In Vitro Evaluation of Anthelmintic Interaction of Plant Species Combinations Putatively Containing Similar Bioactive Macromolecules in Sheep

In the phase of waning chemo-anthelmintic efficacy and general selection by nematode parasites and other pathogenic micro-organisms for single or multiple drug resistance, it is imperative to explore how efficacy of plants possessing anthelmintic activity among other biological activities can be optimized [1-5]. High plant species anthelmintic efficacy, though often lower than that of their orthodox counterparts, has advantage of retaining nematode load below that which will affect production negatively; referred to as “economic threshold” [6].

Non-chemical helminth control methods of this nature will serve as viable alternatives (options), which will reduce recurrent use of relevant chemical anthelmintics or reliance on a few. This, in effect, deters selection for resistant parasites, as has been strongly implicated in the current crisis [7]. Plants, by their very nature, contain a wide variety of bioactive macromolecules belonging to the same and/or different classes of compounds and can be likened to combined remedies setting precedence to anthelmintic therapy [8-12].

Combination anthelmintic therapy or prophylaxis has currently been adopted to retain high drug efficacy and simultaneously recycled those anthelmintics that have failed or currently exert relatively low efficacy [13-16]. Additionally, combination therapy widens spectrum of nematode parasite control within particular and different sites of infection in the gastrointestinal tract hosting them [17,18]. Gastrointestinal nematode infection of livestock poses a huge economic challenge to health and productivity of grazing-livestock globally, relative to those in confinement [7,19-22]. This problem has attained unprecedented level in small ruminants (goats and sheep), in addition to challenges of current control strategies [23]. Given the role of small ruminants as important source of wealth and animal protein to low resource households of Africa, Asia and most developing nations, any perturbation to their productivity or mortality resulting from infection by these parasites will have a huge setback on their income and livelihood [24]. Consistent and progressive failure of chemical anthelmintics, which has been the primary method of nematode parasite control from their inception, demand a review of previous and current modes of application and possibly a fundamental change to re-establish high efficacy [25-28]. Additionally, potential methods of improving ethno botanical anthelmintic efficacy will prove to be a very useful and crucial tool in the control process.

These changes are critical, because they will potentially arrest rapid development of resistant parasites and improve the efficacy span of anthelmintics in use, including subsequent candidates that will be developed. Additionally, research and development of new and more effective chemical candidates have similar high efficacy with preceding ones turned ineffective, as resistant nematode strains still emerge sooner or later [29,30]. Extensive research and development of new anthelmintic candidates could well be out of favour with the economic interest of major pharmaceutical industries, because of the sheer size of small ruminant industry that is mostly affected relative to other domestic ruminant species [7,30]. These challenges raise a lot of concerns, reaffirming dire need to conserve the efficacy of current anthelmintics and concurrently seek avenues of optimization. Existing control strategies not precluded, other viable and sustainable options should be explored, with the former ones serving as important platform for future research, innovation and development. Two principal methods of gastrointestinal parasite control of livestock have been implemented; external animal environmental control strategy that is prophylactic in nature, and internal gastrointestinal control using anthelmintic remedies that are either therapeutic or prophylactic. External strategies of control, some of which include grazing management, use of treated or conserved forages among others, seek to deter build-up of intermediate developing stages of parasites in the animal host environment [21,31]. Internal anthelmintic remedies are either chemical or bioactive principles that combat parasites within the host animal.

The later phase of gastrointestinal parasite control is critical to improve animal health and productivity. Wide-spread application of chemical anthelmintics has been fraught with wide ranging challenges, some of which include emergence of resistant strains to all classes of chemical anthelmintics, lodging of residues in animal products, environmental pollution by excreted un-metabolised chemical anthelmintics and induced resistance by rendering them unduly available to untargeted organisms [1-3,22,32-40]. Innovation of this method of control by adopting combination therapy has been another option of enhancing anthelmintic efficacy. It involves combined administration of two or more chemical anthelmintics, following waning efficacy of any one of them, thus altering and improving pharmacokinetic and pharmacodynamic activities of one or both [13,41-44]. These practices are commendable though resistant strains of gastrointestinal nematodes still emerge with time, when consistently used without alternating with other effective options. It is therefore critical to further explore and diversify other options, in view of attaining this goal. Besides, emergence of resistant gastrointestinal nematode parasites strain because of treatment with particular anthelmintics or anthelmintic combination(s), is irreversible [45]. Naturally, animals forage on various plants and may employ the activity of various bio-compounds in self-cure prevention. Similar practice can be employed using plants possessing/exerting anthelmintic activity.

Combination anthelmintic phytotherapy has been used for a long time, without any sound scientific basis of the bioactive principles involved, and interactions leading to improved efficacy. It is hypothesized that combination of plant species crude extract exerting anthelmintic activity will produce no synergistic or antagonistic effects, and correspondingly observed efficacy, synergy and antagonism will not relate to plant secondary metabolites including alkaloids, condensed tannins and flavonoid content of plant species. The specific objective was to evaluate and identify plant extracts with potential of being used in combination to develop a more effective livestock nematode control remedy. This study also elucidated the quantitative contribution of alkaloids, condensed tannins and flavonoids of component plant species to anthelmintic efficacy.

Sixteen plants that are used traditionally to treat infectious helminths of livestock were selected from available literature and allotted to three main groups (Table 1) following their putative primary anthelmintic principles. It is worthy of note that there may be overlap of phytochemical composition following analyses, but emphasis is laid on suggested primary bioactive principle(s) or macromolecule(s). They consisted of:

• Alkaloids and condensed tannins containing plant species which included Crinum macowanii, Gunnera perpensa, Nicotiana tabacum, Sarcostemma viminale, Vernonia amygdalina, Zingiber officinale, Zizyphus mucronata and Aloe vanbalenii [40,46-56].
• Flavonoids containing plant species comprised of Trema orientalis, Urtica dioica and Zanthozylum capense [53,55,57,58]
• Proteinases and nitrogenous compound containing plant species were made of, Allium cepa, Ananas comosus, Bidens pilosa, Carica papaya and Ricinus communis [37,59-66].

Plant species	Family	Common name
Allium cepa	Amaryllidaceae	Common onion
Aloe vanbalenii	Aloeaceae	Van Balen’s aloe
Ananas comosus	Bromeliaceae	pineapple
Bidens pilosa	Asteraceae	Black-jack
Carica papaya	Caricaceae	Pawpaw
Crinum macowanii	Amaryllidaceae	River lily
Gunnera perpensa	Gunneraceae	River pumkin
Nicotiana tabacum	Solanaceae	Tobacco
Ricinus communis	Euphorbiaceae	Castor oil plant
Sarcostemma viminale	Asclepiadaceae	Caustic vine
Trema orientalis	Cannabaceae	Pigeon wood
Urtica dioica	Urticaceae	Stinging nettle
Vernonia amygdalina	Asteraceae	Bitterleaf
Zanthozylum capense	Rutaceae	Small knobwood
Zingiber officinale	Zingiberaceae	Ginger
Zizyphus mucronata	Rhamnaceae	Buffalo thorn

 

These plants species were categorized and their anthelmintic properties evaluated in three experiments, dubbed sub experiments 1,2 and 3. Samples of plant material were collected from the University of KwaZulu-Natal Botanical garden, some from the National Botanical garden, Pietermaritzburg, others from private gardens, while some were bought from commercial food and vegetable stores in Pietermaritzburg central business district. Voucher samples were deposited at the UKZN Herbarium, Pietermaritzburg.

For each plant, fresh vegetative material was collected, washed, chopped and processed as described in the current section. Those with large and long leaves, were first air dried to reduce moisture content and subsequently oven dried (Oven mark; LABCON, Model 5SOEIB, Maraisburg 1700) to constant weight at 60°C. Oven-dried material of each plant species was milled using an electric centrifuge mill (RETSCH, GmbH and Co.KG, 5657 HAANI, West Germany), fine enough to pass through a 1-mm sieve. Milled plant samples were then put into air-tight labeled plastic containers and stored in boxes, away from light and moisture at room temperature.

A milled sample (4g) Dry Matter (DM) of each plant species was weighed into labeled thimble, fitted into distillation column and extracted in 70% ethanol over a heating unit (GERHARDT BONN, App. Nr 450893). The extraction process was considered complete when the solvent in the thimble carrying unit was apparently free of any coloration. At which point, plant crude extracts were obtained from bottles into which they were drained and concentrated. Bottles, which fell below the 100 ml mark, were made up to standardized volume by adding solvent (70% ethanol) and sealed with parafilm^® (PARAFILM^®AMERICAN NATIONAL CanTM, Neenah, W154956). They were packaged into boxes and stored in a fridge for in vitrodosing of mixed cultured isolated nematode L3 larvae.

Alkaloids were determined following [67]. Five grams ground sample of each plant species vegetative material was weighed into a 250 ml beaker, into which 200 ml of 10% acetic acid in ethanol was added and covered to extract for 4 hours. The solution was filtered using a fine sieve into a beaker of similar capacity to the first and the extract concentrated in a water bath to ¼ its original volume at 100°C. Concentrated ammonium hydroxide was added drop wise to completely precipitate extract and solution allowed to settle. A precipitate was collected and washed with dilute ammonium hydroxide 50:50 volume for volume. The residue was filtered using Whatman^TM 42 filter paper (GE Healthcare UK Limited, Amersham Place Little Chalfont, Buckinghamshire HP7 9NA, UK and Made in China) oven dried under low heat and weighed.

Flavonoids content of selected plant vegetative material was determined following [68]. Ten grams (10g) of oven-dried plant material was milled to pass through a 1-mm sieve, weighed into a 250 ml sterile beaker, and 100ml of 80% aqueous methanol added to it. The content was allowed to stand for 10 hours at room temperature, while being stirred intermittently with a magnetic stirring bar over a magnetic rotor without heat. Each solution was filtered individually through WHATMAN^TM No 42 filter paper. The filtrate of each sample was transferred into pre-weighed 250ml conical flask and evaporated to dryness in a water bath at constant temperature (80°C). Flasks and their contents were allowed to cool and subsequently placed in the desiccators for one hour to rid them of any moisture. Each of them was weighed, and the weight of the sterilized conical flask deducted from that of flask and flavonoid. The difference was computed as a percentage of flavonoid content of different species.

Condensed tannins were analysed following HCl-Butanol proanthocyanidin assay as leucocyanidin equivalent [69,70]. By which, one and a half grams (1.5g) of this material was weighed into pre-weighed filter paper (WHATMAN^TM, number 1, diameter 110mm, Cat number 1001 - 110, GE Healthcare UK limited, Amersham Place, Little Chalfont, Buckinghamshire. HP7, 9NA, UK, and Made in China) and Sohxlet extracted in 1% glacial in petroleum ether to rid them of pigments and fats that could interfere with quantitative determination of condensed tannins. It should be noted that the addition of glacial to petroleum ether serves as antioxidant, and prevents condensed tannins from being oxidized and bound to vegetative material. Weights of maximum 0.2001 g samples of the different plant species were measured into 100ml plastic centrifuge tubes, and 10ml of 70% aqueous acetone added to extract condensed tannins. Centrifuge tubes and their contents were Vortex mixed and placed in an ice bath. Samples were subjected to ultrasonic treatment for 3 minutes in ice cold water and vortex mixed intermittently for 12 minutes, resulting to 4 ultrasonic treatments in all. The content was centrifuged at 5000 rotations per minute (rpm) for 20 minutes at 4°C, and supernatant carefully collected in a glass test tube and stored on ice. Appropriate dilutions of tannin extracts with 70% aqueous acetone were made. Butanol reagent of volume 6 ml (950 ml of butanol and 50 ml of HCl 37% ) and 0.2 ml of ferric reagent (16.6 ml of concentrated HCl 37% diluted to 100 ml with water to make 2 M HCl and 2 g of ammonium ferric sulphate dissolved in it) was added to the tubes and vortex mixed. Tubes were covered and placed in a heating bath adjusted to between 96-100°C for 60 minutes. At the end of the incubation, they were cooled and absorbances measured using BECKMAN DU^®640 Spectrophotometer at visible wavelength of light 550 nm. From each of these absorbances red, was deducted that of an unheated mixture (blank). The method allows for appropriate absorbances between 0.30 and less than or equal to 0.60 to be considered stable and most appropriate. Percentage condensed tannins in each of the plant samples were computed following the formula below:

Percentage condensed tannins in dry matter= A_550nm x 78.26 x D/% dry matter, where: A_550nm= Absorbance at 550 nm; 78.26 = Accumulative factor taking into account: extinction coefficient of leucocyanidin, mass of sample (200g) and other factors except dilution; D=Dilution factor.

Dung was collected from 18 sheep as per rectum, pooled together and thoroughly mixed. Five grams of faecal material was weighed into a Petri dish and incubated at 27°C for 12 days. On day 13, each cultured faecal sample was dosed with 5ml of combined plant extract, or 2.5ml each of the designated combined pair of crude plant species extracts (double concentration; 2 x 2.5ml). Dosed samples were further incubated for one day. The control was watered and left untreated. Larvae (L3) that survived combined dosing were isolated on day 14, including controls following the Baermann method. Fluid of volume 10ml was drawn from the stem of each funnel into a labelled test tube of capacity 15ml and allowed to settle for 15 minutes. Fluid was further drawn from the supernatant using a Pasteur pipette, filled into a MCMASTER slide and surviving L3 larvae counted. Corrected mortality was computed using Abott’s formula, and used as indices of in vitro combined anthelmintic efficacy [71].

Plant species were grouped according to the primary putative anthelmintic principle, and combinations established by permutation. Following these groups, plant species combinations possessing similar classes of bioactive macromolecules were retained as separate sub-experiments to test their efficacious interactions. Sub-experiment one comprised of combinations involving alkaloids and tannins; sub-experiment two was based on combinations of plant species possessing flavonoids; and sub-experiment three was based on plant species containing proteinases and nitrogen compounds. In all three experiments, only sheep faeces were used. Each combination of plant species was evaluated in three replications, each of which was run on faeces collected on the same day. Sub-experiment one had 27 combinations, sub-experiment two 3 and sub-experiment three 10 combinations.

Two approaches were used to compute additive and synergistic anthelmintic effects resulting from in vitro combination therapy. In the first method, expected combined efficacies of any pair of plant species ‘a’ and ‘b’ were computed (a+b)/2 and subsequently deducted from their observed combined efficacy to yield simple synergy. Positive differences measured synergistic effects, whereas negative differences measured antagonistic effects. In the second method, expected efficacy of plant species combination was estimated following Webb’s fractional product method [72]. Following this method, if the efficacies of two plant species “A” and “B” represented by “a” and “b” proportion of worms killed, then expected efficacy of combinations assuming additive effect is computed thus: Efficacy (A+B) = 1 - ((1 - a) x (1 - b)). Synergistic effect is considered to have occurred when the response of combined administration is greater than additive.

Data collected in each of these three experiments was analysed following General Linear Model (GLM) of [73]. Pearson correlation was used to seek possible relationship between anthelmintic efficacy on the one hand and each of alkaloids, flavonoids and condensed tannins as primary putative anthelmintic macromolecules. Additionally, multiple regression analysis was run to seek explanations of the role of various variables including alkaloids, flavonoids and condensed tannins to observed trends of anthelmintic efficacy. Mean separation was done using Student Neuman Keul’s statistic, aided by [73].

Results are presented in table 2 bearing the effects of various combinations of members of each group on efficacy, and in table 3 bearing phytochemical properties.

Observed efficacies of plant species containing alkaloids and tannins were high but not different (P=0.5595) among combinations, with mean combined efficacy of 94.4±0.05% for all plant species combinations. Expected combined ((A+B)/2) efficacies were modest, with mean 86.9%. Simple synergy (differences between observed and expected efficacies) ranged from -9.6±0.05 to 17.5±0.05 % (Table 2), but were generally not different (P= 0.2477) among combinations; with mean of 7.4±0.05%, which was greater (P<0.05) than zero. Webb’s efficacies for combinations were high with a mean 98.3%. Webb’s synergistic effect arising from combinations were not different from each other (P=0.5114), and had a mean of -4.0±0.05%, which is lower than zero.

Alkaloids, condensed tannins and flavonoids were identified and their concentrations evaluated in these plant species. Alkaloid contents were similar (P=0.304), with mean concentration of 38.6±0.68 g/KgDM (Table 3). Concentrations of condensed tannins of different plant species were different (P<0.0001), with mean content of 6.0±0.13 g/KgDM. The trend of tannin content was: Z. mucronata>G. perpensa>N. tabacum>C. macowanii>V. amygdalina>Z. officinale>S. viminale (Table 3). Correspondingly, flavonoid contents were different (P= 0.0006), and had mean content 152.1±0.74 g/KgDM. The trend of flavonoid content was thus: N. tabacum>Z. officinale>V. amygdalina>Z. mucronata>C. macowanii>S. viminale>G. perpensa (Table 3). The order of macro biochemical content for all plant species in this group was, flavonoids> alkaloids>condensed tannins. There was no correlation between combined efficacy and any of alkaloids (r=0.1458; P=0.2543), condensed tannins (r= 0.0059; P=0.9637) or flavonoids (r= -0.0293; P=0.8199). Alkaloids, condensed tannins and flavonoids were all poor predictors of combined efficacy for plant species possessing alkaloids and tannins in a multi-regression analysis as no variables met the criterion of P=0.15.

Observed efficacy (Table 2) of combined plant species were high but not different (P=0.3183) between combinations, with a mean of 94.3±0.54%. Expected efficacies for flavonoid containing plant combinations were modest with a mean of 79.5%. Simple synergies were not different (P=0.2671) among combinations; they ranged from 7.2±0.54% to 22.0±0.54% (Table 2) with mean of 14.8±0.54%. Webb’s efficacies for combinations of these plant species were high but different (P<0.0001) and had mean 95.9 %. Webb’s synergistic effects were not different (P=0.3099) among treatments, but had a mean of -1.6±0.54 %, which differed (P<0.05) from zero.

Quantitatively, content of condensed tannins, alkaloids and flavonoids (Table 3) of these plant species had various relationships. Condensed tannin content were different (P=0.0089) among plant species, with a mean 8.3±0.91 g/KgDM, whereas alkaloid content were not different (P=0.07), and had mean 37.5±1.93 g/KgDM. Additionally, flavonoid content of these plants were different (P=0.0211) among them, with mean 156.4±1.78 g/KgDM. There was no discernible association between anthelmintic efficacy and any of alkaloids (r= -0.1411; P=0.7173), condensed tannins (r=0.3361; P=0.3765) or flavonoid (r= -0.1457; P=0.7084) content of these plant species. Multiple regression analysis of alkaloids, condensed tannins and flavonoid content as predictors of combined efficacy was not significant, as none entered the model at P= 0.15.

Observed efficacies of these plant species were high, but not different (P=0.785) among combinations, with mean 97.9±0.10% (Table 2). Expected efficacies were modest with mean 82.3%. Simple synergies were not different (P=0.7162) among combinations, with a mean of 15.5±0.10%, which is higher (P<0.05) than zero. Webb’s computed efficacy for combinations of these plant species were high (97.0 %) and different from each other (P< 0.0001). Webb’s synergistic effects were not different (P=0.7882) among combinations; with a mean of 0.8±0.10%, which is greater (P<0.05) than zero.

Alkaloid contents were different (P=0.0135) and had mean 35.2±0.78 g/KgDM (Table 3), whereas condensed tannin contents were similar (P=0.4312), with mean 4.5±0.46 g/KgDM. Flavonoid contents were also different (P<0.0001) with mean 232.9±1.24 g/kgDM. There was no correlation between any of alkaloids (r= -0.02774; P= 0.8843), condensed tannins (r= -0.3071; P=0.0987) or flavonoids (r=0.0359; P=0.8505) and observed efficacy. A multi-regressions of alkaloids, condensed tannins and flavonoids as predictors of combined anthelmintic efficacy also gave no relationship. Overall trend of concentration for all three biochemical compounds was flavonoids> alkaloids> condensed tannins (Table 3).

Interaction of simple and Webb synergy in all three sub experiments had a linear relationship. While simple synergies from most combinations were positive, most of Webb’s were oppositely negative (Figure 1). Combinations that yielded the best synergistic effect in SEP 1, were to the positive plane of Webb and included Gun-Nic, Nic-Alo, Sarc-Vern, Vern-Zin, Zin-Alo, Zin-Ziz and Ziz-Alo. Similarly, combinations with the most synergistic effects for SEP 2 included Trem-Urt and Urt-Zan. Correspondingly, combinations of plant species extract that yielded the best synergistic effects from SEP 3 were All-Bid, All-Car, All-Ric, Ana-Car, Bid-Car, and Car-Ric. Sub experiment 3 produced the highest number of combinations with synergistic interaction.

 

Figure 1: Scattered plot of interaction between simple and Webb’s synergies for combinations involving tannin/tannin (dash trend line), flavonoid /flavonoid (solid trend line) and proteases (broken trend line) containing plant species.

Combination of plant species in phytoanthelmintic therapy is expected to improve efficacy and spectrum of cover to that which is higher than for component plant species constituting a pair [14,74]. Two approaches were adopted to evaluate synergism and improved anthelmintic activity arising from combination of these plant species. The first was that of differences between observed and expected efficacies (simple synergy), and the second was computed using Webb’s fractional product method [72]. Contrary to our expectation, there appeared to be synergistic activity from both simple synergy and Webb’s synergy (Figure 1). For the former, these differences were mostly positive, alluding to improved anthelmintic activity in all three sub experiments as a result of constituted combinations. From the later, synergistic activities of most combinations were overwhelmingly negative in all three sub-experiments suggesting antagonistic activity or interactions leading to improved anthelmintic activity but below synergy. Remarkably, graphical trends from interaction between simple and Webb’s synergies had a common gradient, which ranged from negative to positive. In both approaches of evaluating synergism, there was generally improved activity from combination anthelmintic phytotherapy with most candidates occurring below synergy, though better than individual plant species activity, while others exerted positive synergy (Figure 1). The positive candidates (Figure 1) represent those that produced the best synergistic interaction, and hold potential promise for outstanding anthelmintic treatment. While simple synergies were largely positive, Webb’s synergy was mostly negative. Though combined concentration of crude extract was reduced in the current trial to 40%, computed Webb additive effect was virtually at 100%. It is most likely in the current study that reduction in dose might not have accorded adequate room to sufficiently evaluate synergistic effect, as shown in the general trend of additive activities in all three sub experiments. There is, therefore, need to further reduce the dose in order to target less additive effect than that obtained from the current study.

In all three sub experiments, alkaloids, condensed tannins and flavonoids were quantitatively evaluated for all plant species. Given that empirical evidence closely links alkaloids, condensed tannins and flavonoids to exerting anthelmintic activity, a natural expectation would be some level of association of these macromolecules with anthelmintic efficacy in plant species in which they occur [36,46,51,75-85]. Contrary to this expectation, there was no discernible correlation of all three macromolecular compounds with anthelmintic efficacy. In combinations, the collective activity of these macromolecules and others that might not have been identified may be responsible for the observed activity. Additionally, anthelmintic potency of these principles put together, may far exceed or shield that of individual macromolecules including alkaloids, condensed tannins and flavonoids. In attempt to formulate some predictive measure of combined anthelmintic efficacy or interchangeably observed efficacy based on the contribution of alkaloids, condensed tannins and flavonoids in the current study, a multi-regression analysis failed to prove any contribution. This reaffirms collective rather than strong individual contribution of various anthelmintic principles to efficacy, helping in the proposition of very high dosages of these plant species.

Plant species containing alkaloids generally have a wide variety of these macromolecules and/or other related biochemical’s with different chemical structures, some of which are isomers and others have different molecular weights [86-88]. A similar pool of various types of tannins occurred in plant species possessing condensed tannins [89-91]. Flavonoid, containing plant species likewise have a wide variety of different flavonoids and other biochemical’s [88,92], and sometimes different isomers of the same flavonoid occur in the same and also in different plant species [88]. Similarly, the biochemical content of proteases and nitrogen compounds in plant species containing this macromolecular class would have been structurally diverse, in addition to other related anthelmintics [93]. This structural biochemical diversity of alkaloids and tannins, flavonoids and, proteases and nitrogen compounds in different plant species confers on them different properties and biochemical activities [88,89,91-93]. Therefore, a wider pool of biochemical compounds in combinations, different interactions among them or pharmacodynamic activities, and by inference various pharmacokinetic activities [94]. This would occur first, within a similar class of anthelmintic bioactive pool such as alkaloids, condensed tannins and flavonoids in plant species and cumulatively in different plant species constituting various combinations (combined biochemical pool), yielding improved efficacy as targeted and observed in the current study [95,96]. This further strengthens regards for collective rather than for individual anthelmintic activity.

Based on the current study, a much more reduced combined dose is required to properly address synergistic activity. While the other parameters show that there is potentially greater potency in combined anthelmintic phytotherapy, synergy is more negative than positive. Relationship between simple and Webb synergies has highlighted some combinations to have exceedingly higher efficacy than would have resulted from any one of the component plant species individually at the same concentration. It is therefore imperative to ascertain this synergistic interaction in vivo, as these combinations hold a huge potential in nematode control programs. Improved observed efficacy in the current study is generally in accord with its primary objective. Antagonistic and synergistic activities as observe from both correlation matrix and multiregression occur in plant species exerting anthelmintic activities and other relevant antiparasitic activities. The biological activity identified in plant species suggest that various modulating chemical activities occur within each plant species or combinations to enable them exercise their activity without causing harm to livestock that is treated. Additionally, different types of active compounds may occur in the same plant species (Table 1.2) or in different plant species, conferring on them broad spectrum nematode parasite control capacity [95,97]. This biochemical nature, inherently accords advantage to anthelmintic activity. Combination therapy, therefore, yields multicomponent active principle, which exerts different modes of biochemical activity, leaving nematode parasites insufficient capacity to develop resistance against all of them relative to single plant species treatment [74].

Though combination brings together a variety of bioactive anthelmintic principles, in nature, individual plant species harbour different primary biochemical’s including alkaloids, condensed tannins, flavonoids and other compounds (not determined in this study) in different concentrations. Naturally, anthelmintic biochemical variety exists at the level of individual plant species. Some of the bioactive anthelmintic principles including alkaloids, tannins, flavonoids and terpenes among others, are plant secondary metabolites, which when present in diets beyond certain thresholds negate their beneficial effects [98,99]. A variety of these macromolecules in diets have been suggested to mitigate or modulate the negative effects of individual plant species, implicitly by dilution or counteraction of deleterious effects of each other. These interactive and modulatory effects enhance the nutritional and curative benefits to animal production and health [98]. In the same vein, combinations of plant species exerting anthelmintic activity that result to enhanced activity greater than that of individual component species, is an expression of the merits of a much broader positive interaction. Measurable and relatively enhanced anthelmintic efficacy emanating from combination of different plant species has been adopted from its application in chemical or orthodox anthelmintic combination [72].

Improved activity from combination anthelmintic phytotherapy will result to more potent activity relative to that of individual component species, and similar to what obtains in combination chemical anthelmintic therapy [74]. This heightened activity has the likelihood of complete parasite elimination at contact or parasite/biochemical interaction. Remnants of surviving parasites that would not have had any contact or interaction with combined extract will retain their integral vulnerability to subsequent dosing. This is typical of the concept of refugia where in, the surviving portion of parasites in a herd of animals is made to have absolutely no contact or interaction with anthelmintic drug in use, while those with any contact are killed [22]. Efficacious drug disposition of this nature is critical in livestock nematode control programs because of prevention of selection for resistant parasites and build-up of resistant alleles that usually lead to drug failure.

Among different plant species possessing similar biochemical class but with different structural formulae, some of them may exert anthelmintic activity and others not, suggesting that all plant species possessing these tagged bioactive principle classes may not be necessarily anthelmintic in nature, or may not exert this activity to the same extent when it exists [100,101]. Additionally, plant species content of any of alkaloids, condensed tannins and flavonoids does not conclusively translate to anthelmintic biological activity, because not all macromolecules exert this activity [100]. This raises the need for a much finer biochemical profiling, leading to extensive identification of all potential contributors to this anthelmintic trait. There is need to ascertain advantages of combination anthelmintic phytotherapy or chemotherapy offered over either of them individually. The genetic basis of combination anthelmintic phytotherapy or chemotherapy is crucial in nematode parasite control programs. Chemical anthelmintic therapy presents peculiar cases of anthelmintic resistance, wherein selection for resistance against an anthelmintic does not necessarily affect others in the same or different groups because of different mechanisms of action [102,103]. Independent selection for resistance ties with the genetic control of this trait, which is in turn controlled by different alleles [14]. Combination anthelmintic therapy renders genetic selection for resistance difficult, though not impossible, as many alleles will have to be involved in the process. Combination therapy, therefore, affords the opportunity to recycle anthelmintics, which otherwise would have been ineffective or exerting low efficacy individually, the opportunity to exert acceptable levels of efficacy because of the genetic disposition towards this trait. Additionally, component anthelmintics in combination can influence and radically improve pharmacokinetic and pharmacodynamic interactions of the duo resulting to additive and synergistic effects on efficacy [15]. Similarly, plant bioactive anthelmintic principles constitute a pool of various biochemical classes and types of macromolecules, rendering genetic control much more intricate and complicated. Plants by their biological nature require huge genetic alterations in parasites to take place in order for parasites to become resistant; which is farfetched and inherently advantageous in livestock nematode parasite control programs. Ethno veterinary phytochemical combination anthelmintic therapy therefore constitutes an important option in parasite control that should be accorded adequate attention in research, development and treatment.

Combination anthelmintic phytotherapy generally increases the concentration of similar principles in the component species and potential biological activity. Additive anthelmintic effects occurred in all combination. Interaction between simple and Webb’s synergies yielded some combinations with the best synergistic effects. Additionally, combination anthelmintic therapy diversifies anthelmintic bioactive options enhances efficacy and potentially other activities that promote animal health and productivity. By extension, the likelihood of selecting resistant nematodes and related parasites is remote because of involvement of various anthelmintic/antibiotic principles at macro biochemical level in plant combinations. This, in turn, demands a much more extensive work to further test many more plant species in combination in order to validate and provide more benign consumer/ecological friendly anthelmintic options. It implies that anthelmintic based on combination of two plants within the listed groups (alkaloids and/or condensed tannins; flavonoids; and nitrogenous compounds) will help in elimination these parasites that plague the survival of small ruminants.

This research work was realised with the financial support of the University of KwaZulu-Natal. The services of Mrs Young Alison were key to sourcing and identification of some of the plant species used. Mrs Sue van der Merwe of plant Pathology assisted immensely at growing and sustaining tobacco plants and related services. Support was also received from the National Botanical Garden Pietermaritzburg for plant material. Gratitude is also profoundly accorded to Dr. KS Yobo and Mr Richard Burgdorf of the Discipline of Plant Pathology for laboratory space and material assistance. Additionally, Dr Christina Potgieter of University Herbarium Pietermaritzburg was very helpful and instrumental at sourcing and preparation of referral samples. Mr Samuel Khumalo helped in collecting rectal faecal material from both goats and sheep.