PJZ_58_3_1271-1281

INTRODUCTION

The genus Haemonchus has a dynamic physiological nature. It lives in a faunal host parasitic relationship. The 12 known species of Haemonchus infect a wide range of hosts, including goats, sheep, antelopes, giraffes, Cervidae, bovines and wild ruminants (Hoberg et al., 2004a; Hoberg and Zarlenga, 2016b). The most vulnerable hosts are domestic ruminants, i.e., goats, sheep and cattle. The infection rate is greater in tropical, subtropical, warm temperate and summer rainfall regions. Hot and humid environmental conditions play a significant role in the spread of infection (Arsenopoulos et al., 2021).

Pathologically, H. contortus resides in the abomasa of infected host animals. It is actively engaged in sucking blood, which induces anaemia. Sick animal loss their body weight and wool growth. It also effects hormonal levels, the estrous cycle, the absorption of calcium, phosphorus and magnesium, the intake of food, appetite and immunity levels (Besier et al., 2016a; Dutta et al., 2017; Arsenopoulos et al., 2021). Young and adult animals with compromised immune systems are more susceptible to haemonchosis (Zajac, 2006).

According to the 2021-22, economic census, the total populations of sheep, goats and cattle in Pakistan was estimated to be 31.9 million, 82.5 million and 53.4 million, respectively. The overall livestock contribution to the national GDP was 14.04% (FAO, 2022; Pakistan Economic Survey, 2021-22). Progress in the livestock sector can be hampered by infectious diseases. H. contortus is the major parasite involved in enormous economic losses worldwide (Charlier et al., 2020). Due to haemonchosis, 29% reduction in milk and 27% in meat production was estimated in Punjab, Pakistan causing a loss of 8800.09 million PkR, annually (Qamar et al., 2011). The situation has been worsened by the fact that H. contortus has developed resistance against anthelmintic drugs (Bibi et al., 2017). This indicates the limited success of both preventive and treatment measures with the help of commercial anthelmintics (Besier et al., 2016b).

Medicinal plants have the potential to kill endoparasites and can be used as alternatives to commercial anthelmintics (Lanusse et al., 2018; Charlier et al., 2022). These therapies are applied in 75-90% of developing countries, while half of the population in the industrialized world uses them (Robinson and Zhang, 2011). In Pakistan, diverse climatic conditions allow rich biodiversity and medicinal wealth. More than 700 species are used as medicines for various purposes, while the remaining plants are being assessed for the efficacy of their active ingredients (Shinwari, 2010; Alamgeer et al., 2018). Extracts from different parts of the plant have pharmacological effects on different diseases (Moloudizargari et al., 2013).

Peganum harmala, commonly known as the Syrian rue, or harmel is a wild perennial herb. It belongs to the family Nitrariaceae which is distributed in the Middle East and in parts of South Asia, such as India and Pakistan (Miraj, 2016). It is distinguished by its foul smell (Moloudizargari et al., 2013) and is traditionally used as a disinfectant and mosquito repellent and for the treatment of many human diseases.

Withania somnifera, also called winter cherry, or Indian ginseng is a wild annual herb. It belongs to the Solanaceae family and abundantly grows in arid areas of the Mediterranean, tropical Africa, South Africa, Canary and Cape Verde Islands, Afghanistan, Pakistan, India, China, Sri Lanka and Nepal. It is also found in Europe, southern Australia and New South Wales (Paul et al., 2021). It is considered an important medicinal plant (Meena et al., 2020) and is used for treating neurodegenerative diseases (Ale et al., 2021).

Helminth parasitism is the major cause of reduced production in livestock worldwide. Wildly grown P. harmala and W. somnifera are highly esteemed medicinal plants as reported in several studies on various aspects. To exploit their activities as anthelmintics against H. contortus, a detailed in vitro study was conducted to treat haemonchosis.

MATERIALS AND METHODS

Study area selection for plant collection

P. harmala and W. somnifera were collected in May 2022 from the Kala Chitta Range (33°7’34°N and 71°45’73°E), District Attock, Punjab (Pakistan). The study area was selected on the basis of its unique plant biodiversity (Supplementary Fig. 1A, B). The collected plants were washed and taxonomically identified by a plant taxonomist, Dr. Kifayat Ullah, Department of Biological Sciences, International Islamic University, Islamabad (No. FBAS-93) of these plants were preserved at the said department. The washed materials were shade dried for 7 days, the plant materials were subjected to grinding with an electric grinder.

Abomasal working apparatus

The aim of constructing the apparatus was to control the fluctuation in the external laboratory temperature, which possibly affect the accuracy of the adulticidal assay. The apparatus is a specialized chamber designed to generate and maintain a set range of temperatures (25-30°C) and a relative humidity of 70-80%. It works as an artificial abomasal medium, to ensure the viability of temperature-sensitive worms (Supplementary Fig. 2A, B).

In vitro anthelmintic adulticidal assay

Abomasa from freshly slaughtered goats were collected from the local slaughterhouse. Highly agile, live and mature H. contortus were isolated (Supplementary Fig. 3A, B). The parasites were suspended and washed 2-3 times in PBS. A total of 105 worms were distributed into 7 groups, each in triplicate. Five worms were transferred to each Petri dish. All twenty-one Petri dishes were arranged in an adulticidal working apparatus at 25-30°C. The adjusted range was kept constant for 12 h. Under these controlled conditions, the dead worms were easily detected and separated from the viable worms.

The crude aqueous extract (CAE) and crude methanolic extracts (CME) were prepared by standard methods with partial modifications (Gilani et al., 2004). The percentage stock solution was prepared by dissolving 0.5 g or 500 mg of CAE or CME in 100 mL of PBS. The serial dilutions for the 5 tested groups had extract concentrations of 3.125, 6.25, 12.5, 25 and 50 mg/mL. Albendazole was used as a positive control at a concentration of 12.5 mg/mL. For preparing methanolic extract dilutions, 3% DMSO was used. The negative control group did not receive any treatment. The five concentrations of the targeted extracts were applied from groups I to V, whereas the negative and positive control groups were represented by groups VI and VII, respectively.

Worm mortality was observed from 0 to 12 h (Iqbal et al., 2006). The number of dead worms was calculated after interval of 2 h. The criterion for declaring mortality during the experiment was the worm’s static appearance. The final confirmation was made by placing the worms in fresh PBS. If the worm revived in PBS, it was placed back in its respective treatment. The final declaration of worm’s death was made using a microscope. The percentage of adult mortality was calculated using the following formula:

Ovicidal assay

The protocol of Coles (1984) was modified to isolate eggs. Adult female worms were triturated through a mortar and pestle to collect eggs. Eggs were isolated from the debris after filtering through a 100 µm mesh sieve. The egg solution was further diluted by adding some PBS. McMaster slides were then used to count the total number of eggs in the solution (Supplementary Fig. 4). The percentage efficiency of the extract was calculated by using the following formula:

The 48-wellplates were divided into seven groups of three wells each. Approximately 100 eggs were placed in each well of a microwell plate and PBS was added to create a solution of 130 µL with the help of a micropipette. Egg hatching media was prepared by dissolving 1 g of yeast in a mixture of 90 mL of normal saline and 10 mL of Earle’s balanced salt. Then, 20 µL of nutritive media was added to each well. Afterwards, 150 µL of either aqueous or methanolic extracts of both plants were added at the concentrations of 3.125, 6.25, 12.5, 25 and 50 mg/mL in triplicate. Albendazole at a concentration of 12.5µL was used as a positive control, while PBS or 3% DMSO was added to one group as a negative control. All the groups were allowed to incubate for 48 h at 25-30°C. At the end of the incubation period, 1 drop of lugol’s iodine solution was added in each group to stop eggs from further hatching. Under a microscope, all dead or unhatched eggs were counted with the help of McMaster slide. The egg hatching method described by Coles et al. (1992) was used to evaluate the anthelmintic efficacy of the extracts on the viability of the eggs.

Larvicidal assay

An estimated 100 eggs were suspended in 21 wells of a 48-well plate. Twenty microliters of egg hatching media was added to each well. PBS was further added to make a total volume of 200 µL. This mixture was incubated at 25-30°C for 48 h (Soulsby, 1968; Tariq et al., 2008). After completing the incubation period, 200 µL of CAE or CME at concentrations of 3.125, 6.25, 12.5, 25 and 50 mg/mL were added in each well of groups V to I. Albendazole (12.5µL) was added as a positive control in group VII. The negative control group VI was filled with PBS to a total volume of 400 µL.

Larvicidal activity was assessed by counting the number of dead larvae under microscope after 48 h of treatment using McMaster slide (Supplementary Fig. 5). The percentage of extract efficacy was calculated using the following formula:

Statistical analysis

The statistical significance of differences between means was determined by using two-way analysis of variance (ANOVA). Furthermore, Tukey’s test was used to determine differences between concentrations and solvents. LC50 values for each extract and assay were calculated by using probit analysis.

RESULTS

Ovicidal activity

Aqueous and methanolic extracts of P. harmala and W. somnifera at the concentrations of 3.125, 6.25, 12.5, 25 and 50 mg/mL inhibited the egg hatching of H. contortus. The extract potencies of P. harmala (AE=74.3%, ME=79%) and W. somnifera (AE= 72.7%, ME= 90.3%) at 50 mg/mL significantly increased (P < 0.05) in a dose-dependent manner (Table I). AEs of P. harmala and W. somnifera had similar effects i.e., 74.3% and 72.7% in ovicidal assay. However, significant differences were detected in the activity of the methanolic extracts of P. harmala (79%) and W. somnifera (90.3%) (Table I). The negative control group (PBS and 3% DMSO) in the AE and ME tests had very little effect on egg hatching, but albendazole (12.5 mg/mL) effectively inhibited (100%) egg hatching (Table I). The estimated LC50 values for the aqueous and methanolic extracts of P. harmala were 39.50 mg/mL and 26.88 mg/mL, respectively (Table I, Fig. 1). The LC50 values of the aqueous and methanolic extracts of W. somnifera were 31.85 and 24.79 mg/mL, respectively (Table I, Fig. 1). An evaluation of the ovicidal activity clearly demonstrated that the methanolic extracts of both plant species were better (P < 0.05) than the aqueous extracts.

Larvicidal efficacy

Aqueous and methanolic extracts of P. harmala and W. somnifera at different concentrations (3.125-50 mg/mL) inhibited larval development in H. contortus larvae. At 50 mg/mL concentration, the highest activities were observed for the P. harmala extracts (aqueous = 59%, methanolic = 82.7%) and W. somnifera extracts

Table I. Ovicidal and larvicidal activity (Mean efficacy ± SE) of aqueous extract (AE) and methanolic extract (ME) of P. harmala and W. somnifera on H. contortus eggs.

Groups	Concentrations (mg/mL)	P. harmala		W. somnifera
Groups	Concentrations (mg/mL)	AE	ME	AE	ME
Ovicidal activity (%)
Extracts treatments	50	74.33±4.98b	79.00±6.56b	72.67±7.54b	90.33±1.20a
	25	16.00±1.00fgh	74.33±1.67b	46.67±4.18d	61.00±5.69c
	12.5	8.00±1.53hi	24.67±3.84fg	33.00±2.08e	21.67±2.96fg
	6.25	2.67±1.33i	8.33±0.67hi	9.00±2.65hi	11.33±2.73ghi
	3.125	1.33±0.33i	2.33±0.33i	5.00±2.89i	10.00±1.00i
Negative	PBS	1.33±0.33i		1.00±0.58i
	DMSO3%		1.33±0.67i		2.67±0.33hi
Positive	12.5 ABZ	100.00±0.00a	100.00±0.00a	100.00±0.00a	100.00±0.00a
LC50		39.50	26.88	31.25	24.79
Larvicidal activity (%)
Extract treatment	50	59.00±7.21cd	82.67±0.33b	55.67±5.78d	83.67±1.20b
	25	18.00±3.46ghi	70.33±2.96c	50.33±3.18d	58.67±3.53d
	12.5	6.33±1.76jkl	61.33±2.60cd	37.00±3.79e	30.33±5.84ef
	6.25	7.33±2.03ijkl	13.00±3.21hijkl	27.00±1.73efg	14.00±8.54hijk
	3.125	2.33±0.33l	17.67±2.60ghij	19.67±0.88fgh	26.33±11.05efg
	PBS	2.00±1.00l		9.33±2.85hijkl
Negative	DMSO3%		5.33±1.86kl		10.33±5.17hijkl
Positive	12.5 ABZ	100.000±0.00a	100.000±0.00a	100.00±0.00a	100.00±0.00a
LC50		45.60	20.72	35.84	24.38

All the values are expressed as Mean ± SE. The difference in superscripts indicate significant differences (p < 0.05).

(aqueous = 55.7%, methanolic = 83.7%) (Table II). There was a dose-dependent increase in the activity of the extracts. Similarly, the AEs of P. harmala and W. somnifera showed significantly lower activity than the MEs of these plants. PBS and DMSO (3%) were used as negative controls. They have very minor effects on the inhibition of larval development. Albendazole (12.5 mg/mL), the positive control, displayed 100% inhibition of larval development (Table II). The calculated LC50 values for the aqueous and methanolic extracts of P. harmala were 45.09 mg/mL, and 20.72 mg/mL, respectively (Table II, Fig. 1). However, 35.84 mg/mL and 24.38 mg/mL were the LC50 values calculated for the aqueous and methanolic extracts of W. somnifera, respectively (Table II, Fig. 1). According to the results of the larvicidal assay, the ME of both plants had greater (P < 0.05) efficacy than their aqueous counterparts.

Table II. Adulticidal activity (Mean mortality ±SE) of aqueous extract (AE) and methanolic extract (ME) of P. harmala and W. somnifera on adult worms of H. contortus.

Extracts	Concentrations (mg/mL)	Exposure time						LC 50
Extracts	Concentrations (mg/mL)	2 h	4 h	6 h	8 h	10 h	12 h	LC 50
P. harmala
AE	50	0.00±0.00m	0.00±0.00m	2.00± 0.58ghij	4.00±0.58abcd	4.67±0.33ab	5.00±0.00a	40.74
	25	0.00±0.00m	0.00±0.00m	2.00± 0.58ghij	3.00±1.15defg	3.33±0.88cdef	3.67±0.88bcde
	12.5	0.00±0.00m	0.00±0.00m	1.33± 0.33ijkl	1.33±0.33ijkl	2.00±0.58ghij	2.67±0.33efgh
	6.25	0.00±0.00m	0.00±0.00m	1.00± 0.58jklm	1.33±0.33ijkl	1.67±0.33hijk	2.33±0.33fghi
	3.125	0.00±0.00m	0.00±0.00m	0.67± 0.33klm	1.00±0.58jklm	1.67±0.33hijk	1.67±0.33hijk
Control	Negative PBS	0.00±0.00m	0.00±0.00m	0.00± 0.00m	0.00±0.00m	0.33±0.33lm	0.67±0.33klm
	Positive ABZ	0.00±0.00m	0.00±0.00m	2.00± 0.58ghij	2.67±0.33efgh	4.67±0.33ab	5.00±0.00a
ME	50	0.00±0.00m	1.00±0.58jklm	3.33± 0.33cdef	5.00±0.00a	5.00±0.00a	5.00±0.00a	36.04
	25	0.00±0.00m	0.00±0.00m	1.67± 0.33hijk	3.00±0.58defg	4.00±0.58abcd	4.33±0.33abc
	12.5	0.00±0.00m	0.00±0.00m	1.00± 0.00jklm	2.00±0.58ghij	2.33±0.33fghi	2.67±0.33efgh
	6.25	0.00±0.00m	0.00±0.00m	1.00± 1.00jklm	1.67±0.67hijk	2.33±0.33fghi	2.33±0.33fghi
	3.125	0.00±0.00m	0.00±0.00m	1.33± 0.67ijkl	1.33±0.67ijkl	2.00±0.58ghij	2.33±0.67fghi
Control	Negative DMSO3%	0.00±0.00m	0.00±0.00m	0.00± 0.00m	0.67±0.33klm	0.67±0.33klm	0.67±0.33klm
	Positive ABZ	0.00±0.00m	0.00±0.00m	2.00± 0.58ghij	3.33±0.33cdef	5.00±0.00a	5.00±0.00a
W. somnifera
AE	50	0.33±0.33lm	0.33±0.33lm	1.67± 0.33hijk	4.00±0.58abcd	4.33±0.67abc	4.33±0.67abc	40.34
	25	0.33±0.33lm	0.33±0.33lm	1.33± 0.33ijkl	2.33±0.33fghi	3.00±0.00defg	3.00±0.00defg
	12.5	0.33±0.33lm	0.33±0.33lm	1.33± 0.33ijkl	2.00±0.58ghij	2.00±0.58ghij	2.67±0.67efgh
	6.25	0.00±0.00m	0.00±0.00m	0.00± 0.00m	0.33±0.33lm	0.67±0.33klm	1.00±0.58jklm
	3.125	0.00±0.00m	0.00±0.00m	0.67± 0.33klm	0.33±0.33lm	0.67±0.67klm	1.00±0.58jklm
Control	Negative PBS	0.00±0.00m	0.00±0.00m	0.67± 0.33klm	0.67±0.33klm	0.67±0.33klm	0.67±0.33klm
	Positive ABZ	0.33±0.33lm	0.67±0.67klm	2.00± 0.58ghij	4.33±0.67abc	5.00±0.00a	5.00±0.00a
ME	50	0.00±0.00m	1.33±0.33ijkl	3.00± 0.58defg	5.00±0.00a	5.00±0.00a	5.00±0.00a	34.67
	25	0.00±0.00m	0.00±0.00m	1.33± 0.33ijkl	3.33±0.33cdef	3.67±0.33bcde	4.00±0.00abcd
	12.5	0.00±0.00m	0.00±0.00m	1.33± 0.33ijkl	2.33±0.67fghi	2.67±0.33efgh	2.67±0.33efgh
	6.25	0.00±0.00m	0.00±0.00m	0.67± 0.33klm	2.00±0.58ghij	2.33±0.33fghi	2.67±0.33efgh
	3.125	0.00±0.00m	0.00±0.00m	0.00± 0.00m	0.00±0.00m	0.33±0.33lm	1.00±0.58jklm
Control	Negative DMSO3%	0.00±0.00m	0.00±0.00m	0.00± 0.00m	0.33±0.33lm	0.33±0.33lm	0.67±0.33klm
	Positive ABZ	0.00±0.00m	0.67±0.33klm	3.00± 0.58defg	4.33±0.33abc	5.00±0.00a	5.00±0.00a

All the values are expressed as Mean ± SE. The difference in superscripts indicate significant differences (p < 0.05)

Adulticidal effects

The anthelmintic activity of the crude methanolic extracts of both plants against adult H. contortus had significantly higher (P < 0.05) activity at all concentrations from 3.125 to 50 mg/mL as compared to AEs (Table II). ME of P. harmala and W. somnifera at 50 mg/mL concentration resulted in 100% worm mortality after 8 h of exposure. However, after 8 h of exposure, 80% of the worms were killed by AE at the same concentration (50 mg/mL). The lower concentrations (25-3.125 mg/mL) did not show 100% effects even at 12 hrs of extract exposure (Table II). All the extracts had significant (P < 0.05) dose- and time-dependent effects on worms compared to the positive control (albendazole) (12.5 mg/mL). No considerable deaths were observed in the negative controls in the aqueous (PBS) and methanolic (3% DMSO) assays. In adulticidal assay, the estimated LC50 values for the aqueous and methanolic extracts of P. harmala were 40.74 mg/mL and 36.04 mg/mL, respectively Table II, Fig. 1). The LC50 values of the aqueous and methanolic extracts of W. somnifera were 40.34 and 34.67 mg/mL, respectively (Table II, Fig. 1). The assessment revealed that the methanolic extracts of both plant species were better (P < 0.05) than the aqueous extracts.

DISCUSSION

The results of the present study demonstrated that both P. harmala and W. somnifera had significant (P < 0.05) nematocidal activity compared to the negative controls (PBS and 3% DMSO) and the positive controls i.e., albendazole. In this detailed in vitro study, the worms, larvae and eggs were exposed to a set range of temperatures (25-30°C) in a self-structured foster abomasal apparatus. The obtained results were more reliable and easier to record than those obtained under open laboratory conditions. In our analysis, W. somnifera was found to be more effective than P. harmala at all the tested concentrations. The variations in the results were related to the type of plant extract. The anti-haemonchus effects of these extracts are likely to be associated with the presence of secondary metabolites. Presently, the most tested biologically active compounds include terpenes, glycosides, saponins, flavonoids, tannins, and β-carboline alkaloids such as harmine, harmaline, harmalol and harmol (Deore and Khadabadi, 2010; Moloudizargari et al., 2013; Qamar et al., 2017; Spiegler et al., 2017; Danial et al., 2018; Manjusa and Pradeep, 2022). Saddiqe et al. (2019) revealed that flavonoids, tannins, steroids and saponins in W. somnifera extracts are the key secondary metabolites involved in anthelmintic activity. However, harmine, a primary alkaloid in P. harmala, plays a significant role in this regard. The potency of P. harmala is attributed to the presence of abundant β-carboline alkaloids (Mayad et al., 2019). In the present study, ME had better effects than AE. The difference in mortality might be due to differences in polarity and the total phenol and flavonoid contents in the extracts (Saddiqe et al., 2019). The combination of alkaloids from P. harmala and tannins and saponins from W. somnifera extracts could be more effective than either individual effect alone. The present study lacks this aspect but aimed for promising synergistic results in the future.

Ovicidal results revealed that the tested concentrations (50-3.125mg/mL) of both plant extracts induced egg hatch inhibition, statistically (P < 0.05) comparable to that of negative controls and the positive controls of albendazole groups. At 50 mg/mL concentration, ME of W. somnifera showed highest ovicidal efficacy as compared to its AE and ME and AE of P. harmala. Moreover, the highest activity of ME of W. somnifera expressed similar potential owned by the reference drug albendazole. Egg hatch inhibition increases with increased plant concentration. These results can be attributed to the presence of active phytochemicals that act as inhibitors on egg hatching. The variations in their inhibitory effects in the tested groups were due to the nature of the extraction. The extract concentration and type of solvent may determine the level of solubility (Saidulu et al., 2014). The active compounds potentially involved in egg hatch inhibition were suggested to be saponins and tannins (Marie-Magdeleine et al., 2009; Bekiri et al., 2023). The presence of these compounds was detected in maximal concentration in methanol and water during phytochemical screening (Saidulu et al., 2014). A lesser amount of these compounds might also explain the lower inhibitory effects of the P. harmala extract. Saponins and tannins were absent, but alkaloids and flavonoids were abundantly present in its aqueous extracts (Bekiri et al., 2023). Three alkaloids (berberine, harmaline and piperine) in P. harmala are known to cause inhibitory effects on egg hatching (da Silva et al., 2021). We report that P. harmala and W. somnifera extracts exhibit anthelmintic properties, which contradict the findings of Niaz et al. (2015). These authors suggested that the variation in plant effectiveness could be attributed to the differences in the species of host and the parasite.

Another anthelmintic study of three alkaloids (berberine, harmaline and piperine) against gastrointestinal nematodes in goats showed that harmaline had similar effects (80.30%) on egg hatching (da Silva et al., 2021). P. harmala was also found to be very effective against Schistosoma bovis infection. The orally administered capsule of its powder was used to treat infected bovines. The reductions in parasite eggs were similar to those of praziquantal (PZQ) (Niaz et al., 2015). In another study, aqueous extracts of six plants (Peganum harmala, Artemisea herba-alba, Ricinus communis, Olea europea, Allium sativum and Thymus vulgar) were tested for their ability to treat Cryptosporidium parasites. The extract of P. harmala induced maximum oocysts shedding from the intestine of infected mice (Al-Hamairy et al., 2012). Another confirmation of the anthelmintic efficacy of W. somnifera was reported by Miaron et al. (2005). The extract was applied to dorper sheep to control tapeworms and effectively reduced the egg count (88%) in the faecal material.

In case of larvicidal results, the effects of MEs of W. somnifera and P. harmala showed significantly (P < 0.05) greater larvicidal potential than their AEs and the negative controls. However, the activity of MEs was significantly (P < 0.05) lower than albendazole (positive control). The possible difference between the efficiencies of methanolic and aqueous extracts was due to the solubility of various phytochemicals in the solvents. The possible cause of this low activity of aqueous extracts was the poor solubility of various phytochemicals in water (Dhawan and Gupta, 2016). Most of the metabolites are aromatic and fat soluble and dissolve more readily in methanol than in water (Forssten et al., 2010). Extracts containing a high number of these secondary metabolites had a collective effect on the integrity of the tested larvae. The dead, deformed larvae lose their morphological shape by separating their cuticle from the pharynx, bulb and intestinal cells (Chan-Pérez et al., 2016). Tannins are the most studied active compounds investigated for their effects on egg hatch inhibition and larval development in different nematodes and cestodes (Molan and Faraj, 2010).

In the present study, the methanolic extract performed better than the aqueous extracts, which was very close to the findings of Marie-Magdeleine et al. (2010), who revealed that the methanolic extract had the highest efficacy on the larval motility of H. contortus. The results of the present study agree with the findings of Osman et al. (2012), who investigated the anti-cercaria effects of P. harmala extracts (aqueous and methanol) against Schistosoma mansoni.

In adulticidal study, MEs from W. somnifera and P. harmala were significantly (P < 0.05) more effective than their aqueous counterpart after 8 h of extract exposure. However, at first 2 and 4 h of extract exposure, the difference in adulticidal activities of aqueous and methanolic extracts at all tested concentrations (3.125-50 mg/mL) were negligible similar to that of positive (albendazole) and negative controls groups. The deaths of 100% worms in albendazole and AEs (50 mg/mL) were noticed after 10 and 12 h of extract exposure. The extent of lethality in worms depends on the concentration, duration of the extract exposure at any specific concentration and the ambient temperature of the worms. A dose-dependent increase in concentration creates necrotic spots on worm cuticles and this haemorrhage eventually leads to worm paralysis and death (Kirtiman, 2012). The scarred worms that were lighter in weight and color were the extract-affected worms floating on the surface of the extract. The rest of the worms were separated as live worms retained their motility. Rasool et al. (2019) suggested that tannins might be the cause of anthelmintic activity against adult H. contortus. The active phytochemical can directly affect the worm size and fecundity of females (Martínez-Ortíz-de-Montellano et al., 2010). Several reports have suggested that these biologically active compounds interact with proline-rich surface proteins or internal tissues of helminths. However, due to the unequal distribution of proteins, these effects may vary among different species and developmental stages of nematode species (Spiegler et al., 2017).

However, in another study by Osman (2010), the mortality of worms was greater in the presence of aqueous extracts than in the presence of methanolic extracts. Furthermore, the average duration and mortality of the above-mentioned studies involving the extract type and concentration showed similar effects as those described in our study. In comparison with the findings of other nematocidal reports, the plants in the present study also exhibited significant effects on H. contortus. In P. harmala, different forms of harmine have been shown to have significant antileishmanial effects (Lala et al., 2004). Extracts of the aerial parts of P. harmala were found to remove Theileria hirci parasites from infected lambs (Derakhshanfar and Mirzaei, 2008). Several studies contradicting our findings, such as Astulla et al. (2008), examined four alkaloids (harmine, harmaline, vasicinone, and deoxyvasicinone) in the seed extract of P. harmala and found that harmine and harmaline had moderate inhibitory effects on Plasmodium falciparum. Sun et al. (2021) assessed the dose-dependent toxic effects of harmine on Caenorhabditis elegans. It was found that harmine interferes with acetylcholinesterase activity and induces neurotoxic effects on the worm nervous system. The worms quickly lose their structural growth, egg laying ability and life.

Lipi et al., (2010) investigated the effects of aqueous extracts of the leaves and roots of W. somnifera on adult earthworms (Pheritima posthuma). Leaf extract was more effective at inducing worm paralysis than root extract. Its ethanolic extract also exhibited similar vermicidal activity against Pheritima postanauma (Meeran et al., 2019). The traditional nervine and antistress medicine Aśvagandhādyariṣṭa is a fermented liquid of W. somnifera. The formulation also proved to have anthelmintic potential when tested against Eisenia foetida, a human resembling roundworm (Singh et al., 2014). These significant findings validate its use in treating worm infections.

A comparison of these studies confirmed that both P. harmala and W. somnifera can reduce the number of eggs, larvae and worms of H. contortus. The variation in results of these in vitro studies compared with earlier reports is due to the controlled environmental factors, i.e., temperature. Other factors, such as the type of plant secondary metabolites, polarity of the solvent, nature of extraction, extract exposure time and extent of resistance in worms, are also considered important for evaluating the medicinal significance of plant extracts.

CONCLUSION

Aqueous and methanolic extracts of P. harmala and W. somnifera exhibited significant (p<0.05) anthelmintic activity against three life stages of H. contortus. The W. somnifera plant methanolic extract had the overall highest anthelmintic efficacy. On the basis of the LC50, the extracts of W. somnifera were found to be more potent than the other extracts. Eggs, larvae and adult worms exposed to a set range of temperatures (25-30°C) could constitute a novel approach for safe and reliable dose formulation during in vivo trials. The in vitro results suggested that P. harmala and W. somnifera plant extracts could be used as substitutes for the treatment of extremely prevalent H. contortus nematodes.

Declarations

Acknowledgments

The authors are thankful to the local inhabitants of kala chitta range for searching, medicinal plants material. The authors are also appreciating the support of abattoir’s workers for providing abomasa of slaughtered animals and the technical assistance of the personals of respective department to carry out this research work.

Funding

The manuscript is prepared with no external support/ funding.

IRB approval

The Institutional Ethical Review Committee of International Islamic University, Islamabad, Pakistan granted approval of the study (No. IIUI/ORIC/Bioethics/110, October 22, 2024).

Data availability

All the data used in paper is presented in tables and figures and no extra data is available for e-mail request.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20240416105757

Generative AI or AI-assisted Technology Statement

The authors declare that no Genrative AI was used in the creation of this manuscript.

Statement of conflict of interest

The authors have declared no conflict of interest.

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