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

Atividade anti-helmíntica in vitro e in vivo das sementes de Amomum subulatum roxb e Vitex negundo

Abstract

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 (LC₅₀=14.2 µg mL^-1) than V. negundo (LC₅₀= 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.

Keywords:
Amomum subulatum; Vitex negundo; Haemonchus contortus; In vivo and in vitro assay

Resumo

O presente estudo foi desenhado para verificar as propriedades anti-helmínticas de algumas plantas locais. Sementes de Amomum (A.) subulatum e Vitex (V.) negundo em diferentes solventes foram submetidas à análise de atividade anti-helmíntica in vitro (ensaio de motilidade de adultos; AMA e teste de eclosão de ovos; EHA) e in vivo (teste de redução da contagem de ovos nas fezes; TRCOF), usando o Haemonchus (H.) contortus como modelo experimental no protocolo de teste. Os resultados dos testes AMA, EHA e TRCOF foram analisados ​​estatisticamente por meio de regressão linear e teste de Duncan. No teste AMA, na concentração de 50 mg mL-1, o percentual de mortalidade de H. contortus foi maior com o uso de A. subulatum do que com V. negundo, enquanto, no teste EHA, A. subulatum apresentou maior ação ovicida (LC50=14,2 µg mL- 1) do que V. negundo (LC50= 65,7405 µg mL-1). O TRCOF também indicou a melhor eficácia do uso de A. subulatum do que de V. negundo contra a infecção natural de parasitas gastrointestinais (GI). O extrato bruto seco das plantas utilizadas neste estudo apresentou 29,6% a 57,7% de atividade anti-helmíntica. A taxa de redução observada com o uso de A. subulatum (3 g kg-1) foi maior que com o uso de V. negundo (7 g kg-1). Em relação à análise da eficácia dos solventes utilizados para o extrato de plantas, o acetato de etila e o clorofórmio apresentaram maior ação ovicida em vermes adultos (testes in vitro), enquanto o extrato bruto metanólico aquoso apresentou maior eficácia do que os extratos brutos secos em testes in vivo. Consideramos vantajoso documentar o conhecimento indígena relativos aos procedimentos científicos padronizados, para sua validação. Este estudo irá servir de motivação para que os agricultores façam escolhas melhores referentes ao cultivo das plantas indígenas devido às suas diferentes eficácias comprovadas, servindo como alternativa para a abordagem preventiva contra as infecções parasitárias GI.

Palavras-chave:
Amomum subulatum; Vitex negro; Haemonchus contortus; Ensaio in vivo e in vitro

1. 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).

The fruit of Amomum (A.) subulatum, commonly called ‘Bari Ilaichi’ or ‘Heel Kalan’, is a GI treatment of choice for producing carminative, digestive, antiemetic and stomachic effects (Sherpa et al., 2015). A. subulatum seeds are either used as flavoring spice, or as a diuretic, expectorant or cardiac tonic. The seeds of A. subulatum are also used to treat various ailments and conditions such as skin diseases, dyspepsia, cardiac debility, ulcers, hyperacidity, dysentery, wounds, cough, fever, gonorrhea, liver congestion (Sharma et al., 2002; Parmar et al., 2009). Moreover, the seeds of A. subulatum also used to treat digestive disorders, anestrous, genital prolapse, and anhidrosis in animals (Muhammad et al., 2005; Dilshad et al., 2008). For the therapy of agalactia, anorexia and worm infestation, powdered seeds of A. subulatum mixed with molasses, sodium chloride and ammonium chloride were found effective (Hussain et al., 2008). Vitex (V.) negundo (Vitex spp.) is used for the treatment of snakebite, asthma, depression, allergy, skin diseases, malaria, body pains, wounds and different venereal diseases (Neuwinger, 2000). For the treatment of worm infection, powdered seeds of V. negundo mixed with jiggery showed excellent results (Dilshad et al., 2008).

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., 2017, 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.

2. Materials and Methods

2.1. 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.

2.2. 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.

2.2.2. Fractionation

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

2.3. 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.

2.4. 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.

2.5. 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).

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.

Group	Treatment	Dose rate
1	untreated or control
2	levamisole	7.5 mg per kg b.wt.
3	crude powder (CP) of A. subulatum (S.)	0.5 g per kg b.wt.
4	crude powder (CP) of A. subulatum (S.)	1 g per kg b.wt.
5	crude powder (CP) of A. subulatum (S.)	3 g per kg b.wt.
6	crude powder (CP) of V. negundo (S.)	1 g per kg b.wt.
7	crude powder (CP) of V. negundo (S.)	4 g per kg b.wt.
8	crude powder (CP) of V. negundo (S.)	7 g per kg b.wt.
9	*CAME of A. subulatum (S.)	0.5 g per kg b.wt.
10	CAME of A. subulatum (S.)	1 g per kg b.wt.
11	CAME of A. subulatum (S.)	3 g per kg b.wt.
12	CAME of V. negundo (S.)	1 g per kg b.wt.
13	CAME of V. negundo (S.)	4 g per kg b.wt.
14	CAME of V. negundo (S.)	7 g per kg b.wt.

• ^*
  CAME = Crude aqueous methanol extract.

2.6. 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 (LC₅₀). Duncan multiple range (DMR) test was used to compare eggs per gram (Mean+SEM) of faeces.

3. Results

3.1. 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.

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.

Treatments mg mL-1	Amomum subulatum (Seeds)				Vitex negundo (Seeds)
	Mean±SE of dead worms at different hours				Mean±SE of dead worms at different hours
Hours post-exposure	0 hour	2 hours	6 hours	10 hours	0 hour	2 hours	6 hours	10 hours
Levamisole 0.5	0r	10.00±0.00a	10.00±0.00a	10.00±0.00a	0k	10.00±0.00a	10.00±0.00a	10.00±0.00a
PBS	0r	0r	0r	0r	0k	0k	0k	0k
Crude Aqueous Methanolic Extract
1.56	0m	0m	0.33±0.33lm	1.00±0.00 klm	0o	0o	0k	0.67±0.67mno
3.12	0m	0m	1.00±0.00klm	1.67±0.00ijk	0o	0o	0.33±0.33ijk	1.00±0.58lo
6.25	0m	0m	1.33±0.33jkl	2.33±0.33hij	0o	0o	0.67±0.33hk	3.00±0.58fi
12.5	0m	0m	1.67±0.33ijk	3.33±0.33h	0o	0o	1.67±0.33gj	4.00±0.58ef
25	0m	1.67±0.33ijk	3.00±0.58h	6.00±0.58def	0o	1.00±0.58lo	2.33±0.33eh	4.33±0.33de
50	0m	2.67±0.88hi	5.67±0.33efg	7.33±0.67bc	0o	2.00±0.58il	4.00±0.58cde	7.00±0.58c
Chloroform fractions
1.56	0r	0r	0.67±0.67qr	1.67±0.33nq	0k	0k	0k	0.33±0.33jk
3.12	0r	0r	1.33±0.33or	2.67±0.33ko	0k	0k	0.67±0.33ijk	1.33±0.33gj
6.25	0r	0.67±0.33qr	2.00±0.00mq	3.00±0.00jn	0k	0k	1.00±0.00hk	1.67±0.33fi
12.5	0r	1.33±0.33or	2.67±0.67ko	4.00±0.58gk	0k	0k	1.33±0.33gj	2.33±0.33dg
25	0r	1.67±0.33nq	4.33±0.67fj	5.33±0.67efg	0k	0.33±033jk	2.00±0.58eh	3.00±0.58cde
50	0r	3.33±0.88im	5.67±0.88ef	7.67±0.33bc	0k	1.00±0.58hk	3.00±0.58cde	4.67±0.67b
Ethyl acetate fractions
1.56	0r	0r	1.33±0.33opq	2.67±0.33kn	0m	0m	0m	0.33±0.33lm
3.12	0r	0.67±0.33qr	2.33±0.33lo	3.33±0.67jkl	0m	0m	0.67±0.33klm	1.33±0.33ijk
6.25	0r	1.00±0.00pqr	2.67±0.33kn	4.33±0.88hij	0 m	0m	1.33±0.33ijk	2.00±0.00ghi
12.5	0r	1.67±0.33nq	4.33±0.33hij	6.00±0.58ef	0m	0m	1.67±0.33hij	2.33±0.33gh
25	0r	2.00±0.58mp	5.33±0.33fgh	9.00±0.58ab	0m	0.67±0.33klm	2.00±0.00 ghi	4.00±0.58de
50	0r	4.33±0.33hij	7.00±0.58de	10.00±0.00a	0m	1.67±0.33hij	3.33±0.88 ef	6.00±0.58b
Petroleum spirit fractions
1.56	0n	0n	0n	1.00±0.00klm	0k	0k	0k	0.33±0.33jk
3.12	0n	0n	0n	1.67±0.33ijk	0k	0k	0k	0.67±0.33ijk
6.25	0n	0n	0.67±0.33lmn	2.33±0.33ghi	0k	0k	0k	1.33±0.33ghi
12.5	0n	0n	1.33±0.33jkl	2.67±0.33fgh	0k	0k	0.67±0.33ijk	2.00±0.58efg
25	0n	0n	2.00±0.58hij	3.33±0.33def	0k	0k	1.33±0.33ghi	2.67±0.33de
50	0n	0n	3.00±0.58efg	4.67±0.67bc	0k	0k	2.67±0.67de	4.33±0.33b
Aqueous fractions
1.56	0k	0k	0k	0.33±0.33jk	0l	0l	0l	0.33±0.33kl
3.12	0k	0k	0k	0.67±0.33ijk	0l	0l	0l	1.33±0.33ijk
6.25	0k	0k	0k	1.33±0.33ghi	0l	0l	0.67±0.33jkl	1.67±0.33hij
12.5	0k	0k	0k	2.33±0.33ef	0l	0l	1.00±0.58ijkl	2.67±0.67fgh
25	0k	0k	1.33±0.33ghi	2.67±0.33de	0l	0.33±0.33kl	1.67±0.33hij	3.67±0.88ef
50	0k	0k	2.33±0.33ef	5.00±0.58b	0l	0.67±0.67jkl	3.00±0.58fg	5.67±0.67bc

• Motility 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.

In EHA, the CAME of A. subulatum was considered to be better ovicidal based on its lower LC₅₀ (14.1773 µg 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 dose-dependent manner (Table 3).

Table 3
In vitro effect of seeds of Vitex negundo and Amomum subulatum and LC₅₀ of the different fractions and crude aqueous methanol extracts on hatching (%) of Haemonchus contortus eggs.

Treatments	fraction concentrations mg mL-1					LC50 µg mL-1
	1.2	12	120	1200	12000
Albendazole	79	64	40	5	0	0.0345
Amomum subulatum (Seeds)
CAME	41	39	28	19	6	14.1773
Choloroform fraction	38	37	33	27	13	19.1514
Ethyl acetate fraction	45	37	28	17	4	19.6203
Aqueous fraction	44	42	33	23	17	21.4279
Petroleum spirit fraction	45	37	33	27	13	254.9207
Vitex negundo (Seeds)
CAME	77	66	46	31	12	65.7405
Aqueous fraction	73	61	40	34	14	39.7628
Petroleum spirit fraction	75	58	48	36	18	62.5137
Ethyl acetate fraction	77	66	50	39	17	93.3188
Choloroform fraction	89	67	49	36	26	192.0218

3.2. 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 post-treatment 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).

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.

4. 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).

Phytochemical studies of A. subulatum fruits have unveiled essential oils (Lawrence, 1970), anthocyanins (Lakshmi and Chauhan, 1976), a flavanone, chalcone (Rao et al., 1976) and aurone (Lakshmi and Chauhan, 1977). Lawrence (1970) has reported 1, 8-cineol (more than 70%) dominating 3% essential oil and varying amounts of terpinyl acetate, limonene, terpineol, terpinene, and sabinene from A. subulatum seeds. The essential oil extracted from A. subulatum fruits possess antimycotic activity (Mishra and Dubey, 1990). Phytoconstituents of A. subulatum like volatile oils, glycosides, flavonoids and terpenoids (Defeudis et al., 2003) are being used due to their hepatoprotective and antioxidant activities (Jafri et al., 2001). The gastroprotective effect of A. subulatum is attributed to petroleum ether fractions and essential oil that decreases gastric motility (Bonjar, 2004). Rao et al. (1976) reported gastroprotective activity of A. subulatum’s phenolic fractions i.e., flavanones, anthocyanins or aurones. Bonjar (2004) reported the antibacterial activity of A. subulatum. Kumar (2016) determined the aqueous and ethanolic extracts of A. subulatum Roxb. rhizomes. The concentration-dependent anthelmintic results showed that only 100 mg/mL ethanolic extracts of A. subulatum were effective against parasites. He reported that the anthelmintic activity of the ethanolic extracts was much higher than the aqueous extracts. In a study conducted by Alam et al. (2020), all the doses of ethanolic extract of A. subulatum showed dose dependent anthelmintic activity similar to our study.

The genus Vitex is supposed to have hormone-like properties. Vitex megapotamica is found to be a source of 20-hydroxy ecdysone which is known as an insect molting hormone (Subramanian and Misra, 1979). The antiarthritic effect was reported from the leaves of plants of genus Vitex (Chaturvedi and Singh, 1965). Subramanian and Misra (1979) reported two new flavonoid glycosides from the stem bark of V. negundo. Hypoglycemic, anti-inflammatory, hypouricaemic and analgesic effects have been reported from V. negundo extracts (Umamaheswari et al., 2007). Flavonoid-rich fractions of its seeds have anti-oxidant and antiandrogenic properties (Das et al., 2004). Vitex negundo has also been used as hepato-preventive, anti-histaminic and CNS depressant (Gupta et al., 1999). Zhong et al. (1996) reported significant analgesic activity of aqueous extract from the seeds of V. negundo.

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.

5. 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 short-term prophylactic approach to cultivate in the grazing area which may reduce the severity of GI parasitism in the livestock population.

Acknowledgements

Authors are very thankful to Prof. Dr. Neil A. Karrow, Department of Animal Biosciences, University of Guelph, Guelph, Ontario, Canada for English editing of the manuscript.

References

• ABBOTT, W.S., 1925. A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, vol. 18, no. 2, pp. 265-267. http://dx.doi.org/10.1093/jee/18.2.265a
  » http://dx.doi.org/10.1093/jee/18.2.265a
• AHMAD, S., SAJID, M.S., KHAN, M.K., NADEEM, R., RIZWAN, H.M., MALIK, M.A. and YOSUF, F., 2020. Effect of trace element supplementation on the gastrointestinal parasites of grazing sheep of Multan district, Pakistan. Journal of Animal and Plant Science, vol. 30, no. 1, pp. 72-80.
• ALAM, K., PATHAK, D., SETHI, V.A. and HODA, Q., 2020. Evaluation of anthelmintic activity of Amomum subulatum fruits extract. World Journal of Advanced Research and Reviews, vol. 7, no. 2, pp. 141-145. http://dx.doi.org/10.30574/wjarr.2020.7.2.0287
  » http://dx.doi.org/10.30574/wjarr.2020.7.2.0287
• BADAR, N., IQBAL, Z., SAJID, M.S., RIZWAN, H.M., JABBAR, A., BABAR, W., KHAN, M.N. and AHMED, A., 2017. Documentation of ethnoveterinary practices in district Jhang, Pakistan. Journal of Animal and Plant Sciences, vol. 27, no. 2, pp. 398-406.
• BADAR, S.N., IQBAL, Z., SAJID, M.S., RIZWAN, H.M., SHAREEF, M., MALIK, M.A. and KHAN, M.N., 2021. Comparative anthelmintic efficacy of Arundo donax, Areca catechu, and Ferula assa-foetida against Haemonchus contortus. Brazillian Journal of Veterinary Parasitology, vol. 30, no. 2, p. e001221. http://dx.doi.org/10.1590/s1984-29612021028 PMid:34076046.
  » http://dx.doi.org/10.1590/s1984-29612021028
• BONJAR, G.H.S., 2004. Antibacterial screening of plants used in Iranian folkloric medicine. Fitoterapia, vol. 75, no. 2, pp. 231-235. http://dx.doi.org/10.1016/j.fitote.2003.12.013 PMid:15030933.
  » http://dx.doi.org/10.1016/j.fitote.2003.12.013
• CHATURVEDI, G.N. and SINGH, R.H., 1965. Experimental studies on the anti-arthritic effect of certain indigenous drugs. The Indian Journal of Medical Research, vol. 53, pp. 71-80. PMid:14266449.
• CHIRAG, M.M., HITESH, B.P. and SHAILESH, K.M., 2013. Animal husbandry practice to contaminants and residues of chemical in animal origin foods and health hazard. International Journal of Molecular Veterinary Research, vol. 3, no. 1, pp. 55-61. http://dx.doi.org/10.5376/ijmvr.2013.03.0010
  » http://dx.doi.org/10.5376/ijmvr.2013.03.0010
• DAS, S., PARVEEN, S., KUNDRA, C.P. and PEREIRA, B.M.J., 2004. Reproduction in male rats is vulnerable to treatment with the flavonoid-rich seed extracts of Vitex negundo. Phytotherapy Research, vol. 18, no. 1, pp. 8-13. http://dx.doi.org/10.1002/ptr.1352 PMid:14750193.
  » http://dx.doi.org/10.1002/ptr.1352
• DEFEUDIS, F.V., PAPADOPOULOS, V. and DRIEU, K., 2003. Ginkgo biloba extracts and cancer: a research area in its infancy. Fundamental & Clinical Pharmacology, vol. 17, no. 4, pp. 405-417. http://dx.doi.org/10.1046/j.1472-8206.2003.00156.x PMid:12914542.
  » http://dx.doi.org/10.1046/j.1472-8206.2003.00156.x
• DILSHAD, S.M.R., NAJEEB-UR-REHMAN, IQBAL, Z., MUHAMMAD, G., IQBAL, A. and AHMAD, N., 2008. An inventory of the ethnoveterinary practices for reproductive disorders in cattle and buffaloes, Sargodha district of Pakistan. Journal of Ethnopharmacology, vol. 117, no. 3, pp. 393-402. http://dx.doi.org/10.1016/j.jep.2008.02.011 PMid:18384987.
  » http://dx.doi.org/10.1016/j.jep.2008.02.011
• GORAYA, K., IQBAL, Z., SAJID, M.S., MUHAMMAD, G., AIN, Q. and SALEEM, M., 2013. Diversity of flora used for the cure of equine diseases in selected peri-urban areas of Punjab, Pakistan. Journal of Ethnobiology and Ethnomedicine, vol. 9, no. 1, p. 70. http://dx.doi.org/10.1186/1746-4269-9-70 PMid:24283263.
  » http://dx.doi.org/10.1186/1746-4269-9-70
• GUPTA, M., MAZUMDER, U.K. and BHAWAL, S.R., 1999. CNS activity of Vitex negundo Linn. in mice. Indian Journal of Experimental Biology, vol. 37, no. 2, pp. 143-146. PMid:10641133.
• HAMAD, K.K., IQBAL, Z., SINDHU, Z.D., ABBAS, R.Z., KHAN, A., MUHAMMAD, G. and EPPERSON, B., 2014. Combination of Nicotiana tabacum and Azadirachta indica: a novel substitute to contain prevalence of levamisole and ivermectin-resistant Haemonchus contortus in ovine. Pakistan Veterinary Journal, vol. 34, no. 1, pp. 24-29.
• HUSSAIN, A., KHAN, M.N., IQBAL, Z. and SAJID, M.S., 2008. An account of the botanical anthelmintics used in traditional veterinary practices in Sahiwal district of Punjab, Pakistan. Journal of Ethnopharmacology, vol. 119, no. 1, pp. 185-190. http://dx.doi.org/10.1016/j.jep.2008.06.034 PMid:18657602.
  » http://dx.doi.org/10.1016/j.jep.2008.06.034
• IQBAL, Z., LATEEF, M., JABBAR, A., AKHTAR, M.S. and KHAN, M.N., 2006. Anthelmintic activity of Vernonia anthelmintica seeds against trichostrongylid nematodes of sheep. Pharmaceutical Biology, vol. 44, no. 8, pp. 563-567. http://dx.doi.org/10.1080/13880200600896512
  » http://dx.doi.org/10.1080/13880200600896512
• IQBAL, Z., LATEEF, M., JABBAR, A., MUHAMMAD, G. and KHAN, M.N., 2005. Anthelmintic activity of Calotropis procera (Ait.) Ait. F. flowers in sheep. Journal of Ethnopharmacology, vol. 102, no. 2, pp. 256-261. http://dx.doi.org/10.1016/j.jep.2005.06.022 PMid:16085379.
  » http://dx.doi.org/10.1016/j.jep.2005.06.022
• JABBAR, A., ZAMAN, M.A., IQBAL, Z., YASEEN, M. and SHAMIM, A., 2007. Anthelmintic activity of Chenopodium album (L.) and Caesalpinia crista (L.) against trichostrongylid nematodes of sheep. Journal of Ethnopharmacology, vol. 114, no. 1, pp. 86-91. http://dx.doi.org/10.1016/j.jep.2007.07.027 PMid:17826017.
  » http://dx.doi.org/10.1016/j.jep.2007.07.027
• JAFRI, M.A., FARAH, JAVED, K. and SINGH, S., 2001. Evaluation of the gastric antiulcerogenic effect of large cardamom (fruits of Amomum subulatum Roxb). Journal of Ethnopharmacology, vol. 75, no. 2-3, pp. 89-94. http://dx.doi.org/10.1016/S0378-8741(00)00398-6 PMid:11297839.
  » http://dx.doi.org/10.1016/S0378-8741(00)00398-6
• KAHIYA, C., MUKARATIRWA, S. and THAMSBORG, S.M., 2003. Effects of Acacia nilotica and Acacia karoo diets on Haemonchus contortus infection in goats. Veterinary Parasitology, vol. 115, no. 3, pp. 265-274. http://dx.doi.org/10.1016/S0304-4017(03)00213-9 PMid:12935742.
  » http://dx.doi.org/10.1016/S0304-4017(03)00213-9
• KUMAR, T., 2016. Development and evaluation of herbal anthelmintic formulation Hisar: Guru Jambheshwar University of Science and Technology, 121 p. PhD thesis.
• LAKSHMI, V. and CHAUHAN, J.S., 1976. Chemical examination of seeds of Amomum subulatum. Journal of the Indian Chemical Society, vol. 53, p. 633.
• LAKSHMI, V. and CHAUHAN, J.S., 1977. Structure of a new Aurone glycoside from Amomum subulatum seeds. Indian Journal of Chemistry-Section B, vol. 15, pp. 814-815.
• LAWRENCE, B.M., 1970. Terpenes in two amomum species. Phytochemistry, vol. 9, no. 3, p. 665. http://dx.doi.org/10.1016/S0031-9422(00)85711-8
  » http://dx.doi.org/10.1016/S0031-9422(00)85711-8
• MIN, B.R. and HART, S.P., 2003. Tannins for suppression of internal parasites. Journal of Animal Science, vol. 81, no. 14, pp. 102-109.
• MISHRA, A.K. and DUBEY, N.K., 1990. Fungi toxicity of essential oil of Amomum subulatum against Aspergillus flavus. Economic Botany, vol. 44, no. 4, pp. 530-533.
• MUHAMMAD, G., KHAN, M.Z., HUSSAIN, H.M., IQBAL, Z., IQBAL, M. and ATHAR, M., 2005. Ethnoveterinary practices of owners of pneumatic-cart pulling camels in Faisalabad (Pakistan). Journal of Ethnopharmacology, vol. 97, no. 2, pp. 241-246. http://dx.doi.org/10.1016/j.jep.2004.11.008 PMid:15707760.
  » http://dx.doi.org/10.1016/j.jep.2004.11.008
• NEUWINGER, H.D., 2000. African traditional medicine: a dictionary of plant use and applications. Germany: Medpharm Scientific Stuttgart.
• O’GRADY, J. and KOTZE, A.C., 2004. Haemonchus contortus: in vitro drug screening assays with the adult life stage. Experimental Parasitology, vol. 106, no. 3-4, pp. 164-172. http://dx.doi.org/10.1016/j.exppara.2004.03.007 PMid:15172224.
  » http://dx.doi.org/10.1016/j.exppara.2004.03.007
• PARMAR, M.Y., SHAH, P., THAKKAR, V. and GANDHI, T.R., 2009. Hepatoprotective activity of Amomum subulatum Roxb against ethanol-induced liver damage. International Journal of Green Pharmacy, vol. 3, no. 3, pp. 250-254.
• RAO, C.B., RAO, T.N. and SURYAPRAKASAM, S., 1976. Cardamonin and alpinetin from the seeds of Amomum subulatum. Planta Medica, vol. 29, no. 4, pp. 391-392. http://dx.doi.org/10.1055/s-0028-1097682 PMid:959382.
  » http://dx.doi.org/10.1055/s-0028-1097682
• RAUL, S.K., PADHY, G., DHAVALA, M. and SHRAVANI, B., 2012. Comparitive study of anthelmintic activity of Vitex negundo, Moringa oleifera, Tamarindus indica on Indian earthworm Phertima posthuma Drug Invention Today, vol. 4, no. 7, pp. 407-408.
• RAZA, M.A., YOUNAS, M. and SCHLECHT, E., 2014. Prevalence of gastrointestinal helminths in pastoral sheep and goat flocks in the cholistan desert of Pakistan. Journal of Animal and Plant Sciences, vol. 24, no. 1, pp. 127-134.
• RIZWAN, H.M., SAJID, M.S., IQBAL, Z. and SAQIB, M., 2017. Point prevalence of gastrointestinal parasites of domestic sheep (Ovis aries) in district Sialkot, Punjab, Pakistan. Journal of Animal and Plant Sciences, vol. 27, no. 3, pp. 803-808.
• RIZWAN, H.M., SAJID, M.S., IQBAL, Z. and SAQIB, M., 2019. Association of phytomineral with gastrointestinal parasites of grazing sheep in Sialkot district, Punjab, Pakistan. Pakistan Journal of Agricultural Sciences, vol. 56, no. 2, pp. 459-468.
• SAHARE, K.N. and SINGH, V., 2013. Antifilarial activity of ethyl acetate extract of Vitex negundo leaves in vitro. Asian Pacific Journal of Tropical Medicine, vol. 6, no. 9, pp. 689-692. http://dx.doi.org/10.1016/S1995-7645(13)60119-4 PMid:23827144.
  » http://dx.doi.org/10.1016/S1995-7645(13)60119-4
• SHARMA, G., SHARMA, E., SHARMA, R. and SINGH, K.K., 2002. Performance of an age series of Alnus-cardamom Plantations in the Sikkim Himalaya: productivity, energetics and efficiencies. Annals of Botany, vol. 89, no. 3, pp. 261-272. http://dx.doi.org/10.1093/aob/mcf035 PMid:12096738.
  » http://dx.doi.org/10.1093/aob/mcf035
• SHERPA, M.T., MATHUR, A. and DAS, S., 2015. Medicinal plants and traditional medicine system of sikkim: a review. World Journal of Pharmacy and Pharmaceutical Sciences, vol. 4, no. 2, pp. 161-184.
• SINGH, S., ANSARI, N.A., SRIVASTAVA, M.C., SHARMA, M.K. and SINGH, S.N., 1985. Anthelmintic activity of Vernonia anthelmintica. Indian Drugs, vol. 22, no. 10, pp. 508-511.
• SUBRAMANIAN, P.M. and MISRA, G.S., 1979. Flavonoids of Vitex negundo. Journal of Natural Products, vol. 42, no. 5, pp. 540-542. http://dx.doi.org/10.1021/np50005a019
  » http://dx.doi.org/10.1021/np50005a019
• TABASSAM, S.M., IQBAL, Z., JABBAR, A., SINDHU, Z.D. and CHATTHA, A.I., 2008. Efficacy of crude neem seed kernel extracts against natural infestation of Sarcoptes scabiei var. ovis. Journal of Ethnopharmacology, vol. 115, no. 2, pp. 284-287. http://dx.doi.org/10.1016/j.jep.2007.10.003 PMid:18023309.
  » http://dx.doi.org/10.1016/j.jep.2007.10.003
• TABUTI, J.R.S., DHILLION, S.S. and LYE, K.A., 2003. Ethnoveterinary medicines for cattle (Bos indicus) in Bulamogi county, Uganda: plant species and mode of use. Journal of Ethnopharmacology, vol. 88, no. 2-3, pp. 279-286. http://dx.doi.org/10.1016/S0378-8741(03)00265-4 PMid:12963156.
  » http://dx.doi.org/10.1016/S0378-8741(03)00265-4
• UMAMAHESWARI, M., ASOKKUMAR, K., SOMASUNDARAM, A., SIVASHANMUGAM, T., SUBHADRADEVI, V. and RAVI, T.K., 2007. Xanthineoxidase inhibitory activity of some Indian medical plants. Journal of Ethnopharmacology, vol. 109, no. 3, pp. 547-551. http://dx.doi.org/10.1016/j.jep.2006.08.020 PMid:17014977.
  » http://dx.doi.org/10.1016/j.jep.2006.08.020
• WILLIAMSON, E.M., OKPAKO, D.T. and EVANS, F.J., 1998. Pharmacological methods in phytotherapy research: selection, preparation and pharmacological evaluation of plant material Chichester: John Wiley & Sons.
• ZHONG, S.T., QIU, G.Y. and LIU, Y.B., 1996. Comparative studies on pharmacological activity of the fruits of Vitex trifolia L. var. simplicifolia Cham, Vitex trifolia L., Vitex negundo L. and Vitex negundo L. var. cannabifolia. Pharmacology of Clinical Chinese Materials and Medicine, vol. 1, no. 12, pp. 37-39.

Publication Dates

• Publication in this collection
  03 June 2022
• Date of issue
  2024

History

• Received
  06 Mar 2022
• Accepted
  12 May 2022