A Review of Ethnoveterinary Knowledge, Biological Activities and Secondary Metabolites of Medicinal Woody Plants Used for Managing Animal Health in South Africa

Globally, the use of ethnoveterinary medicine as remedies for animal health among different ethnic groups justify the need for a systematic exploration to enhance their potential. In addition, the increasing popularity and utilisation of woody plants remain common in traditional medicine, which may be attributed to their inherent benefits. The current review was aimed at analysing ethnoveterinary surveys, biological activities, and secondary metabolites/phytochemical profiles of the woody plants of South Africa. Eligible literature (period: 2000 to 2020) were retrieved from different databases such as Google Scholar, PubMed, Sabinet, and Science Direct. Based on the inclusion and exclusion criteria, 20 ethnoveterinary surveys were eligible and were subjected to further analysis. We identified 104 woody plant species from 44 plant families that are used in the treatment of different diseases in animals, particularly cattle (70%) and goats (20%). The most mentioned (with six citations) woody plants were Terminalia sericea Burch. ex DC and Ziziphus mucronata Willd., which were followed by plants with five (Cussonia spicata Thunb., Pterocarpus angolensis DC and Vachellia karroo (Hayne) Banfi & Galasso) or four (Acokanthera oppositifolia (Lam.) Codd, Cassia abbreviata Oliv., and Strychnos henningsii Gilg) individual mentions. The most dominant families were Fabaceae (19%), Apocynaceae (5.8%), Rubiaceae (5.8%), Anacardiaceae (4.8%), Combretaceae (4.8%), Euphorbiaceae (4.8%), Malvaceae (4.8%), Rhamnaceae (4.8%), and Celastraceae (3.8%). Bark (33%), leaves (29%), and roots (19%) were the plant parts dominantly used to prepare remedies for ethnoveterinary medicine. An estimated 20% of woody plants have been screened for antimicrobial, anthelmintic, antioxidant, and cytotoxicity effects. Phytochemical profiles established a rich pool of valuable secondary metabolites (phenolic, flavonoids and condensed tannins) that may be responsible for the exerted biological activities. Overall, the significant portion of woody plants lacking empirical evidence on their biological effects indicates a major knowledge gap that requires more research efforts.


Introduction
Globally, many animals, especially cattle, goats, and horses, play diverse role in human life, ranging from being a source of food, income and cultural wealth, touristic attraction, and job creation [1][2][3][4][5][6]. The rearing of animals is well-embedded in the culture of many ethnic groups, which justifies the popularity of indigenous knowledge and practice for managing the health and well-being of animals [5,[7][8][9][10][11]. Relative to ethnobiology/ethnobotany,

Materials and Methods
The literature search strategy was facilitated using keywords such as woody plants, ethnoveterinary medicine, livestock, and animal health. In addition, phytochemical, antioxidant, phenolic, and antibacterial effect were examples of terms used to generate data for the biological activity and phytochemical aspects of this review. These keywords were used singly and in combination to identify suitable literature from several databases, namely Google Scholar, Pubmed, Science Direct, and Sabinet. We focused on peer-reviewed papers published from 2000 to 2020 on South African woody plants.
Screening of the research outputs from the databases was conducted in two stages. Firstly, the title and abstract of the papers were screened against the inclusion criteria. A publication needed to provide the Latin name for the woody plant to be eligible for inclusion. Articles reporting on ethnoveterinary uses, biological activities, and phytochemical analyses of the woody plants fulfilled the inclusion criteria (e.g., specified time duration, woody species, and South African studies). Review papers and studies not involving South African medicinal woody plants were excluded. Based on the selection criteria, 20 papers were selected and were analysed in order to generate an inventory of woody plants (Table 1). Subsequently, analyses on plant families, mode of preparation, plant parts used to treat animals/livestock diseases, biological activities, and phytochemicals were conducted. Based on the significance of scientific names [31,32], the botanical names were validated via multi-databases, such as PlantZAfrica (http://pza.sanbi.org/ (accessed on 29 September 2021)), The Plant List (http://www.theplantlist.org/ (accessed on 29 September 2021)), and The World Flora Online (http://www.worldfloraonline.org/ (accessed on 29 September 2021)). Structured questionnaire and general conversation 30 Mkwanazi et al., [42] KwaZulu-Natal 5 4 1 1 Structured questionnaire Not specified Moichwanetse et al., [43] North West 25 18 8 5 Face-to-face, semi-structured interview 15 Moyo and Masika [44] Eastern Cape  2  2  1  1  Structured  questionnaire  59 Mthi et al., [45] Eastern Cape 6 6 4 3 Semi-structured questionnaire, observations and guided field trip 48 Mwale and Masika [46] Eastern Cape 9 7 1 1 Structured questionnaire 54 Ndou [47] North West 31 17 8 7 Semi structured face-to-face interview 21 Ramovha

Overview of Eligible Literature and Ethnoveterinary Studies
The eligible studies were conducted in five of the nine provinces in South Africa. These included the Eastern Cape (45%), Limpopo (30%), North West (15%) Mpumalanga (5%), and KwaZulu-Natal (5%) provinces ( Table 1). The majority of these aforementioned provinces are regarded as predominantly rural-based, which may explain the continuous dependence on woody plants for veterinary needs. In a recent review of the ethnoveterinary plants of South Africa, McGaw et al., [15] indicated a similar distribution pattern relating to the use of medicinal plants as remedies for animals/livestock in South Africa. In Pakistan, a rich ethnoveterinary knowledge was recorded in communities residing in remote areas with limited access to conventional veterinary services, which often forced the inhabitants to rely on the natural resources within their immediate environment to meet the health needs of their livestock [14].
The data collection methods included the use of questionnaires, interviews, field observations, and Rapid Rural Appraisal (Table 1). Data on ethnoveterinary medicine was collected from diverse participants such as farmers, cattle headers, indigenous knowledge holders, and traditional healers. In terms of number, the number of participants ranged from 15 [43] to 180 [33] while about 30% of the studies had no indication of the sample size involved in the ethnobotanical survey. Given that the primary focus of these surveys was not on woody plants, varying portions (11-100%) of woody species were identified in the recorded plants (Table 1). In the survey in the Eastern Cape by Dold and Cocks [34], approximately 40% of the 53 recorded plants with ethnoveterinary value were woody plant species. A similar trend was evident in other ethnoveterinary surveys in the Eastern Cape [35,41,51] and Limpopo [48] provinces. In North West province, the portion of woody plants ranged from 26% [47,52] to 32% [43]. In an attempt to understand the basis for the selection and utilisation of plants by local communities, several theories and hypotheses exist [53]. In the current situation, the ecological apparency hypothesis likely accounts for the use of woody plants for ethnoveterinary medicine among local communities. Even though South Africa is recognized as being rich in biodiversity and diverse vegetation-types, it remains highly susceptible due to rapid development, habitat loss, and overexploitation [16]. Increasing evidence supported the dynamic nature of the existing vegetation in South Africa, which is associated with the effects of climate change [54]. On this basis, it is often difficult to predict the pattern for the use of woody plants in ethnoveterinary medicine.

Inventory of Woody Plants with Ethnoveterinary Uses
The high reliance on plants for managing livestock/animals among local communities, especially in developing countries, remains a common trend [14,55]. This popularity has often been attributed to the limited access to convention veterinary drugs and the existence of vast indigenous knowledge for managing livestock in rural communities [47,56,57]. Several studies have revealed that traditional medicines are mostly used because they are regarded as effective and readily available as well as accessible. As often indicated by participants in ethnoveterinary surveys, dependence on traditional medicines is common because western veterinary facilities are inaccessible and are too costly for resource-poor livestock farmers [34,37,45].
Based on the 20 eligible studies from the literature, we generated 104 woody plants with diverse ethnoveterinary uses in South Africa (Table 2). Terminalia sericea Burch. Ex DC and Ziziphus mucronata Willd were the most common plants, with six mentions. Furthermore, Cussonia spicata Thunb., Pterocarpus angolensis DC., and Vachellia karroo (Hayne) Banfi & Galasso (five citations) and Cassia abbreviata Oliv. and Strychnos henningsii Gilg (four citations) were popular within the 20 analysed studies from the literature. On the other hand, the majority (86%, i.e., 89 plants) of the 104 woody plants had limited (1-2) mentions.               Ndou [47] In terms of plant families (Figure 1), most of the identified plants were from the Fabaceae (19%), Apocynaceae (5.8%), Rubiaceae (5.8%), Anacardiaceae (4.8%), Combretaceae (4.8%), Euphorbiaceae (4.8%), Malvaceae (4.8%), Rhamnaceae (4.8%), and Celastraceae (3.8%) families. Even though 44 families were recorded, the majority (estimated 64%) of the families were represented by one woody plant. Based on the analysis of approximately 4576 vascular plants representing 192 families (from the 254 African families) with medicinal value in sub-Saharan African, the dominance of Fabaceae remains evident in African traditional medicine [58]. Furthermore, Fabaceae was the most represented plant family for plants used against cattle diseases in South Africa [59]. Plant parts used to prepare herbal remedies included bark, leaves, fruits, roots, and flowers ( Figure 2). However, the most commonly used plant parts for remedy preparations were bark (33%) followed by leaves (29%) and roots (19%). The dominance of plant parts such as bark and roots may not be sustainable overtime, as their indiscriminate harvesting is often of great conservation concerns for the survival of the plant [26]. Thus, conscious efforts remain essential to ensure good harvesting practices and the long-term sustainability of these valuable woody plants.

Overview of Animals/Livestock and Diseases
As shown in Figure 3, cattle were the major (61%) animal/livestock treated with the woody plants. In South Africa, the importance of cattle among different cultural groups cannot be overemphasized [1,59]. Van der Merwe et al., [52] documented the use of ethnoveterinary medicinal plants in cattle by the Setswana-speaking people in the Madikwe area of the North West Province. The most important diseases treated were retained placenta, diarrhoea, fractures, fertility enhancement, general gastrointestinal problems, and pneumonia. A high proportion of the woody plants were used for diarrhoea. Some of the plants documented during the study are used elsewhere in the Eastern Cape to treat different livestock diseases. These include Vachellia karroo, Vachellia tortilis, Cussonia spicata, Rhoicissus tridentata, and Ziziphus mucronata.

In Vitro Biological Screening of Woody Plants
The increasing incidence of drug resistance in most pathogenic bacteria and parasites that cause economic loss in animals/livestock production calls for the development of new sources for medication [60,61]. Among to the 104 woody plants with ethnoveterinary uses in South Africa (Table 2), approximately 20% were screened for their relevant biological activities (e.g., antibacterial, anthelmintic, and antioxidant) and safety (cytotoxicity) level. However, the current review included woody plants that have been screened for biological activities without evidence of their ethnoveterinary use in South Africa. This approach may increase the success rate of bio-prospecting for therapeutic woody plants for ethnoveterinary needs in South Africa [62]. As highlighted by Eloff [63], no statistically significant difference was observed in the antimicrobial activity of plants with ethnobotanical knowledge when compared to randomly selected plants. Hence, the most promising biological activity may not correlate with the most popular plants with existing ethnobotanical knowledge [62,64].

Antibacterial Activity
Even though the antibacterial effects of 39 woody plants were reported (Table 3), approximately 56% of the 39 plants lacked ethnoveterinary applications in the eligible studies that were recorded (Tables 1 and 2). In terms of the assay-type, approximately 95% of the studies were conducting using the micro-plate dilution method, which is considered as a more robust and reliable assay relative to agar diffusion [63,64]. Based on the recorded antibacterial activity (Table 3), Gram-positive bacteria were more dominant (57% of the 14 organisms) than Gram-negative bacterial strains. Although a diverse range of bacterial strains was tested, the relevance and justification for their selection were unclear in most of the studies. Researchers need to be cognizant of the bacteria type in order to demonstrate the clinical relevance of the anti-bacterial effect of the tested plant extracts [64,65].
Leaves/aerial parts (77%) and bark (17%) were the most common parts of the woody plants that were evaluated for their antibacterial activity. Remarkable differences in the antibacterial effect of woody plant parts were evident in Leucosidea sericea [69], Schotia brachypetala Sond, Searsia lancea (L.f.) F.A.Barkley (Rhus lanceas), and Ziziphus mucronata Willd [70]. Particularly in Schotia brachypetala and Ziziphus mucronata, the leaf extracts had remarkable antibacterial effects against the tested bacterial strains while the bark extracts were ineffective. Furthermore, the type of solvent used for extracting the plant parts strongly influenced the resultant antibacterial response (Table 3). Despite the popularity of water as the most commonly used solvent in traditional medicine, water extracts often exhibit weaker antibacterial effects relative to many organic solvents [63].

Anthelmintic Activity
As highlighted by Aremu et al., [74], evaluating anthelmintic potential is often conducted using (i) developmental and behavioural assays (DBA) and (ii) colorimetric assays (CA). Following treatment and incubation with plant extracts, the assays measure the survival and/or reproductive potential (DBA) or metabolic response using the appropriate marker (CA). A total of 48 woody plants have been tested for their anthelmintic activity, which was mainly (90%) assessed using DBA (Table 4). However, only 42% of these woody plants had existing indigenous knowledge related to the management of animal health among local communities in South Africa. Alsophila dregei (Kunze) R.M.Tryon, Leucosidea sericea, and Sclerocarya birrea were identified as the most commonly evaluated woody plants in terms of their anthelmintic effect. Using predefined anthelmintic effect categories [69], the organic solvent extracts of Leucosidea sericea had high (minimum lethal concentration, MLC = 0.26-0.52 mg/mL) anthelmintic activity against Caenorhabditis elegans [69]. Likewise, Fouche et al., [75] demonstrated that Maerua angolensis stem extract exerted 65% inhibition, which was noteworthy among all of the evaluated woody plants. Furthermore, extracts from Heteromorpha trifoliata, Maesa lanceolate, and Leucosidea sericea using an egg hatch assay (e.g., of DBA) exhibited significant anthelmintic activity against Haemonchus contortus and killed 100% of the parasites when administered at the dosages of 12.50, 6.25, and 3.13 mg/mL [76]. Fouche et al., [75] investigated the acetone extracts of various woody plants for their anthelmintic activity against Haemonchus contortus, and the stem of Maerua angolensis had a mean inhibition rate of 65%, which was noteworthy compared to the other plants tested and included in the review.
Caenorhabditis elegans (63%), Haemonchus contortus (35%), and Trichostrongylus colubriformis (2%) have been the widely used organisms for assessing the anthelmintic effects of woody species (Table 4). In recent times, the use of free-living nematodes, particularly, have remained common due to their inherent benefits [74,77,78]. Caenorhabditis elegans is regarded as the best representative of a large phylum that contains several parasites [78]. However, the use of Caenorhabditis elegans as a test organism has resulted in limited success in terms of the discovery of valuable new leads [62,77,79]. Hence, Caenorhabditis elegans should only serve as a screening tool for the rapid identification of promising plant extracts that will be further subjected to more appropriate test model(s).
The type of solvents used for plant extraction has a critical influence on the anthelmintic effect of woody plants (Table 4). For instance, the ethyl acetate extract of Combretum apiculatum exhibited strong lethality, killing 70-80% of nematodes (Caenorhabditis elegans) while the water extract had a 10-20% killing rate at 1 mg/mL [80]. Furthermore, Searsia lancea hexane extract had higher (50%) in vitro anthelmintic activity against Caenorhabditis elegans than the methanol and water extracts did [70]. The in vitro anthelmintic efficacy of several woody plants against Caenorhabditis elegans revealed that ethanol extracts possessed higher anthelmintic activity than water extracts [71].
Contrary to the majority of studies focusing on an single test organism (Table 4), Shai et al., [81] evaluated the anthelmintic activity of Curtisia dentata against parasitic (Trichostrongylus colubriformis and Haemonchus contortus) and the free-living (Caenorhabditis elegans) nematodes. The acetone and dichloromethane extracts were active against all of the nematodes at concentrations as low as 160 µg/mL. This finding clearly highlights the anthelmintic potential of Curtisia dentata, which requires further experiments, especially in terms of its in vivo response.

Antioxidant Activity
Antioxidants are free radical scavengers and often possess the ability to reverse or repair the damage caused by free radicals in animal cells [82]. Recently, there has been increasing interest in determining the antioxidant potential of plants used for medicinal purposes [83]. It is generally known that damages caused by reactive oxygen species are often a contributing factor to many diseases [84]. As shown in Table 5, the antioxidant potential of the 24 woody plants have mainly been evaluated via in vitro assays including ABTS-2,2azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), DPPH-1,1-diphenyl-2-picryl-hydrazyl, and FRAP -ferric reducing antioxidant power. Relative to the inventory in Table 2, only six woody plants show antioxidant activity.
Given that these bio-analytical assays differ in terms of reaction mechanisms, oxidant, and target species as well as reaction conditions [82], it is often beneficial to evaluate plant extracts in multi-assays. Based on the DPPH assay, the most promising (EC 50 < 5 µg/mL) antioxidant activity was exerted by woody plants such as Alsophila dregei, Apodytes dimidiata, Brachylaena discolor, Burkea africana, Clausena anisata, Combretanum zeyheri, Millettia grandis, Strychnos mitis, Volkameria glabra, and Zanthoxylum capense. Similar noteworthy antixodant effects was observed in the ABTS assay for Burkea africana and Combretum zeyheri [85]. However, moderate antioxidant activity ranging from 68-579 µg/mL was demonstrated among the eight evaluated woody plants. These aforementioned antioxidant tests were in vitro-based, thereby limiting the clinical relevance of the current findings. Hence, it will be pertinent to establish the in vivo antioxidant activity of woody plants with noteworthy response.

Cytotoxicity
The safety of medicinal plants remains essential toward the drive to incorporate these valuable natural resources as part of healthcare for animals. Evidence of the cytotoxicity levels for 39 woody plants were recorded in the current review (Table 6). Approximately 51% of these woody plants have explicit applications in South African ethnoveterinary medicine (Table 2). Particularly, the safety of different organs from three plants namely Calpurnia aurea, Maesa lanceolata, and Sclerocarya birrea were assessed in more than one study [66,70,75,76].
According to the United States National Cancer Institute (NCI), the criteria for the cytotoxicity of crude extracts, extracts with an LC 50 value that is less than 20 µg/mL are classified as cytotoxic. On this basis, Apodytes dimidiate, Brachylaena discolour, Calpurnia aurea, Elaeodendron croceum, Maesa lanceolata, and Strychnos mitis exerted varying degrees of cytotoxicity (LC 50 = 3.32-19.9 µg/mL), and caution needs to be taken during their utilisation for ethnoveterinary medicine [66,67,76]. Furthermore, McGaw et al., [70] assessed the cytotoxicity activity of the hexane, methanol, and water extracts of the selected woody plants against the larvae of Artemia salina (brine shrimp). From the results, the water extracts from Searsia lancea and Ziziphus mucronata leaves displayed strong lethality to the tested organism. On the other hand, moderate cytotoxicity was demonstrated by the acetone and water extracts of Vachellia nilotica bark against Vero monkey cell assays, and these extracts exhibited toxic effects on the cells with LC 50 = 33.2 µg/mL and LC 50 = 27.8 µg/mL, respectively. This was closely-followed by the acetone extracts from Tetradenia riparia, leaf with LC 50 = 51.3 µg/mL [86].

Phytochemical Analysis of Plants Used for Ethnoveterinary Purposes
Phytochemical screening is important when investigating medicinal plants given that bioactive compounds can be responsible for their resultant biological activities [87,88]. In particular, the phenolic compounds in plants serve as defense mechanisms against pathogens and may be explored for therapeutic purposes [89]. The 20 woody plants recorded exhibit a diverse range of phytochemicals (Table 7), an indication of their potential benefits as ethnoveterinary medicine. For instance, 12 selected woody plant extracts had a rich source of phenols that ranged from 100 to 428 mg GAE/g, and Lippia javanica had the highest phenolic content while Englerophytum magaliesmontanum had the lowest content [85]. In addition, the flavonoid content varied from 6-159 mg QE/g, as contained in Ehretia rigida (lowest) and Leucaena leucocephala (highest). Olaokun et al., [90] quantified the total phenolic and flavonoid contents in Curtisia dentata and Pittosporum viridiflorum. The results indicated that Curtisia dentata extract yielded the higher phenolics (125.12 mg/g GAE) and flavonoids (27.69 mg/g GAE) compared to the extract from Pittosporum viridiflorum.      [85] In recent times, increasing evidence from several studies on polyphenolic compounds from medicinal plants support their biological and pharmaceutical importance in maintaining animal health and overall productivity [91]. For example, betulinic acid and lupeol were successfully isolated from Curtisia dentata [81], which is one of the woody plants recorded in our inventory (Table 2). Subsequently, both compounds demonstrated a moderate degree of an anthelmintic effect (200 and 1 000 µg/mL) against parasitic nematodes. However, the relatively higher concentration required for the compounds to be effective limits their clinical relevance as an anthelmintic for livestock.

Conclusions
The current review entailed an overview of the role and contributions of woody plants in ethnoveterinary medicine in South Africa. We highlighted the richness of South Africa's flora as a medicinal resource and the effectiveness of woody plants used in ethnoveterinary medicine. Terminalia sericea and Ziziphus mucronata were the most commonly utilised woody plants based on existing indigenous knowledge. The extensive utilisation of some plant parts (e.g., bark and roots) remain a major concern due to the potential detrimental effects of the indiscriminate harvesting of such parts may have on the survival and sustainability of the woody plants. The majority (80%) of woody plants with indigenous knowledge related to their applications in the management of animal health remain poorly evaluated in terms of their biological efficacy and phytochemical composition. Nevertheless, some of the woody plants (e.g., Alsophila dregei, Cussonia spicata, Indigofera frutescens, Leucosidea sericea, and Maesa lanceolata) have demonstrated promising biological activities, mainly in antibacterial and anthelminthic assays. Given the pre-dominantly in vitro based assays currently being utilised, there is an urgent need to evaluate woody plants with promising biological effect in appropriate in vivo models. The test organisms need to have direct relevance to prevailing health challenges facing livestock in rural areas where the use of woody plants have been widely documented. In terms of the phytochemical profiles, South African woody plants have a rich pool of chemicals with potential therapeutic effects.