A Systematic Review on Comparative Analysis, Toxicology, and Pharmacology of Medicinal Plants Against Haemonchus contortus

Background: Haemonchus contortus is an important pathogenic nematode parasite and major economic constraint of small ruminants in tropics and subtropics regions. This review is an attempt to systematically address the; (a) efficacy of different plants against H. contortus by in vitro and in vivo proof; (b) toxicology, mechanism of action, and active phyto-compounds involve in anti-haemonchiasis activity; (c) and comparative analysis of plant species evaluated both in vitro and in vivo. Methods: Online databases (Google Scholar, PubMed, Scopus, and ScienceDirect) were searched and published research articles (1980–2020) were gathered and reviewed. Results: A total of 187 plant species were reported belonging to 59 families and 145 genera with Asteraceae and Fabaceae being frequently used. Out of the total plant species, 171 species were found to be evaluated in vitro and only 40 species in vivo. Twenty-four species were commonly evaluated for in vitro and in vivo anti-haemonchiasis activity. Among the reported assays, egg hatching test (EHT) and fecal egg count reduction (FECR) were the most widely used assays in vitro and in vivo, respectively. Moreover, sheep were the frequently used experimental model in vivo. After comparative analysis, Lachesiodendron viridiflorum, Corymbia citriodora, Calotropis procera, and Artemisia herba-alba were found highly effective both in vitro and in vivo. L. viridiflorum inhibited enzymatic activities and metabolic processes of the parasite and was found to be safe without toxic effects. C. citriodora was moderately toxic in vivo, however, the plant extract produced promising nematicidal effects by causing muscular disorganization and changes in the mitochondrial profile. Additionally, C. procera and A. herba -alba despite of their high anti-haemonchiasis activity were found to be highly toxic at the tested concentrations. C. procera caused perforation and tegumental disorganization along with adult worm paralysis. Nineteen compounds were reported, among which anethole and carvone completely inhibited egg hatching in vitro and significantly reduced fecal egg count, decreased male length, and reproductive capacity of female in vivo. Conclusion: This review summarized different medicinal plants owing to nematicidal activities against H. contortus eggs, larvae, and adult worms. Plants like L. viridiflorum, C. citriodora, C. procera, and A. herba-alba, while compounds anethole and carvone having promising nematicidal activities and could be an alternative source for developing novel drugs after further investigation.


INTRODUCTION
Haemonchus contortus is the causative agent of haemonchiasis usually known as "twisted barber" pole worm or stomach worm (Saminathan et al., 2015), and a common blood feeder of small ruminants. The parasite is present throughout the tropical and subtropical regions of the world where it is a major constraint for profitable production of sheep and goats (Zenebe et al., 2017). Haemonchiasis is characterized by severe anemia, leading to a serious impairment of the animal, severe economic losses, and acute disease outbreaks with high death rate particularly in young animals (Selemon, 2018). Among the parasitic diseases, gastrointestinal nematode infections remain one of the main causes of impaired production in small ruminants (Hounzangbe-Adote et al., 2005). According to the pharmaceutical companies the annual cost of antiparasitic compounds is proposed to be tens of billions of dollars worldwide (Wolstenholme et al., 2004). However, the annual treatment cost of H. contortus has been estimated to be 26 million USD in Kenya, 46 million USD in South Africa, and 103 million USD in India (Peter and Chandrawathani, 2005).
To overcome the major economic losses in agriculture, it is essential to enhance the control of key parasitic diseases (Gilleard, 2006). For this purpose, various approaches are being in use to control parasitism, including biological control, pasture management, dietary management, vaccination, and the use of anthelmintic chemicals. Most widely used practice being followed nowadays is the use of anthelmintic chemicals. Unfortunately, regular and indiscriminate administration has posed a variety of problems including emergence of resistance in nematode parasites, e.g. multi resistant H. contortus has been reported. Furthermore, the commercially available anthelmintics drugs are somewhat costly and smallholder farmers are unable to expend meager income for purchasing of drugs to carry on regular treatment (Irum et al., 2015).
As a result, there is a dire need to develop alternative anthelmintic approaches from natural flora which can be less toxic, biodegradable, environmental friendly, and to cover the most challenging problem of parasite resistance issue (Carvalho et al., 2012). Worldwide, different plant species have been reported and evaluated for natural bio-products to control the parasitic infections and reduce the dependency on conventional chemotherapy. Testing for biological activity in vitro and in vivo has to be done after a purification process in order to exclude interference with accompanying compounds and reference standards for quality control of herbal medicines largely depend on isolated compounds with documented purity (Bucar et al., 2013). The compounds from plants have also been found to be synergistic enhancers in that though they may not possess any anthelmintic properties alone, but when used concurrently with standard drugs they enhance the activity of the drug. The synergistic effect of the association of an anthelmintic drug and plant extracts against resistant pathogens leads to new choices for the treatment of infectious diseases. Also synergy between bioactive plant product and antiparasitic will confront problems of toxicity and overdose since lesser concentrations of two agents in combination are required, due to these reasons, there is need for continuous exploration of multidrug resistance modulating principles from plants sources (Aiyegoro and Okoh, 2009). The herbal medicines however, suffer from lack of standardization parameters. The main limitation is the lack of standardization of raw materials, processing methods and the final products, dosage formulation, and the non-existence of criteria for quality control (Sachan et al., 2016).
Recently, the interest of researchers in exploring the antiparasitic properties of ethnoveterinary medicinal plants is increasing and this field of research is inundated with ethnopharmacological studies. This review is aimed to gather fragmented literature about the; (a) efficacy of different plants against H. contortus by in vitro and in vivo proof; (b) toxicology, mechanism of action, and active phyto-compounds involve in anti-haemonchiasis activity; (c) and comparative analysis of plant species evaluated both in vitro and in vivo. Moreover, the study also highlights existing knowledge gaps in the present research and provides future recommendations to fulfill those gaps.

METHODOLOGY
The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Moher et al., 2009). No protocol was followed for conducting this systematic review. The PRISMA check list is provided in the supporting information section (Supplementary Table S1).

Databases and Searching Criteria
To find the published literature, a systematic search was performed using different databases, including Google Scholar, PubMed, Scopus, and ScienceDirect. Research articles published in English language from 1980 to 2020 were gathered for this systematic review. Key words such as: anthelmintic activity, nematicidal activity of plants, medicinal plants used for H. contortus, in vitro/in vivo efficacy of plants against H. contortus, active compounds in plants, mechanism of plant extract inhibition and toxicity of plants. "Anthelmintic AND Hemonchus contortus", "Natural nematicidal OR antihaemonchiasis NOT synthetic", "Natural in vitro OR in vivo anthelmintic". Bibliographies of research articles were also searched and relevant references were extracted and downloaded. Moreover, to support the findings of the review further literature search was conducted and relevant articles were included.

Inclusion/Exclusion Criteria
Research articles describing (a) in vitro/in vivo efficacy of medicinal plants against H. contortus, (b) containing full information regarding plant name, country name, extract, concentration, inhibition, time exposure, and assay type, (c) original research articles, (d) and published in English language were included in this systematic review. While articles with (a) epidemiological and molecular dataset of H. contortus, (b) antiparasitic activities other than H. contortus, (c) synthetic drugs/chemicals tested against H. contortus, and (d) language other than English were excluded.

Data Extraction
Endnote (Thomson Reuters, San Francisco, CA, United States) was used to compile the articles. Researchers very carefully extracted all the data from the selected articles including author (s) name, country name, plant name, family name, plant part used, plants' life form, extract used, concentration, time exposure, inhibition, and year of publication. Data were arranged into tables and figures. Chemical structures of the compounds were drawn using MarvinSketch (18.24.0) (https://chemaxon.com/products/marvinn) and Inkscape (0. 92) (https://inkscape.org/) was used to further refine and improve the resolution of each chemical structure. PubChem (https://pubchem.ncbi.nlm.nih.gov) was also used to attain the IUPAC name (s) of pure compounds reported in this review.

Quantitative Analysis
Jaccard Similarity Index (JI) Jaccard similarity index was calculated to determine the similarity between the two sets of studies reported in this review. One set of study is the "in vitro pharmacological validation of medicinal plants" and the other one is the "in vivo pharmacological validation of medicinal plants". JI was calculated by using the formula (Kayani et al., 2015): Where "a" is the total number of plant species used in vitro, "b" is the total number of plant species used in vivo as anthelmintic against H. contortus, and "c" is the number of plant species common to both in vitro and in vivo studies.

RESULTS
We identified a total of 1,480 published articles through literature search. After removing duplicates, and irrelevant articles, a total of 108 articles were selected for this review (Figure 1). Quality assessment of the selected articles was performed and summarized as author name, species/compound(s) stated in the article, plant source, species authentication, quality control as well as chemical analysis reported (Table 1).

Taxonomic Clarification
According to the modern botanical nomenclature, 53 reported plant species have a synonym issue. Plant accepted names are mentioned in table (Table 1; Supplementary Tables S2, S3), while the synonym mentioned in the original articles were put into brackets. In addition, taxonomic corrections regarding the author of those plants and their family names were also revised.        frequently used part (50%) followed by seeds (11.3%), roots (8%), and whole plants (7%). Among other plant parts stems, flowers, barks, shoots, fruits, bulbs, peel, fibers, pulp, and latex were included (Supplementary Table S2).
Different solvents including n-hexane, aqueous, ethanolic, hydro-alcoholic, methanolic, and others were used in extracts preparation ( Figure 2). Among all, the methanol extract was the predominant one. Bioassays reported in this review included, egg  Table S3).

Comparative Analysis and Toxicity of Common Plant Species
Plant species, which were common in both in vitro and in vivo studies, were compared to discriminate their efficacy against H. contortus. A total of 24 plant species were found to be commonly used both in vitro and in vivo ( Table 2).
After comparative analysis based on minimum concentration and maximum efficacy, the identified plant species with promising anthelmintic activity in vitro against H. contortus were Lachesiodendron viridiflorum (Kunth) P.G.Ribeiro, L.P.Queiroz and Luckow (syn. Piptadenia viridiflora (Kunth) Benth.), Cymbopogon schoenanthus (L.) Spreng., Allium    sativum L., Lippia origanoides Kunth (syn. Lippia sidoides Chem.), Artemisia absinthium L., Cymbopogon citratus (DC.) Stapf, Eucalyptus staigeriana F. Muell. ex F.M. Bailey, Artemisia herba-alba Asso, and Corymbia citriodora (Hook.) K.D. Hill and L.A.S. Johnson in order of their appearance. While A. herbaalba, C. procera, C. citriodora, Lysiloma latisiliqum (L.) Benth., and Euphorbia helioscopia L. were reported with high efficacy in vivo. Among the plants, L. viridiflorum was found highly effective both in vitro and in vivo with no observed toxic effects. C. citriodora was moderately toxic in vivo, but with promising nematicidal activity in vitro and in vivo. Additionally, C. procera and A. herba-alba despite of their high antihaemonchiasis activity were found to be highly toxic at the tested concentrations (Supplementary Table S4). However, five species namely; Annona squamosa L., Artemisia maritima L., Artemisia capillaris Thunb., Castela tortuosa Liebm., L. latisiliquum, and Seriphidium brevifolium (Wall. ex DC.) Ling and Y.R. Ling were not evaluated for their toxicological effects. Mostly non-toxic and low-toxic extracts were orally administered. The LC 50 value of most plant species was missing and was not calculated.
From the results, it is evident that the effects of different plant extracts were dose and time dependent. Moreover, the comparative analysis also revealed that plant species were more effective in vitro than in vivo against various stages of the parasite.

Phyto-Compounds With Anti-Haemonchiasis Activity
In this review, 19 compounds were reported to be assessed for in vitro activity against different life stages of H. contortus. While only 3 compounds were found to be evaluated for in vivo activity using gerbil (Meriones unguiculatus) as an animal model. Based on minimum concentration and maximum nematicidal activity, Cinnamaldehyde, anethole, and carvone were highly active and completely inhibited egg hatching of the parasite at the tested concentrations of 0.085, 0.085, and 0.366 mg/ml, respectively. Carvacrol inhibited the larval development at a minimum concentration of 1 mg/ml, though the effectiveness of thymol and anethole against larvae development was also significant, but relatively at high concentrations i.e., 10 and 20 mg/ml, correspondingly.
Carvacrol and carvacryl acetate at 2 mg/ml showed 100% larval motility inhibition as compared to other compounds. Similarly, carvacryl acetate (0.2 mg/ml) and citronellal (2 mg/ ml) totally reduced the motility of adult parasites in vitro. However, citronellal was moderately toxic in mice. Anethole and carvone significantly reduced fecal egg count, decreased male length, and reproductive capacity of female at 50 mg/kg concentration in vivo (Table 3).

Jaccard Similarity Index
The JI was used to compare the two sets of data (i.e., in vitro and in vivo studies) to determine the similarity of the studies reported in this review. The result revealed 12.8% similarity between the two data sets ( Figure 5).

Anti-haemonchiasis Medicinal Plants, Their Families, and Habit
Extensive use of Asteraceae, Fabaceae, Lamiaceae, and Euphorbiaceae, users' reliability, and antiparasitic activity could be attributed to the presence of potent phytochemicals such as saponins, tannins, flavonoids in Asteraceae (Carvalho et al., 2013), saponins, essential oils in Lamiaceae (Raja, 2012), phenolic compounds, alkaloids, and hemagglutinins in Fabaceae (Żarnowski et al., 2001), and alkaloids and terpenoids in Euphorbiaceae (Mwine and Van Damme, 2011). Mechanism of action of these compounds against different developmental stages of H. contortus is still unknown, however, probably these may inhibit both egg embryonation and direct effects on the larvae (Al-Shaibani et al., 2008). Athanasiadou et al. (2001) attributed the antiparasitic activity of condensed tannins to their ability to bind with the cellular membrane proteins, which results in the unavailability of nutrients to the larvae, causing starvation, and death (Athanasiadou et al., 2001). It seems that complexities of these compounds enable them to interrupt various molecular targets of different developmental stages of the parasite. Saponoids usually act by binding to surface molecules (proteins/sterols) inducing inhibition of the protein expression, and/or lysis of the cell (Bruneton, 1993). The expression of surface proteins of nematodes is stage specific (Rhoads and Fetterer, 1994).
Furthermore, Asteraceae is being the first and Fabaceae is the third largest terrestrial plant families all over the world and this could be another possible reason for such an extensive utilization of these families (Tariq et al., 2017). Plant species of Fabaceae are able to fix nitrogen, which leads to protein deposition in leaves and seeds (Molares and Ladio, 2012). Species of Lamiaceae could be easily cultivated and propagated, moreover, they are mostly utilized due to their strong aroma and ability to survive in severe hot weather because of their essential oils (Raja, 2012). Euphorbiaceae extensive use for different medicinal purposes may be attributed to its global distribution and mode of adaptation in the worst dry conditions because of the succulent nature of its species and crassulacean acid metabolism (CAM) pathway ability. Plants of this family possess a wide array of secondary metabolites and tendency of mutation load due to their exposure to a wide range of environmental conditions (Mwine and Van Damme, 2011).
Herbs were frequently used form of life against H. contortus eggs, larvae, and adult worms as compared to trees and shrubs. The dominancy of herbs over other forms of life could be attributed to their easy availability and high efficacy against different ailments as compared to shrubs and trees . Herbs are widely used in folk medicines all over the globe and contain a large number of active compounds responsible for their high efficacy and, therefore, are preferred by the scientists and traditional healers (Tariq et al., 2017).
Leaves were reported as the widely used part during pharmacological validation of medicinal plants against H. contortus. Leaves contain a variety of chemical compounds     and due to their easy harvesting and less harmful effect on plant life, make them as the first choice of herbalists Tariq et al., 2017).
Medicinal plants owing to their potential of having a significant source of bioactive compounds that may lead to the development of novel drugs (Azwanida, 2015;Ali et al., 2020). Scientists have analyzed and evaluated the effect of various kinds of solvents, for the purpose to extract these bioactive compounds from various plant parts (Altemimi et al., 2017). Extraction is the separation of medicinally active portions of a plant, using selective solvents through standard procedures (Azwanida, 2015). The purpose of extraction is to separate the soluble plant metabolites, leaving behind the insoluble cellular marc (residue) (Azwanida, 2015). Methanol was the most preferred solvent for plant extraction possibly owing to its polar nature that ensures the release of several bioactive compounds from plants. It has been scientifically proven that highly polar solvents should be used to extract different bioactive compounds with high accuracy (Altemimi et al., 2017). Fruitful results of active compound in plants mainly depend upon the solvent used for herbal formulation.
The results revealed that more studies were conducted to evaluate in vitro anthelmintic activities of medicinal plants as compared to in vivo. In vitro validation of medicinal plants provides the proof of reliability of these plants against H. contortus. In veterinary parasitology several in vitro techniques are broadly used for analysis of nematicidal activity of drugs/plant extracts prior to in vivo testing (Sangster and Gill, 1999). There are several positive aspects of in vitro assays prior to in vivo including less time consuming, less expensive, need for a smaller number of animals, and permitting the evaluation of the efficacy of different anthelmintic compounds throughout the life cycle of the parasite (Demeler et al., 2013). Based on the reliable results obtained from in vitro analysis further selection of extract/pure compound for in vivo evaluation can be carried-out (Zips et al., 2005). In vivo studies are mainly conducted to evaluate the mechanism of action of the desired extract/compound, the immune response of the host animal, toxicity levels, as well as the in vivo effectiveness. Although there are many advantages of in vivo studies, but there are also some shortcomings including more time consuming, expensive, and lower precision and reproducibility (Lacey et al., 1990). These limitations should be taken into account and highlight the significance of pharmaceutical/pharmacokinetic studies for the industrial development of new anthelmintic products against H. contortus. The research to find effective and natural anthelmintics has been highly inundated with in vitro studies, hence, it is suggested to evaluate the plant extracts/compounds in vivo in future.

Pharmacological Action
Pharmacological activity of an extract/compounds/drug depends on how the candidate interacts with enzymes, proteins, nucleic acids, biomolecules, and different types of receptors (Roy, 2011). Pharmacological activity is an important phenomenon to know the precise target of the drug/extract/compound with anthelmintic efficacy against the parasite or other organism/ pathogen under observation (Figure 6).
A. sativum ethanolic extract inhibits the motility of the H. contortus through a destructive and inhibitive effect on the enzyme acetylcholinesterase (AChE). The enzyme rapidly hydrolyzes acetylcholine (neurotransmitter) and thus, limits and terminates the cholinergic synaptic transmission (Taylor, 1990;Lee, 1996). Inhibition of ACheE leads to the accumulation of acetylcholine, thereby interrupting the neuromuscular transmission causes paralysis of musculature. Due to muscular discoordination/paralysis food swallowing and movement through the digestive system is stopped. The parasites enter the state of starvation and energy deprivation and thus, unable to survive inside the host (Kaur and Sood, 1982;Opperman and  Chang, 1992). The antiparasitic activity of A. sativum may also be attributed to the sulfur containing compounds (e.g., ajoene and allicin) which can possibly form disulphide bonds with free thiol groups, and thus, inhibit enzymes or other proteins, which are important for survival of the parasite (Krstin et al., 2018). Crude aqueous extract of Achillea millefolium L. has profound anthelmintic activity, this could be due to the presence of several key chemical constituents one of which is eugenol. It is reported that eugenol can cause alteration of cell shape, membrane blebs , restrict cell growth, swelling, and collapsing cell membrane and arrest cell division (Ueda-Nakamura et al., 2006). The compound artemisinin of A. absinthium can inhibit vital enzymes of metabolic cascade by forming covalent bonds and resulting in irreversible inhibition of the enzyme (s) activities. The enzymes include S-adenosyl-methionine synthesase (SAMS), spermidine synthase (SpdSyn), L-lactate dehydrogenase (LDH), pyruvate kinase, and ornithine aminotransferase (OAT) . Artemisinin is also reported to disrupt mitochondrial membrane potential, cause cytochrome c release into the cytoplasm (Jia et al., 2016), and inhibit the electron transfer and oxidative phosphorylation of mitochondria, with final activation of caspase-3-mediated apoptosis (Li et al., 2005). Additionally, crude aqueous extract of A. absinthium can induce ultrastructural changes such as tegumental damage, nephridial canal epithelium lining and intrauterine eggs destruction, lipid accumulation, glycogen depletion, and finally worm paralysis and death (Beshay, 2018). Similar anthelmintic effects of tegumental disorganization/perforation and adult worm paralysis were also observed for C. procera aqueous and ethanolic extracts (Khalil et al., 2016). Bromelain extracted from Ananas comosus (L.) Merr. stem has been found as potent anthelmintic against gastrointestinal nematodes (Stepek et al., 2004;Domingues et al., 2013). Bromelain removes and digests the cuticle layer of nematodes resulting in immobility and death of the parasites (Stepek et al., 2004;Stepek et al., 2006). Azadirachta indica A. Juss. leaves contain condensed tannins (CT) (Sakti et al., 2018), which facilitate diffusion of flavonoids by binding to the cuticle proteins (Kerboeuf et al., 2008). Flavonoids and CT inhibit secretion of key enzymes (e.g., esterase, tyrosin kinase, and nonspecific cholinesterase) that may cause fatal intracellular instability, neuromuscular disorganization, energy depletion, paralysis and death of parasites (Hoste et al., 2006;Kerboeuf et al., 2008).
Essential oils of Corriandrum sativum L., C. citratus, C. schoenanthus, E. staigeriana and L. origanoides were found highly effective against H. contortus in vitro. Essential oils may acquire this efficacy owing to a mixture of different chemical constituents whose interaction can result in compounds that inhibit or disorganize vital functions from the initial stages of development onward, interrupt with parasite metabolic activities, and interfere with drive mechanisms due to possible destructuring of the nervous system (Oka et al., 2000). Furthermore, essential oils can alter the permeability and cause depolarization of cytoplasmic membrane by interacting and disrupting the chemical structures of lipids, polysaccharides, and phospholipids (Bakkali et al., 2008). Phytochemical profile of Cocos nucifera L. revealed the presence of alkaloids, flavonoids, phenols, triterpenes, and condensed tannins among others (Lima et al., 2015). The compounds like flavonoids have antioxidant activities, while condensed tannins have shown antiparasitic activities by binding to proteins present in the cuticle, oral cavity, esophagus, and cloaca, thus inducing chemical and physical damage in the parasite (Costa et al., 2010). The efficacy of plant compounds may be attributed to the fact that they inhibit or retard the growth, maturation damage, suppress appetite or reduce procreative ability, which are all the causes of mortality. Moreover, the considerable activity of plants extracts may be due to the additive or synergistic relationship among different major components which can interact with multiple molecular targets in various developmental stages of the parasite to produce a pharmacological effect (Marie-Magdeleine et al., 2009). H. contortus exposed to C. citriodora essential oils demonstrated ultrastructural changes, such as formations of vacuoles, disorganization of muscular layer, and changes in the mitochondrial profile. These changes suggest the loss of homeostasis and loss of motility due to muscular disorganization of the parasite . E. helioscopia has monoterpens, which are lipophilic and can penetrate through cell membrane, induce expansion, increase permeability and disturb membrane structure and membrane embedded enzymes (Cox et al., 2000;Samy and Ignacimuthu, 2000;Cox et al., 2001). The nematicidal activity may also be attributed to the presence of tannins, which on the surface of nematodes can form complexes with proteins and result in alteration of metabolic pathways (Min et al., 2004;Ahmed, 2010) and enzymes (cysteine proteinases), which can damage the cuticle and kill the nematode parasites (Stepek et al., 2004;Stepek et al., 2006). Similarly, saponins, amino acids, and sterols can disturb proteins structure (Mabusela et al., 1990) therefore, affecting growth and reparation of nematode body. While compound muzigadial and ajoene has anti-feedant activity and inhibit proliferation of arterial smooth muscle cells and protein prenylation (Ferri et al., 2003;Mohanlall and Odhav, 2009). L. latisiliquum leaves forage utilization presented ultrastructural changes; for instance, disturbance of intestinal muscular cells/ tissues and cytoplasmic vacuolization, suggesting that different secondary metabolites of leaves may provoke these changes. The alterations to the intestinal cells may be due to the ingestion of active compounds by the parasite, and the resulting direct contact between the bioactive compounds and the intestinal cells. The cytoplasmic vacuolization described can be interpreted as signs of disturbances in cellular functions, possibly due to imbalance of fluid exchanges between the intestinal and pseudocoelomic space and/or between the muscle and the pseudocoelomic space (Martínez-Ortiz-de-Montellano et al., 2019).
The high efficacy of Nicotiana tabacum L. against H. contortus could be attributed to the presence of nicotine, a ganglion stimulant (Bowman and Rand, 1980). Nematode muscles are known to contain excitatory neuromuscular junctions containing ganglion type nicotinic receptors with acetylcholine as their neurotransmitter (Neal, 2020). Any ganglion stimulant would tend to activate theses neuromuscular junctions causing a spastic paralysis in the worms leading to their death and expulsion from the host (Nouri et al., 2016). Among the reported plants with promising anti-haemonchiasis activity, L. viridiflorum leaves are rich in flavonoids and its anthelmintic action of egg hatching inhibition could be due to the effect on enzymatic activity and metabolic processes in helminthes (Kerboeuf et al., 2008).
Different protein contents e.g., proteases, ribosomal proteins, chitinases etc. isolated from Spigelia anthelmia L. were effective against different life stages of H. contortus (Araujo et al., 2017). Proteases can cause severe damage to the cuticle by disrupting cuticular proteins of the parasite (Liang et al., 2010), in larvae the proteases may hydrolyze/digest the proteins necessary for larval migration (Araujo et al., 2017), and also degrade the egg membrane during egg hatching (Mansfield et al., 1992). Chitinases were also identified which can degrade chitin present in the egg shell (Rogers and Brooks, 1977) and larva, inhibiting their development and leading to death (Rocha et al., 2015).
Comparative analysis also revealed that plant extracts/essential oils were more effective in vitro than in vivo against various stages of the parasite. Similar differences between in vitro and in vivo results with plant treatments have been previously reported (Peneluc et al., 2009;Nogueira et al., 2012) and might be related with bioavailability of plant chemical constituents in different parts of the ruminant gastrointestinal tract (Athanasiadou et al., 2007;Eguale et al., 2007b). Furthermore, adult nematodes may also be more resistant to the active components, or rumen microbiota may reduce the activity of metabolites (Nogueira et al., 2012), and other aspects, such as ruminal pH. Mostly, in gerbils the efficacy of plant extracts was reported to be comparatively low than the activity observed in sheep. Using rodents for evaluation of plants anthelmintic activity has some drawbacks, firstly the habitat for nematodes is quite different in rodents and small ruminants, hence, association between habitat and drug site absorption define the higher or lower drug activity (Hennessy, 1997). Secondly, different efficacy obtained in rodents and sheep can be described by mechanism of distribution and biotransformation of the drug in a monogastric and polygastric animal species. However, efficacy test on rodent nematodes can help researchers deduce the prescriptions to be used on sheep and goats (Camurça-Vasconcelos et al., 2007). The studies concerning isolation and purification of plant compounds responsible for antiparasitic acitivities are few and insufficient. Most of the studies reported the presence of key components of the plants which do not provide any information about the effective antiparasitic compounds and their mechanism of action. Therefore, pure comounds isolated from plants should be given focus for in vitro, in vivo evaluation, toxicology, and pharmacology studies in future research, this will provide baseline information for developing new ecofriendly and cost effective drugs with lesser side effects.

Toxicity Evaluation
Safety issues of herbal medicines have been remained a big question and scientists are being interested in herbal medicines for decades. The notion that "natural" equals "safe" is apparently deceptive, since natural products comprise pharmacologically active compounds which, when taken in high doses or in specific conditions, can be detrimental to health. Bromelain of A. comosus is non-toxic and considered safe without any adverse effects and has shown good absorption and therapeutic benefits (Maurer, 2001). Moreover, no alteration in body weight, food, and water consumption was observed. The enzymes, urea, and creatinine levels of serum were also unaltered and no significant difference was observed (Dutta and Bhattacharyya, 2013). A. millefolium aqueous extract oral and intraperitoneal administration produced no significant biochemical and histopathological changes in Wistar rats (Cavalcanti et al., 2006). No relevant signs of toxicity were observed for longer periods of exposure, however, slight changes in blood glucose and cholesterol levels, and liver weight were detected, neither correlated with dose or period of exposure nor suggestive of toxicity (Cavalcanti et al., 2006). C. sativum was safe and no effects on hematological profile, histology, relative organ weights, and plasma markers of damage vital organs were found. However, a significant body weight loss was observed due to reduction in food intake (Patel et al., 2012), which is suggestive of the disturbances in carbohydrates, proteins, and fats (Ecobichon and Klaassen, 2001). C. citratus was safe and produced no toxic effects when tested against mice peritoneal macrophages (Santoro et al., 2007). Similarly, C. schoenanthus depicted no toxicological effects on hepatic and renal parameters in lambs (Katiki et al., 2012).
The ethyl acetate extract of C. nucifera presented no acute oral toxicity at the tested doses. However, the intraperitoneal and intramuscular administration was toxic (Tayler et al., 2020). Moreover, the hemoglobin level fallen below the normal limit after 8 days, suggesting that the extract could have negative effects if used for longer periods of time (Tayler et al., 2020). Similarly, E. staigeriana essential oil when administered orally was non-toxic, while intraperitoneal administration did not depict similar results (Macedo et al., 2010). Traditionally, when a substance administered orally and show LD 50 value equal to 1000 mg/kg is considered to be safe or less toxic (Garner et al., 1961). The observed difference in toxicity may be attributed to the fact that after oral administration, the extract may be poorly absorbed, detoxified by the liver (Hayes and Loomis, 1996) or degraded by the stomach and gut digestive enzymes, however, during intraperitoneal administration the absorption is systematic and toxicity is stronger and appear earlier (Hayes and Loomis, 1996;Obici et al., 2008).
E. helioscopia was safe and produced no physiological alternations of vital organs and the biochemical parameters were also unchanged at the tested doses (Saleem et al., 2016). S. anthelmia and N. tabacum did not affect the body weight and animal behavior and were considered to be non-toxic (Ribeiro et al., 2017;Andjani et al., 2019).
Low toxicity of A. sativum and L. viridiflorum was observed on human HaCat and mammalian macrophages cells, respectively, (Krstin et al., 2018;de Melo et al., 2020). Mice became dead after oral administration of C. citriodora essential oil and citronellal suggesting toxicity of the plant species .
Prolonged use of A. absinthium and A. herba-alba lead to neurotoxicity and infertility by affecting the reproductive system, respectively (Almasad et al., 2007;Lachenmeier, 2010). A. indica poisoning affect was dose and time dependent and histopathological analysis showed that the testicles, liver, and kidneys were the organs affected (Deng et al., 2013). C. procera latex was found to be toxic at the tested doses, animals developed signs of nervousness, salivation, urination, dyspnea, tachycardia, and loss of condition. Severe pathological changes in intestines, heart, liver, kidneys, lungs, and brain were also observed (Mahmoud et al., 1979). A slight change (decrease) in the serum biochemical profile was also observed. This decrease in serum zinc, iron, and copper concentration (Al-Qarawi et al., 2001) might be due to continue (i) interference of adult parasites in the abomasum with digestibility and absorption of nutritive substances as a results of existing damage to the abomasal mucosa and its digestive function, or (ii) effects of unknown toxic principles elaborated by the worm.

CONCLUSION AND FUTURE RECOMMENDATIONS
Mostly, in vitro studies have been performed to evaluate the antihaemonchiasis activity of plants. In vitro studies have a key role in initial screening however, these studies provide no information of bioavailability, toxicity, and in vivo efficacy of tested extract/ compound. Hence, in future in vivo studies by using suitable animal models should be carried out to understand the pharmacokinetics and pharmacodynamics of the tested extract/ compound. Most of the in vivo studies provide no evidence about toxicity and mechanism of action of the medicinal plant/compound, this is the most neglected aspect and strongly suggested to researchers to evaluate toxicity levels and pharmacological action of the tested plant/compound. Mentha x vilosa Huds., Anthemis nobilis L (syn. Chamaemelum nobilis (L.) All.), Lantana camara L., Trichilia claussenii C. DC., Croton macrostachyus Hochst ex Delile, Lavandula officinalis (Chaix and Kitt.), Coleus maculosus subsp. edulis (Vatke) A.J.Paton (Plectranthus punctatus (L.f) L'Her.), Maesa lanceolata Forssk., and Foeniculum vulgare Mill. among others were highly effective in vitro against different life stages of the parasite, however, these plant species are not tested for in vivo efficacy. These plants should be evaluated for in vivo antihaemonchiasis activity along with phytochemical profile, toxicological effects, and pharmacological activity. Most of the reported studies provide no information regarding time exposure and LC 50 values of medicinal plant/compound used for in vitro evaluation. Time exposure and LC 50 are very important parameters to understand the accurate efficacy of medicinal plants, therefore, it is recommended to provide this information in future studies. Plants contain a number of different compounds, which act synergistically to perform an activity, only few compounds have been isolated and tested in vitro/in vivo against H. contortus. It is recommended to identify/isolate individual compounds and evaluate their activity, this will provide more precise and in depth information of antihaemonchiasis potential of the medicinal plant under observation. Plant compounds cinnamaldehyde, thymol, and carvacrol have revealed high efficacy in in vitro studies, it is recommended to further investigate these compounds for in vivo activity. Carvone and anethole have shown promising anti-haemonchiasis potential in vitro and in vivo, however, their toxicity levels and pharmacological effects are unknown and should be investigated in future studies. L. viridiflorum has revealed high efficacy both in vitro and in vivo and has no adverse/toxic effects after oral administration and considered to be safe, it is recommended to pharmaceutical industries to further investigate this plant species because it could be an alternative candidate for drug development against H. contortus.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
MA and RA conceptualized the idea. RA, MR, and SM searched the literature and drafted the manuscript. RA, MR, SM, SN, and SA drafted the figures and tables. MA, SNK, and SM supervised the whole process, provided comments on the initial draft and helped in editing. All authors critically revised the manuscript and approved the final version.