergia capensis
| Meliaceae | PRU/120587/1/Adebayo SA | Cape ash | Headaches, backaches and cough [ 58] | 120 | 4.0 |
Erythrophleum lasianthum
| Fabaceae | PRU/120588/1/Adebayo SA | Thornless tree | | 350 | 11.6 |
Harpephyllum caffrum
| Anacardiaceae | PRU/120589/1/Adebayo SA | Wild plum or bush mango | | 120 | 4.0 |
Kigelia africana
| Bignoniaceae | PRU/120590/1/Adebayo SA | Sausage tree | Analgesics, fever rheumatism [ 61] | 60 | 2.0 |
Melianthus comosus
| Melianthaceae | PRU/120591/1/Adebayo SA | Honey flower | | 260 | 8.7 |
Peltophorum africanum
| Fabaceae | PRU/120592/1/Adebayo SA | African/weeping wattle | | 100 | 3.3 |
Pittosporum viridiflorum
| Pittosporaceae | PRU/120593/1/Adebayo SA | Cheesewood | | 140 | 4.6 |
Plumbago auriculata
| Plumbaginaceae | PRU/120594/1/Adebayo SA | Plumbago | Headaches and malaria relief [ 58] | 80 | 2.7 |
Polygala fruticosa
| Polygalaceae | PRU/120595/1/Adebayo SA | Butterfly bush | | 570 | 19.0 |
Ptaeroxylon obliquum
| Ptaeroxylaceae | PRU/120596/1/Adebayo SA | Sneezewood | | 200 | 6.7 |
Rhus chirindensis
| Anacardiaceae | PRU/120597/1/Adebayo SA | Red currant | | 200 | 6.7 |
Sclerocarya birrea
| Anacardiaceae | PRU/120598/1/Adebayo SA | Marula | Anti-inflammatory, fever [ 62] | 150 | 5.0 |
Tecomaria capensis
| Bignoniaceae | PRU/120599/1/Adebayo SA | Cape honey suckle | | 170 | 5.7 |
Terminalia phanerophlebia
| Combretaceae | PRU/120600/1/Adebayo SA | Lebombo cluster leaf | Aches, wounds and infections [ 41, 63] | 210 | 7.0 |
Trichilia dregeana
| Meliaceae | PRU/120601/1/Adebayo SA | Thunder tree | Stomach ailment and backaches [ 58] | 70 | 2.3 |
Terminalia prunioides
| Combretaceae | PRU/120602/1/Adebayo SA | Lowveld cluster leaf | Abdominal pains, backaches [ 41] | 170 | 5.7 |
Tulbaghia violacea
| Alliaceae | PRU/120603/1/Adebayo SA | Wild garlic | Pain relief and fever [ 64] | 660 | 22.0 |
Warburgia salutaris
| Canellaceae | PRU/120604/1/Adebayo SA | Pepper-bark tree | Headaches, influenza and fever [ 65] | 150 | 5.0 |
Zanthoxylum capense
| Rutaceae | PRU/120605/1/Adebayo SA | Small knobwood | | 220 | 7.3 |
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 (IC
50) 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 % CO
2. 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 × 10
5 cells/mL (100 μL) in 96-well microtitre plates and incubated at 37 °C and 5 % CO
2 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 CO
2 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 LC
50 values by the MIC values (SI = LC
50/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.
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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.