In vitro and in vivo anthelmintic response of the seeds of Amomum subulatum roxb and Vitex negundo

The current study was designed to check the anthelmintic activities of some local plants. Seeds of Amomum (A.) subulatum and Vitex (V.) negundo in different solvents were subjected to in vitro (adult motility assay; AMA and egg hatch assay; EHA) and in vivo (faecal egg count reduction test; FECRT) anthelmintic activity testing protocols using Haemonchus (H.) contortus as an experimental model. The results of AMA, EHA, and FECRT were statistically analysed through linear regression and Duncan multiple range test. In AMA test, at 50 mg mL -1 concentration, the percent mortality of H. contortus was higher in A. subulatum than V. negundo , whereas, in EHA test, A. subulatum was proven better ovicidal (LC 50 =14.2 µg mL -1 ) than V. negundo (LC 50 = 65.7405 µg mL -1 ). The FECRT also indicated the better efficacy of A. subulatum than V. negundo against natural infection of gastrointestinal (GI) parasites. The crude powder of plants used in this study showed 29.6% to 57.7% anthelmintic. The reduction rate was found higher for A. subulatum (3 g kg -1 ) as compared to V. negundo (7 g kg -1 ). Reagrding efficacy analysis of solvents used for plants extract, ethyl acetate and chloroform were found better in increasing ovicidal activity in adult worms ( in vitro testing), whereas, the crude aqueous methanol was found better than the crude powders in in vivo testing. It will be beneficial to document the indigenous knowledge to standard scientific procedures for their validation. This study will help to motivate the farmers to make a better choice of cultivation of the indigenous plants because of their varying efficacies as an alternative preventive approach against the GI parasitic infections.

In Pakistan, several medicinal plants have been documented from various regions.People from different parts of the country are using these medicinal plants for the treatment of different diseases including parasitic diseases and transfer this knowledge from generation to generation (Goraya et al., 2013;Badar et al., 2017Badar et al., , 2021)).Still, these plants are used by native healers and, they don't have any knowledge about the active ingredients.In the present study, we select the A. subulatum Roxb and V. negundo L plants which are being used by native healers for the treatment of parasitic infection (Badar et al., 2017).The objective of the study was to check the anthelmintic efficacy of A. subulatum Roxb and V. negundo L through in vitro and in vivo assays against Haemonchus contortus models.

Selection and procurement of plants
The two seed plants viz; A. subulatum Roxb (family Zingiberaceae) and V. negundo L. (family Verbenaceae) were selected during a documentation survey of the herbs as candidates to treat the animals for helminths infection (Badar et al., 2017).The selected plants' seeds were purchased from a local market and also directly harvested from the fields.

Extraction and fractionation of seeds 2.2.1. Extraction
Grinding of seeds was done after drying, and crude aqueous-methanol extracts (CAME) were prepared following the guidelines described by Tabassam et al. (2008).Ground powder was soaked in an aqueous solvent (aqueous-methanol 30:70) for three days and passed through muslin cloth and filter paper.The whole procedure was repeated thrice.Afterwards, the combined filtrate was evaporated to get CAME using rotary evaporator at 40°C at low pressure.

Fractionation
Crude extract was further processed for fraction with organic solvents e.g., ethyl acetate, petroleum spirit and

Introduction
Helminths especially gastrointestinal (GI) nematodes are highly abundant due to easy mode of transmission, wide variety of hosts and vectors, and low level of awareness which cause huge economic losses to livestock producers (Raza et al., 2014;Rizwan et al., 2017).Ethnoveterinary Medicine (EVM) has been used for many years and is widely used in many areas of the world including Indo-Pak as a traditional medicine in curing animal diseases.Use of EVM is of great significance in modern veterinary medicine, especially in sub-continent countries due to easy accessibility and low cost (Iqbal et al., 2006;Goraya et al., 2013;Hamad et al., 2014;Rizwan et al., 2019;Ahmad et al., 2020).
Anthelmintic resistance shown by nematodes against all chemical groups, and chemical residues in animals in underdeveloped countries such as Pakistan is a great problem (Muhammad et al., 2005;Iqbal et al., 2005;Jabbar et al., 2007;Hussain et al., 2008;Tabassam et al., 2008;Chirag et al., 2013).Due to this factor, scientists from developed countries have also been screening plants for their anthelmintic properties (Tabuti et al., 2003).Studies and EVM surveys have revealed effective and extensive use of some plants with anthelminthic properties (Jabbar et al., 2007;Hussain et al., 2008;Goraya et al., 2013;Badar et al., 2017).
Brazilian Journal of Biology, 2024, vol.84, e261768 3/9 Efficacy of medicinal plants against parasites chloroform.All the crude extracts were fractionated by using organic solvents such as chloroform, petroleum, ethyl acetate and spirit (Williamson et al., 1998).Distill water (20 mL) was used to dissolve 20 g of crude extract in separating funnel.In the next step, the petroleum spirit (60 mL) was added to the funnel followed by vigorous shaking and kept undisturbed for 30 minutes for the separation phase.Petroleum layer containing soluble part of the extract was separated and petroleum ether (60 mL) was added followed by vigorous shaking and separation phase process until getting a clear layer of petroleum spirit.The solvent was evaporated using a rotary evaporator to get spirit petroleum fractions.The ethyl acetate was used to fractionalize the remaining part of the extract following the same procedure as described above.

Parasitological procedures 2.3.1. Adult Motility Assay (AMA)
The efficacy of different fractions and the crude extracts was accessed by taking Haemonchus (H.) contortus (live) from abomasum of slaughtered sheep as described by Singh et al. (1985).The worms were separated in petri dishes (minimum 10 worms in each) and exposed to CAME, ethyl acetate fraction, petroleum spirit fraction and chloroform fraction at 1.56, 3.12, 6.25, 12.5, 25 and 50 mg mL -1 concentrations.Levamisole at 0.55 mg mL -1 and phosphate buffer saline (PBS) were used as a positive and negative controls, respectively.All treatments were conducted in three replicates at room temperature.Observation made for the accuracy of experiment was motility inhibition at 0, 2, 4, 6, 8, 10, 12 and 24 hours intervals.For each treatment, the number of survived and dead worms was noted.

Egg Hatch Assay (EHA)
The CAME, ethyl acetate, petroleum spirit and chloroformfractions of crude extract were used to check anthelmintic activity keeping Albendazole as a positive control.Briefly, 24 multiwell plates were used for each solvent with 5 dilutions i.e., 12, 1.2, 0.12, 0.012 and 0.0012 mg mL -1 in triplicate.250 eggs were in 1.5 mL of water were poured in each well.Following 36 hours incubation of plates at 28°C, eggs percent hatching (live or dead larvae) was counted using an inverted microscope.

Faecal Egg Count Reduction Test (FECRT)
At day 0, 14 different groups of sheep were formed which are one to four months in age and were previously infected with H. contortus.Group was selected using a complete randomization method based on their live weights.Layout plan for crude powder and CAME of Seeds of A. subulatum and V. negundo given to different groups of sheep naturally infected with H. controtus is given in Table 1.The faecal egg counts were done on days 0, 4, 8 and 12 post-treatments from each animal as given by Badar et al. (2021).

Statistical analyses
All measurements were summarized as Mean ± SE. Adult mortality was determined by Abbott's formula and probit equation was used to determine the association of Probit of kill and log concentration of treatment (Abbott, 1925).For egg hatch test, linear regression was used to determine the lethal concentration 50 (LC 50 ).Duncan multiple range (DMR) test was used to compare eggs per gram (Mean+SEM) of faeces.

In vitro anthelmintic activity
In AMA, after 10 hours post-exposure of H. contortus with 50 mg mL -1 of A. subulatum, 73.3% mortality was reported.The reference drug (Levamisole) revealed 100% worm mortality within 2 hours post-exposure.Among the fractions of A. subulatum, ethyl acetate produced the most effective anthelmintic activity.However, V. negundo extract in ethyl acetate did not show up to the mark anthelmintic activity.The CAME, chloroform and petroleum spirit fractions of both plants were found least responsive.The results of AMA for various treatments of fractions of A. subulatum and V. negundo are summarized in Table 2.
In EHA, the CAME of A. subulatum was considered to be better ovicidal based on its lower LC 50 (14.1773µg mL -1 ) than that of V. negundo.The eggs exposed to aqueous fraction of V. negundo and chloroform fraction of A. subulatum showed more pronounced response, whereas, petroleum spirit fraction of both plants' extract was effective in dosedependent manner (Table 3).

In vivo anthelmintic activity
The anthelmintic and dose dependant efficacies of crude powder (CP) and methanol extract of both plants were studied.Efficacy of CP and methanol extracts of study plants based on FECRT at days 0, 4, 8 and 12 posttreatment is shown in Figure 1.Anthelmintic efficacy was recorded from 29.6% to 57.5%.Using A. subulatum at the rate of 3 g kg -1 showed a higher reduction (57.5%) in faecal egg counts compared to V. negundo which was 8 g kg -1 .The CP and methanol extracts showed different anthelmintic efficacies.The effects of both plants' treatments were statistically significant (P<0.05)from the untreated control.The variations in the egg per gram (EPG) of the Levamisole treated groups with those treated with plant extracts were statistically different (P<0.05).

Discussion
There is increasing evidence in support of the hypothesis that bioactive secondary phytochemicals are relatively abundant and are likely to initiate drug discovery.Due to the difference in their polarities, these phytochemicals have variable affinities for different solvents.Different chemical compounds are present in each fraction according to the solvents used.Active compounds present in the fractions are the main responsible agents for biological activities.So, there is a need to check the efficacies of fractions of different botanical compounds to be used as anthelmintics (Hamad et al., 2014).
The change in anthelmintic efficacies of different fractions might be due to the change in parasitic targets and plant chemistry.Several tropical legumes such as Lespedeza (L.) cuneata (50 g CTs kg −1 ) has also shown some promising results in reducing total faecal egg output, faecal egg counts (FECs) by 57-100%, and the number of nematodes (Teladorsagia sp., Trichostrongylus and Haemonchus) in goats (Min and Hart, 2003).A higher level of dried Acacia karoo leaves can significantly reduce FECs and H. contortus load in goats (Kahiya et al., 2003).The anthelmintic targets may vary with the parasitic stages.O'Grady and Kotze ( 2004) have discussed that mechanism-based assays for anthelmintic effects showed a potential problem with the whole-organism screening.These assays can be applied easily to the free-living parasitic stages such as larval development, larval motility assays and egg hatch, while the eventual use of the anthelmintics will be directed to the host dependant parasitic stages.In an in vitro assay, 0.16 mg/mL inhibitory concentration (IC50) of ethyl acetate extract of V. negundo leaves was recorded (Sahare and Singh, 2013).Ethanol extract of V. negundo leaves at 50 mg/mL requires less time to cause paralysis and death of parasites as compared to ethanol extract of roots of the Tamarindus (T.) indica (Raul et al., 2012).
Synthetic anthelmintic drugs show unwanted effects on the animal body like urticaria, gastrointestinal disturbance, bronchospasm, dizziness, in coordination, paraesthesias, and vertigo.In pregnanet animals those with compromised hepatic and renal function these drugs are contraindicated.The formulation of anthelmintic drugs from A. subulatum and V. negundo will be safe, cost effective, natural and probably will not show any such unwanted effects.Still there is need to determine the main active ingridients from these plants which are responsible for the anthelmintic activity.

Conclusions
The phytotherapeutic approaches have been found promising in vitro and in vivo for the remedies of the GI parasites in general and H. contortus in specific.It is recommended to document the indigenous knowledge after subjected to standard scientific procedures for their validation.Both the study plants (A.subulatum and V. negundo) showed anthelmintic activity comparable to the standard therapy, however, among various fractions, ethyl acetate fractions at 50 mg mL -1 of A. subulatum and CAME at 50 mg mL -1 of V. negundo were found the most effective against H. contortus in vitro.In vivo trials indicated the CAME at 3 g kg -1 of A. subulatum and crude powder at 8 g kg -1 of V. negundo as the most promising dose.The study plants may initially be recommended as a shortterm prophylactic approach to cultivate in the grazing area which may reduce the severity of GI parasitism in the livestock population.

Figure 1 .
Figure 1.Effects of various solvents extracts of Vitex negundo and Amomum subulatum on naturally infected mixed species of gastrointestinal nematodes' eggs in sheep feces.Eggs per gram (EPG) is the measure of efficacy of extract present on y-axis whereas, day in which effect comes out along with dose is mentioned on x-axis.

Table 1 .
Layout plan for crude powder and crude aqueous methanol extract of seeds of Amomum subulatum and Vitex negundo given to different groups of sheep naturally infected with Haeminchus controtus.

Table 2 .
Comprative efficacy of crude aqueous methanol extracts, chloroform fractions, ethyl acetate fractions, petroleum spirit fractions and aqueous fractions of Amomum subulatum and Vitex negundo with Levamisole on the survival of Haeminchus contortus of sheep.
efgMotility of worms were observed for 24 hours at every 2 hours interval.However, data of 0, 2, 6 and 10 hours has been given which deemed enough to meet the objectives of the experiment.Values having common alphabet superscripts indicate non-significant results at 95% confidence interval.Brazilian Journal of Biology, 2024, vol.84, e261768 5/9Efficacy of medicinal plants against parasites

Table 3 .
In vitro effect of seeds of Vitex negundo and Amomum subulatum and LC 50 of the different fractions and crude aqueous methanol extracts on hatching (%) of Haemonchus contortus eggs.