In vitro Anthelmintic Activity of Aqueous and Ethanolic Extract of Senna italica (Caesalpiniaceae) on Three-stages of Haemonchus contortus

A phytochemical screening and in vitro anthelminthic activity of aqueous and ethanolic extract of Senna italica on Haemonchus contortus were conducted. Polyphenol, tannin and flavonoid contents were determined by using gallic acid and rutin. egg hatching inhibition test was carried out on fresh eggs; larval mortality test was conducted on infective larvae (L3) and adult worm mortality test was conducted. Eggs, larvae and adults worms were incubated in aqueous and ethanolic extract of S. italica at different concentrations (0.1; 0.3; 0.5; 0.7 and 1 mg/mL). All extracts of plants showed an effect on all stages of H. contortus with high efficiency variations depending on the dose used. The inhibition of eggs hatching rate increased from 8.67±1.53% to 65.67±1.15% and from 24.67±1.53% to 80±1.73% respectively for aqueous and ethanolic extract of S. italic. The larval mortality rate increased from 12.22±2.34% to 56.67±4.9% after 24 h and from 45.28±4.11% to 91.25±3.73% after 48 h for aqueous extract and from 30.07±2.84% to 70.83±6.31% after 24 h and Short Research Article Yongwa et al.; JPRI, 32(3): 25-34, 2020; Article no.JPRI.54819 26 from 48.79±3.73% to 96.25±4.79% after 48 h for ethanolic extract. After 24 h of exposure to aqueous and ethanolic extract of S. italica, the adult mortality rate varies from 55.56±9.62% to 83.33±9.62% for aqueous extract and from 61.11±9.62% to 88.89±9.62%.These in vitro results confirm the use of S. italica in traditional medicine.


INTRODUCTION
In Tropical Africa, breeding of small ruminants is hampered by gastro-intestinal nematodes causing the reduction of the production potential [1]. Among the disease that hinders the survival and productivity of sheep and goats, gastrointestinal nematode infection ranks highest on a global scale, with Haemonchus contortus being of overwhelming importance [2]. Haemonchus contortus is the most important nematode parasite of small ruminants, causing severe anaemia and high mortality in all classes of livestock [3]. It is one of the nematode species that dominate the parasitic spectrum of small ruminants in Africa in general and south of the Sahara in particular [4]. The infestation rate of different species of Haemonchus ranged from 50 -85% [5]. The principal diagnostic feature of haemonchosis is anaemia, induced by the blood feeding nature of adults and larval stage. The average blood loss has been calculated as 0.05 mL/parasite/day [6]. Since many years, the control of haemonchosis is generally achieved by the use of synthetic anthelmintic. The frequent use of these anthelmintic over the years has inevitably led to the development of drug resistance. The emergence of resistance to anthelmintic drugs which is now a worldwide phenomenon [7] and the increasing awareness of consumers about drug residues that potentially enter the food chain have stimulated investigation into alternative anthelmintic such as medicinal plants. The treatment of gastrointestinal parasites of small ruminants by medicinal plants is a common practice in rural areas [8]. This calls into question the use of these anthelmintic. There is therefore an urgent need to seek innovative alternative solutions, in order to ensure a more sustainable control of this parasitism. Hence, new therapeutic approaches will be necessary by the use of medicinal plants, since they are accessible at all times and inexpensive [9]. The therapeutic properties of plants are strongly linked to their phytochemical components. Among a number of phytochemical constituents present in plants, saponins, polyphenols, tannins and flavonoids are kwon for their anthelmintic activity [10].
Among the therapeutics' plant, Senna italica family Caesalpiniaceae is known by traditional healers for the virtues of deworming animals. This plant is used in the treatment of fever; jaundice; venereal diseases and biliary crises and against intestinal worms. In South-Africa, S. italica is used for their antibacterial activity and efficacy against sexually transmitted disease [11]. In Cameroon, this plant is used in the treatment of hepatitis; gastroenteritis; jaundice; sexually transmitted diseases as well as stomach aches. The present study is carried out to screen for phytochemical properties and evaluate the in vitro activity of the aqueous and ethanolic extract of S. italica on H. contortus.

Phytochemical Analysis
The overall phenolic content was assessed using Folin-Ciocalteu reagent [13]. The absorbance was measured at 765 nm using a spectrophotometer and the results were expressed as mg of gallic acid equivalents (GAE) per gram of extract (mg GAE/g). The flavonoid content was determined by applying aluminum chloride colorimetric method described by Barros et al. [14] and expressed as mg of rutin equivalents (RE) per gram of extract (mg RE/g). The condensed tannin content was analyzed by using the vanillin assay described by Ba et al. [15]. The results were expressed as mg of catechin equivalents (CE) per gram of extract (mg CE/g). The saponin constituent was quantified according to the modified method described by Jamuna et al. [16]. A 0.01 g of extract was added to 10 ml of distilled water and the mixture was vigorously shaken for 30 mins. The height of moss was measured by using a graduated ruler and quantified according to the following formula: Saponin (mg/g) = [(0.432) *(height of moss in cm after 15 s) + 0.008] / (weight of extract in gram).

Collection of adults of Haemonchus contortus
The abomasa of goats and sheep were bought from markets in Ngaoundere. These abomasa were transported to the laboratory of Zoology of the University of Ngaoundere. Using the protocol described by Kabore [8] for obtaining females adults of H. contortus based on the presence of the vulva. The abomasa were incised in order to collect all worms and transferred in PBS solution.
Females of H. contortus were isolated under binocular microscope at objective ×10.

Anthelmintic bioassay on adults Haemonchus contortus
Solutions of the ethanolic extract of roots of S. italica were prepared with PBS for 04 concentrations (0.04; 0.06; 0.08 and 0.1 mg/ml). Worms which presented good mobility were used for this test. One worm was introduced in well containing 500 µL of solution for 03 columns of 06 wells. The PBS was used as negative control and a positive control consisted of albendazole. The test solution were concomitantly prepared and incubated at 37°C for 24h. Worm mortality was checked by observing under the binocular microscope. After shaking worm, immotile and fully elongated worms were considered to be dead. The mortality rate was determined after 24 h. The mortality rate was calculated using the formula below: Where ND is the number of dead worms in each well and each concentration. NT is the total number of worms in each well and in each concentration.

Parasite donor goat
The abomasa of goats were obtained from the abattoir of the "Marché bantay" of Ngaoundere town after necropsy of small ruminants. Adult female worms were identified using morphometric and morphologic characteristics according to Ahmed et al. [17]. After collecting female H. contortus, they were crushed to liberate eggs [18]. Eggs obtained were cultured in petri dishes at room temperature for seven days [19]. The culture media constituted of 3 ml of sterile liquid of feaces prepared from 3 g of faeces removed from the rectum of parasites free goat, to which was added charcoal. At the end of the 8 th day, infective larvae were harvested. About 4500 larvae were estimated by counting the number of larvae contained in 0.1 mL of well homogenized solution of infective larvae. After five repetitions of counting, the mean number of larvae in 0.1 mL of solution was determined and the volume containing 2500 larvae were deduced, measured and inoculated into a wormfree goat. This goat served as H. contortus egg donor for the in vitro trials.

Recovery of nematode eggs
After the pre-patent period of 21 days, 3 g of faeces were collected directly collected from the rectum of the donor goat. According to the procedure described by Wabo et al. [20], faeces were homogenized in a mortar by adding 60 mL of salt (Na Cl 40% W/V). The solution was cleaned of organic debris by filtration through a 250 µm mesh-size sieve into a beaker and finally poured into four conical tubes until the formation of a meniscus at the top. Three minutes later, slides and cover slides containing the eggs were rinsed with distilled water into 100 mL beaker. The beaker was allowed to stand for 30 minutes for the sedimentation of the eggs at the bottom. To completely remove the salt solution, eggs were washed three times by siphoning out 90 mL of solution and replacing with the same amount of distilled water each 30 minutes. Finally the supernatant was removed and the remaining solution containing eggs was used in the assay.

Egg Hatch Assay (EHA)
The in vitro EHA was based on the method described by Coles et al. [21]. The infective larvae were obtained by stool culture from feces of goats (goat donor) previously infested by H. contortus larvae. The feces were collected directly from the goat's rectum. The eggs contained in the fecal matter were subsequently placed in stool culture at room temperature for 7 days. At the end of this culture time, the larvae were extracted from the faecal mass by the Baermann device, the principle of which is based on the hygrotropism of the larvae.

Evaluation of larvicidal activity of Senna italica
Larval mortality assay using L 3 larvae was performed according to the method described by Wabo pone et al. [20]. Aqueous and ethanolic extract of Senna italic and positive control made by albendazole were dissolved in PBS. Twenty larvae were distributed into each well of a 24-flatbottomed microtitre plate at different concentration (0.1; 0.3; 0.5; 0.7 and 1 mg/mL). PBS was used as negative control. The plates were incubated for 24 and 48 h at 27°C. The experiment was replicated three times for each extract on the same plate. After the incubation's period, the number of dead larvae was counted under the microscope based on their stranght shaped, their immobility and the presence of holes. The percentage of mortality (Mt%) was determined using the following formula: Mt (%) = ( Number of dead larvae Number of larvae in culture ) × 100

Statistical Analysis
The 50% inhibitory concentrations (IC 50 ) for eggs hatching rates were calculated using linear regression equations drawn after transformation of the eggs hatching inhibition rate to probit according to the decimal logarithm of concentrations. While the 50% lethal concentrations (LC 50 ) for L 3 larvae was determined using linear regression equations drawn after transformation of larval mortality rate to probit according to the decimal logarithm of concentrations. Comparison of the mean inhibition percentage of eggs hatching and mean percentage of larval mortality at different concentrations with control was performed by two-way analysis of variance (ANOVA). Statistical analyses were performed using the software SPSS version 17.0 software. The post hoc statistical significance test employed was LSD, differences between the means were considered significant at P < 0.05. The 50% inhibitory concentration (IC 50 ) and lethal concentration (LC 50 ) were determined.

Phytochemical Screening
The quantification of phytochemical metabolites of aqueous and ethanolic of roots of S. italica was carried out to evaluate the chemical families present in the plant extracts and which might be involved in the anthelmintic activity. Polyphenols; flavonoids, tannins and saponins were quantified; the results of these assays are presented in Table 1. It appear from this table that polyphenols and tannins content in aqueous and ethanolic extracts are highest compared to flavonoids and saponins. The Table 1 also revealed that saponins are absent in both extracts of the plant. In a study performed by Barbosa et al. [22] the same compounds have been found in roots, seeds, bark and leaves of S. italica and described as the main active ingredient. Dabai et al. [23] have found in leaves of Senna rugosa, alkaloids; steroids and flavonoids but noted the absence of tannins in all the different extracts of the plant. These differences would be due to certain factors such as the harvest season and geographical location. According to Adoum et al. [24], the geographical location of plants can affect its bioactive constituents induced by factors such as climate and soil. In a study conducted by Rajesh et al. [10], the activity of a plant extract depends on the availability of secondary metabolites like tannins; saponins; polyphenols; triterpens and flavonoids. The aqueous and ethanolic extract of roots of S. italica contains almost the same secondary metabolites namely polyphenols; flavonoids and tannins which might be toxic for the worms and responsible for the observed anthelmintic activity.

Anthelmintic Activity of Senna italica on Adults Haemonchus contortus
The in vitro study is a way to evaluate the anthelmintic activity of the aqueous and ethanolic extracts of S. italica on adults H. contortus (     Albendazole, a synthetic anthelmintic used as a positive control in our in vitro tests is a commonly used drug whose mechanism of action has been demonstrated by many researchers. Alvarez et al. [33] have highlighted two main mechanisms that cause the destruction of nematode parasites. The first is the diffusion of the anthelmintic product through the outer surfaces such as the eggshell and larval cuticles and the second, diffusion through the intestinal cells.

Anthelmintic Activity of Senna italica on Mortality of L 3 of Haemonchus contortus
According to several authors [30,34,35], the anthelmintic activity of plants is attributed to the secondary metabolites present in plants respectively polyphenols; flavonoids and tannins.
To this end these plants metabolites may have work in combination or singly to cause inhibition of egg hatch, larval mortality and adult mortality that was observed in this work.
Indeed, the secondary metabolites present in the different plant extracts used in this study could have attached on eggs, larvae or adults and cause the desired effects. The effectiveness of plant extracts on both egg hatch inhibition and larval paralysis and adult death may be easier to diffuse through egg shells and cuticles of larvae and adults worms. According to Athanasiadou et al. [36], the secondary metabolites of plant extracts could bind to available free proteins to render them unusable as nutrients and cause larval death in eggs or mortality of larvae and adults. Indeed, the important anthelmintic effect obtained with extracts of S. italica in our study would probably be due to the fact that it is more concentrated in active principles.
The findings of this present study showed that aqueous and ethanolic extract of S. italica exhibited evidence of in vitro anthelmintic activity against eggs larvae and females adults of H. contortus.

CONCLUSION
In conclusion, this work focused on the in vitro activity of the aqueous and ethanolic extracts of Senna italica on Haemonchus contortus. It appears from the results of the present study that aqueous and ethanolic extract of S. italica exhibited evidence of in vitro anthelmintic activity on eggs; infective larvae L 3 and against adults of H. contortus. However, further in vivo studies are required to evaluate the bioactivity of the extracts of the above plant and to investigate the potential presence of toxic effects in order to determine the minimum non-lethal doses for the treatment of gastrointestinal helminths.

CONSENT
It is not applicable.

ETHICAL APPROVAL
All authors hereby declare that "principles of laboratory animal care" (NIH publication no. 85-23, revised 1985) were followed, as well as specific national laws where applicable. All experiments have been examined and approved by the Animal Ethical Committee of the Ngaoundere Regional Health Authority, Cameroon.

ACKNOWLEDGEMENT
All materials used in this study were kindly donated to Prof. Dr. D. Ndjonka by the Alexander von Humboldt Foundation (AvH). This donation is greatly appreciated.