African Herbal Remedies with Antioxidant Activity: A Potential Resource Base for Wound Treatment

The use of traditional herbal remedies as alternative medicine plays an important role in Africa since it forms part of primary health care for treatment of various medical conditions, including wounds. Although physiological levels of free radicals are essential to the healing process, they are known to partly contribute to wound chronicity when in excess. Consequently, antioxidant therapy has been shown to facilitate healing of such wounds. Also, a growing body of evidence suggests that, at least, part of the therapeutic value of herbals may be explained by their antioxidant activity. This paper reviews African herbal remedies with antioxidant activity with the aim of indicating potential resources for wound treatment. Firstly, herbals with identified antioxidant compounds and, secondly, herbals with proven antioxidant activity, but where the compound(s) responsible for the activity has not yet been identified, are listed. In the latter case it has been attempted to ascribe the activity to a compound known to be present in the plant family and/or species, where related activity has previously been documented for another genus of the species. Also, the tests employed to assess antioxidant activity and the potential caveats thereof during assessment are briefly commented on.


Introduction
Human cells are continuously exposed to exogenous oxidants as well as to those produced endogenously during normal physiological processes. Antioxidants form part of protective mechanisms that exist in human cells to scavenge and neutralize these oxidants. Oxidants such as the reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in several diseases [1,2]. Antioxidant defenses are defective in these diseases and therefore it is possible to limit oxidative damage and ameliorate disease progression with antioxidant supplementation [3].
With reference to wounds, antioxidants play pivotal roles that consequently restore normalcy to injured skin. Basal levels of ROS and other free radicals are essential in almost all phases of the wound healing process (Figure 1) [4]. During haemostasis, ROS regulates the constriction of blood vessels to limit loss of blood. Furthermore, ROS facilitates the migration of neutrophils and monocytes from surrounding blood vessels towards the injury site. The presence of ROS and other free radicals in the wound vicinity during the inflammatory phase of the healing process is also required for infection control and general maintenance of sterility. Finally, ROS promotes the proliferation of keratinocytes, endothelial cells, and fibroblasts, thereby enhancing angiogenesis and collagen deposition. However, uncontrolled release of ROS could cause oxidative stress, resulting in cellular and tissue damage, thereby causing delayed healing [1].
To keep ROS within physiological levels, antioxidants serve as electron donors, thereby preventing them from capturing electrons from other molecules which ultimately leads to their destruction [4]. Both nonenzymatic antioxidants such as glutathione, ascorbic acid, and -tocopherol, as well as enzymatic antioxidants like catalase and peroxiredoxin, have shown potential to normalize high ROS levels and thus stimulate healing [4]. By normalizing ROS, antioxidants can enhance their physiological roles and thereby accelerate the wound healing process. Naturally occurring antioxidants are generally favoured over their synthetic counterparts, as the latter are suspected to cause or promote negative health effects [5]. This has resulted in the restricted use of synthetic antioxidants in several countries [6].
This review provides a comprehensive list of African medicinal plants and isolated compounds with antioxidant activities, with the aim of highlighting the continent's rich herbal resource base for possible management of wounds and allied conditions. Previous reviews have listed a number of these African medicinal plants with antioxidant properties [7][8][9]. The present work has therefore aimed to expand the list to include medicinal plant species with antioxidant properties that are used in different African countries including those from Madagascar and Mauritius. For the sake of inclusivity, plants that have been shown to contain compounds that hold the potential of being novel antioxidants are also considered. In addition, those with anti-inflammatory properties were also included due to an earlier observation that the anti-inflammatory activities of the same extracts could be explained, at least in part, by their antioxidant properties [10]. Additional efforts were also made to include information, where available, on their vernacular names, their regional distribution, and medicinal use and plant parts used for these preparations or for the isolation of the antioxidant ingredient(s). Table 1 lists medicinal plants that have been investigated and have confirmed antioxidant and/or antiinflammatory activity and that contain compounds which are known to have such activities. Table 2 on the other hand lists medicinal plants that have confirmed antioxidant activity but the compounds responsible for their antioxidant property have not yet been identified.

Tests Used to Assess Antioxidant Activities of African Medicinal Plant Extracts
A variety of test systems were employed to assess the antioxidant properties of the medicinal plant extracts and compounds listed in Tables 1 and 2. A comprehensive list of the methods used in antioxidant activity determination, as well as their merits and demerits, has already been published [343][344][345][346]. The methods used in the determination of antioxidant activity of natural products and isolated compounds result in varied outcomes when the same samples are tested in different laboratories and by other researchers [347]. Furthermore, results of different methods cannot be correlated, as contradictory results are usually obtained. Hence, although several assays are available, none of them is capable of accurately and completely determining the antioxidant activity of a test substance because of the complex nature of the redox-antioxidant system in vivo ( Figure 2). Based on this complexity, antioxidants are broadly classified as (i) inhibitors of free radical formation, (ii) free radical scavengers, (iii) cellular and tissue damage repairers, and (iv) signalling messengers [347]. The inhibition of free radical formation could protect against oxidative damage by suppressing the formation of active ROS/RNS. This typically involves reduction or inhibition of substrates required for free radical formation such as metal ions like iron (Fe) and copper (Cu). The sequestration of these metal ions by antioxidant compounds like ellagic acid and glutathione is known to suppress formation of  Used for malaria, jaundice.
Large-leaved forest bushwillow Leaf South Africa Antioxidant and antibacterial activities.
Also tannins showed inhibitory effect on Fe 2+ -induced lipid peroxidation and radical scavenger activity. Used for pneumonia, syphilis, abdominal pain and conjunctivitis.

Not signalized
Root-bark and seeds Cameroon Anti-inflammatory activity.
Used as an antimalarial. Used as tonic to boost the immune system.
Fenugreek Seeds Ethiopia, Morocco Protective effect against Oxidative stress during ischemia-reperfusion.
It is hypolipidemic, and is also used to treat boils and to improve appetite.
Free phenolics and Vit C.

Cameroon
Central Africa Antiradical and antioxidant activities.
Used against snakebite and to treat rheumatism, headache and stomach disorders.

Round-leaf buchu
Leaves, stems South Africa Hydroxyl radical ion scavenging ability.
Used for stomach problems, kidney and urinary track diseases.

Oval-leaf buchu
Leaves, stems South Africa Anti-inflammatory activity.
Used to treat benign prostatic hyperplasia, prostatitis, diabetes, inflammation of the colon, gums, and mucous membranes. Leaves chewed to relieve stomach complaints.
Essential oils and flavonoids [84,188,331,332] 20 Evidence-Based Complementary and Alternative Medicine  hydrogen peroxide (H 2 O 2 ) and other free radicals [348,349]. Furthermore, increasing evidence suggests a relationship between metal overload and several chronic diseases through the induction of oxidative stress [350]. Therefore, inhibition of free radical formation using metal ions as targets could be useful therapeutically. Antioxidant assays designed for this purpose include the cupric and ferric reducing antioxidant power (CUPRAC/FRAP). These methods measure the ability of antioxidants to reduce cupric (Cu 2+ ) and ferric (Fe 3+ ) ions, respectively. Another mechanism by which antioxidants act is through the suppression of oxidative stress by directly scavenging active free radicals. Most commonly reported antioxidant assays such as 2,2 -azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS), 2,2 -diphenyl-p-picrylhydrazyl radical (DPPH), oxygen radical absorbance capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC), total oxyradical scavenging capacity (TOSC), and total radical antioxidant parameter (TRAP) are focused on testing the ability to scavenge free radicals. Furthermore, there are diverse cellular antioxidant assays that assess the ability of antioxidant compounds and substances to protect cells against excessive free radical generation. Such assays involve the use of a fluorescent compound such as 2,7-dichlorofluoroscein to determine the ability of test samples to quench intracellularly generated free radicals and inhibit radical formation and lipid peroxidation [345].
There are also numerous reports of the ability of antioxidants to repair damaged tissues and improve healing. Topical application of kojic acid and deferiprone, two compounds with the ability to scavenge free radicals, enhanced healing of wounds in rats [351]. Also, the mitochondria-targeted antioxidant, 10-(6 -plastoquinonyl) decyltriphenylphosphonium, accelerated wound closure, stimulated epithelialization, granulation tissue formation, and vascularization, and lowered lipid peroxidation in mice [352]. Moreover, an antioxidant peptide (cathelicidin-OA1) promoted wound healing in a mouse model with full-thickness skin wounds, accelerated reepithelialization and granulation tissue formation by enhancing the recruitment of macrophages to the wound site, and induced cell proliferation and migration [353]. Some antioxidants have also been reported to contribute to healing by enhancing the activity of endogenous antioxidant compounds and enzymes. The induction of the nuclear factor E2-related factor 2-(Nrf2) mediated antioxidative pathway by a rhomboid family protein (RHBDF2) promoted healing of injured tissues, suggesting a relationship between antioxidant gene induction and healing [354]. Niconyl-peptide enhanced wound healing and protected against hydrogen peroxideinduced cell death by increasing the expression of Nrf2 expression in human keratinocytes [355].
The most common tests used to determine the antioxidant activity of samples included the assessment of the ability to scavenge free radicals such as DPPH, ABTS + [16,19,35,62,85,94,98,99,139,158,175,184,187,266,282,302,[356][357][358][359][360][361][362][363][364], or the hydroxyl radicals [79,188,267,365,366], as well as the hydroperoxyl radicals by the Briggs-Rauscher reaction [104]. The ability of the extracts to chelate metal ions was also determined as further indication of their ability to contribute in the reduction of free radicals such as the hydroxyl radical [114]. In addition, assessment of the ability of these medicinal plant extracts to protect against lipid peroxidation was also included, which in turn was measured by the malondialdehyde-thiobarbituric acid (MDA) test [320,367], the modified thiobarbituric acid reactive species (TBARS) assay [18,22], or conjugated diene 22 Evidence-Based Complementary and Alternative Medicine formation [367]. Moreover, lipid peroxidation was assessed using the fluorescent probe, diphenyl-1-pyrenylphosphine (DPPP) [188], or using the inhibition of Cu(2+)-mediated oxidation of human low-density lipoprotein (LDL) [187,367]. The ability of extracts to protect against damage to DNA using the Comet assay was also employed [114,188].
The antioxidant capacity of the medicinal plant extracts was determined using either the TEAC or FRAP assays [11,85,302,313,321,368]. The ability of extracts to modulate the gene expression of the antioxidant enzymes, such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase, and glutathione peroxidase (GPx), was also used as a measure of their antioxidant properties [293]. The photochemilumiescence (PLC) assay is a more recent antioxidant capacity assessment method and was employed for the evaluation of antioxidant capacity of baobab fruit pulp extracts [369].
Anti-inflammatory properties of these extracts were assessed by their ability to inhibit 5-lipoxygenases [94,370,371] or cyclooxygenase (COX-1 and COX-2) activities [65,275,317,372,373]. Using the former [374] and the latter [264,331] methodologies, respectively, a great number of South African medicinal plant extracts were screened for their anti-inflammatory properties. The effect of medicinal extracts on the biosynthesis of different prostaglandins was assessed as a measure of their anti-inflammatory effect [239,337,375]. Extracts of Podocarpus species were shown to inhibit the activities of the COX enzymes [317]. Once again, using this test, the anti-inflammatory properties of the aqueous and ethanolic extracts of 39 plants used in traditional Zulu medicine were screened [376]. The Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) assay which utilizes the CAM's capillary system in bred hen eggs was also used to assess the anti-inflammatory activity through antiangiogenic effects of the ethanol and aqueous extracts of Drosera rotundifolia and D. madagascariensis [155].
The antioxidant and anti-inflammatory abilities of the herbal extracts were further assessed by evaluating their ability to control the production of ROS produced by oxidative burst in neutrophils stimulated with L-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) [21,246]. The inhibition of neutrophils elastase was used as a measure of antiinflammatory property and it was proposed that the presence of flavonoids such as hyperoside, quercetin, and isoquercitrin in D. rotundifolia [377] and five flavonoid compounds in two Polypodium species (P. decumanum and P. triseriale) [378] were thought to contribute to this anti-inflammatory activity. These and other in vitro tests were used to assess the antioxidant properties of three Ghanaian species: Spathodea campanulata, Commelina diffusa, and Secamone afzelii [63].
Inflammation is a complex mechanism with many pathways. Several extracts derived from medicinal plants have been shown to modulate or inhibit the activities of mediators of inflammation. For instance, kolaviron, a bioflavonoid compound isolated from the seeds of Garcinia kola, has been reported to possess anti-inflammatory and antioxidant activities via its effects on COX-2 and inducible nitric oxide synthase (iNOS) by inhibiting the expression of nuclear factor kappa B (NF-B) [115]. Quercetin is a flavonoid molecule ubiquitous in nature and functions as an antioxidant and anti-inflammatory agent. Dose-and time-dependent effects of quercetin have been investigated on proinflammatory cytokine expression and iNOS, focusing on its effects on NF-B signal transduction pathways in lipopolysaccharidestimulated RAW 264.7 cells by using real time polymerase chain reaction (RT-PCR) and immunoblotting. Curcumin, a yellow pigment of turmeric, has been shown to exhibit antiinflammatory activity. Curcumin has been found effective in the treatment or control of chronic inflammatory conditions such as rheumatism, atherosclerosis, type II diabetes, and cancer [203]. Calixto et al. reported that the antiinflammatory action of active spice-derived components results from the disruption of the production of various inflammatory proteins (e.g., cytokines such as tumour necrosis factor-alpha (TNF-), iNOS, and COX-2) [379].
Animal studies were also conducted to assess the antioxidant properties of several medicinal extracts. The antioxidant potential of Hypericum perforatum, containing many polyphenolic compounds, was evaluated on splanchnic artery occlusion (SAO) shock-mediated injury [477] and also against elevated brain oxidative status induced by amnestic dose of scopolamine in rats [126]. Some medicinal plant extracts were tested for their ability to protect against carbon tetrachloride-, 2-acetylaminofluorene-(2-AAF-), and galactosamine-induced liver as well as aflatoxin B1-(AFB1-)induced genotoxicity. Using this test, it was found that an extract of Garcinia kola seeds [116,478,479], a decoction of Trichilia roka root [270], extracts of Entada africana [442], and Thonningia sanguinea [98,480] possessed protective abilities. The antioxidant properties of plant extracts against potassium bromate (KBrO(3))-induced kidney damage showed the ability of G. kola seed extract to protect the kidneys [481].
Not identified [469] 38 Evidence-Based Complementary and Alternative Medicine Used to treat bladder ailments, wounds, impotence and sterility, and to help in childbirth.

Crude Extracts of African Medicinal Plants with Confirmed Antioxidant Activities
The antioxidant properties of a larger proportion of African medicinal plants listed in Tables 1 and 2 were tested using either aqueous or organic plant extracts. After confirming antioxidant properties, a correlation was proposed between this property and the general groups of antioxidant compounds that are present in these extracts. No further attempts were made to isolate the specific compounds that may have contributed towards this property. Flavonoids in Aloe barbadensis [32], chromone glycosides in A. claviflora [35], essential oils in Artemisia abyssinica, and Juniperus procera [79] as well as Helichrysum dasyanthum, H. felinum, H. excisum, and H. petiolare [94], proanthocyanidins in Burkea africana bark [175], polyphenols in extracts of Crataegus monogyna [321], saponins, and alkaloids in extracts of Leucosidea sericea [210,211] are all considered as major compounds that have contributed to the antioxidant properties of these plants. Reports on a number of Barleria species, which includes B. albostellata, B. greenii, and B. prionitis, have indicated their anti-inflammatory [212] and antioxidant capacities [213]. Unlike the isolated compounds, most of the plants listed for possessing antioxidant activity, including extracts of Agerantum conyzoides, Euphorbia hirta, Kigelia africana, and Nauclea latifolia, have been shown to possess wound healing ability [494][495][496]. Furthermore, studies have focused on screening a vast number of plants, used in a specific region, so as to determine their antioxidant properties, Mali [357], South Africa [19,188,267,364], Cameroon [182,313], Algeria [85], Ghana [98], Burkina Faso [266], Madagascar [23], and Mauritius [293], and anti-inflammatory properties, South Africa [168,264,374,376] and West Africa [400].

Discussion and Conclusion
The use of traditional herbal remedies as alternative medicine plays a significant role in Africa since it features extensively in primary health care. The search for natural antioxidants, especially from plant sources, as a potential intervention for treatment of free radical mediated diseases is an important research field, especially for those in developing countries. Many polyphenols, including phenolic acids, flavonoids (anthocyanins and anthoxanthins), tannins, and lignans, are known to act as antioxidants and protect against various pathological conditions such as coronary artery disease and wounds, in addition to their anti-inflammatory, antimicrobial, and anticancer activities [214][215][216].
Flavonoids are a large group of compounds containing several hydroxyl groups on their ring structures and include isoflavonoids and isoflavonoid glycosides, flavones, and flavone glycosides, flavonols and flavonol glycosides, anthocyanins, chalcones and dihydrochalcones, aurones, flavonones and dihydroflavonols, and flavans and biflavonyls. To date, approximately 9000 different flavonoids have been identified from plant sources [217]. Great interest has been dedicated to the antioxidant properties of flavonoids that may function as potent free radical scavengers, reducing agents, and protectors against peroxidation of lipids [208,218]. Reviews have been published documenting numerous studies on antioxidant efficacy of flavonoids and phenolic compounds as well as on the relationship between their antioxidant activities, as hydrogen donating free radical scavengers, in relation to their chemical structures. The importance of the unsaturation in the C ring of quercetin compared to catechin in the increased antioxidant activity of the former has been presented [216,[219][220][221][222][223]. Also, the importance of the position and number of hydroxyl groups on the phenolic rings in increasing or decreasing the antioxidant properties of these compounds has been emphasized [216,[219][220][221][222][223].
Antioxidants may exert their protective effects via different mechanisms at different stages of the oxidation process. There are those that are able to inhibit the production of free radicals via their ability to chelate transition metal ions and those that are able to quench and stabilise free radicals [229,230]. Additionally, they are further subdivided into categories according to their functions [230]. Such classification of the newly isolated antioxidant compounds from African medicinal plant extracts is warranted to better understand their antioxidant properties.
It should be noted that the antioxidant activity of the extracts and compounds listed in this review was mostly determined using either single assays or in vitro analysis. It is therefore possible that some of these extracts and compounds may not show antioxidant activity when alternative testing methods are used. Furthermore, although in vivo studies are encouraged, most studies cited used in vitro assays. As antioxidant activity in vitro does not necessarily translate to activity in vivo, due to pharmacokinetic and pharmacodynamic processes that occurs in vivo, it is possible that samples may not be active when tested in animals. Activity of such samples should therefore be confirmed using animal models.
Additionally, attempts should be made to identify the compounds responsible for the proven antioxidant properties where not yet done, and in cases where they have been isolated, their wound healing properties should be investigated. If the activity of the compounds and plants identified in this review is confirmed in vivo, they could serve as viable sources for the treatment of wounds in future.

Conflicts of Interest
The authors declare that they have no conflicts of interest.