Chemical Substances and in-Vivo Antiplasmodial Activity of Ageratum Conyzoides in Plasmodium Berghei Infected Mice

Malaria afflicts millions of people globally, particularly in tropical Africa; it is transmitted to humans through a bite of an Anopheles mosquito. Phytochemical, acute toxicity and in-vivo antiplasmodial activity of the leaves of Ageratum conyzoides were examined to study its effects on Mice that have been infected with the malaria parasite. Phytochemical screening of the methanol extract revealed the presence of secondary metabolites such as terpenoids, flavonoids, alkaloids, steroids and chromene. The LD50 was established at ˃ 1000 mg/kg body weight of mice. The methanol extract of A. conyzoides displayed intrinsic prophylactic and curative anti-malaria activity. At 200 mg/kg and 100 mg/kg body weight of mice, the extract revealed the highest percentage inhibition (83 and 61) for the prophylactic and curative study respectively. The acute toxicity study showed that A. conyzoides extract is relatively safe within the study administered doses. The methanol extract of the prophylactic study against Plasmodium berghei revealed an increase in the level of significance at administered portions of 100, 200 and 400 mg/kg in comparison with 0.2 ml distilled water and 10 mg/kg chloroquine. The methanol extract of the therapeutic study against Plasmodium berghei revealed a slight increase in the level of significance at administered doses of 100 and 200 mg/kg, however, no significant effect was observed for 400 mg/kg compared to the negative control and reference drug. The outcome implies that methanol leave extract of A. conyzoides possesses meaningful antiplasmodial activities and could be a promising source of novel antimalarial. DOI: https://dx.doi.org/10.4314/jasem.v23i10.7 Copyright: Copyright © 2019 Ifijen et al. This is an open access article distributed under the Creative Commons Attribution License (CCL), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dates: Received: 10August 2019; Revised: 17 September 2019; 17 October 2019

Malaria is produced by a parasite called plasmodium, which is transferred by means of bites of contaminated mosquito (Okethwangu et al., 2019). The parasite reproduces in the liver of a human body and there-after taint the red blood cells (Malawi, 2002) . Between 10 -15 days after a mosquito bite, malaria indication such as fever, headache, and vomiting initiate notice in an infected person (Malawi, 2002). Malaria is a mosquito-borne protozoan illness that is a main public health distress throughout the world. The group of plasmodia identified to infect humans are; P. falciparum, P. vivax, P.ovale, P. malariae and P. knowlesi (Ortiz-Ruiz et al., 2018). The valuable antimalaria action of the two plant-based drugs against Plasmodium falciparum; quinine and artemisinin have produced much curiosity to discover other plants properties for their possible anti-malaria efficacy (Mishra et al., 2009). The need to search and build up more effective anti-malaria drugs that are low-cost and readily obtainable to people in developing countries like Nigeria and some other countries in the sub-Sahara regions has necessitated this study. Ageratum is derived from the Greek word 'Ageras', meaning non-aging, referring to the permanence of the whole plant. Conyzoides, on the other hand is derived from 'Konyz' the Greek term of 'inutahelenium' which the plant look like (Novaes et al., 2013;Okunade, 2002). The weed of A. conyzoides has been known for centuries, for its therapeutic properties and has been employed for treatment of different health conditions, such as wounds and burns, for antimicrobial and antimalaria activities, for many bacteria and fungi infections, antitetanus, arthrosis, headaches and dyspnea, pneumonia, analgesic, anti-inflammatory, antiasthmatic, antispasmodic and haemostatic effects, stomach ailments, gynaecological diseases, leprosy and other skin diseases (Santos et al., 2016). A wide range of chemical compounds including flavonoids, chromenes, alkaloids, cumarins, benzofurans, terpenoids and sterols, have been isolated from this species. Extracts and metabolites from this plant have been established to have some insecticidal and pharmacological actions (Santos et al., 2016;Ukwe Chinwe et al., 2010). In eastern Nigeria, aqueous extracts of leaves ageratum conyzoides is utilized by herbalists and individuals to treat malaria. In light of the above, this study was set to establish the pharmacological basis for the apparent anti-malaria activity of leaves of A. conyzoides. In Nigeria, various tribes have diverse names for it. For instance, Igede of the middle belt, Yorubas of the south west, and Igbos of the south east of the country call it "Ufuopioko", "Imiesu", and "Nriewu" respectively (Agbafor et al., 2015;Ukwe Chinwe et al., 2010). The need to search and build up a more effective anti-malaria drug that is low-cost and readily obtainable to people in developing countries like Nigeria and some other countries in the sub-Sahara regions has necessitated this study. The aim of the study was to evaluate the phytochemical screening and antiplasmodial activity of A. conyzoides in mice.

Collection and preparation of ageratum conyzoides leaves:
The plant A. conyzoides was collected fresh from Adolo community, Benin City, Nigeria. Identification of the leaves was carried out at the Department of Plant Biology and Biotechnology, University of Benin and authenticated by Dr Akinnibosun Henry Adewale of the Department of Plant Biology and Biotechnology, University of Benin, Benin City. A voucher specimen was prepared and herbarium specimen number UBHA344 was deposited. The leaves were rinsed with water after which they were air-dried and ground into a powder with the aid of a mechanical machine. It was stored in an airtight container until ready for use.

Extraction of Crude Powdered Sample:
The fresh leaves of A. conyzoides were rinsed, shade dried and pulverized to coarse powder. The powdered plant material (900g) was extracted with 98% methanol using soxhlet extractor to obtain crude methanol extract. The extract was concentrated to dryness using a rotary evaporator at reduced pressure. The concentrated extract was weighed and the percentage yield calculated based on the initial weight of the crude powdered sample. The extract was stored in an air-tight container and kept in the refrigerator at 4 o C until further experiment.  (Lorke, 1983) with Modifications. Groups of naïve ICR mice (5 mice of each) were given orally by gavage with 100, 500 and 1000 mg/kg. The mice were observed for signs of toxicity which include but not limited to paw licking, salivation, stretching of the entire body, weight loss, weakness, respiratory distress and death in the first 4 h and subsequently daily for 7 days.
Anti-malaria drug: Standard anti-malaria drug, chloroquine diphosphate salt was used to study in vivo drug susceptibility of PbANKA. The drug was freshly prepared in distilled water and administered orally by gavage (Franke-Fayard et al., 2008) (Ifijen et al., 2019). Drug dose, expressed in mg/kg of body weight, was adjusted at the time of administration according to the weight of each mouse. The dose of 10 mg/kg was based on the ED90 of this drug on PbANKA infected mice.
Estimation of Percentage Parasitaemia: Percentage parasitemia was estimated at the end of the observational period using the formula % parasitemia = .

RESULTS AND DISCUSSION
The qualitative and quantitative phytochemical screening of the aqueous extract of A. conyzoides revealed the presence of alkaloids, saponin, tannin, phenol, flavonoid, terpenoid, eugenol, steroid and glycoside (Table 1). The plant flavonoids which are phenolic compounds are known to serve as flavouring ingredients of plant leaves. They have been found to possess an antioxidant potential in animals. Earlier reports revealed that plant phenolic compounds including flavonoids are potent antioxidants with reported anti-mutagenic and anti-carcinogenic effects (Middleton, 1998). Alkaloids have been implicated in the anti-malaria activity of many plants (Hamann, 2001;Nafiu et al., 2013) Triterpenoid and steroid saponins have been found to be detrimental to several infectious protozoans such as P. falciparum (Gebrehiwot et al., 2019). The presence of secondary metabolites, alkaloids and flavonoids in A. conyzoides has been implicated in the antiplasmodial effects of some herbal medicines (Phillipson and Wright, 1991). The behavioural pattern of acute toxicity study of the animals showed no abnormal behaviour within 24hours of A. conyzoides extract exposure, and no mortality was observed all through the study, this showed that A. conyzoides extracts have no toxic effect across the administered doses 100, 500 and 1000 mg/kg (Table 3). A. conyzoides extract prophylactic study against P. berghei showed an increase in the level of significance for the graded administered doses (100, 200 and 400 mg/kg) of A. conyzoides in comparison with 0.2 ml Distilled water and 10 mg/kg Chloroquine of the parasitemia count. The result presented in Table 4 shows that the extract exhibited good anti-malaria properties. 100, 200 and 400 mg/kg were observed to have exhibited more sensitivity, specifically at 100 and 400 mg/kg. They were observed to be even more sensitive than the control groups with respect to the reduction of Plasmodium berghei as recorded from the observed percentage inhibition (10 mg/kg Chloroquine, 100 mg/kg, 200 mg/kg and 400 mg/kg of the extracts) at 76 %, 64 %, 83 % and 81 % (  (Table 4). This was slightly different from the observation of Ukwe et al (Ukwe Chinwe V et al., 2010) as the Chloroquine percentage inhibition was higher than that of the Methanol extract (5mg/kg Chloroquine, 100mg/kg, 200mg/kg and 400mg/kg of the extract) at 82.60%, 43.91%, 56.52%, and 71.74% respectively. Curative study of A. conyzoides extract gainst Plasmodium berghei induced malaria showed a slight increase in the level of significance in 100 and 200 mg/kg of Ageratum conyzoides.
However, no significant effect was observed for 400 mg/kg compared to the negative control and reference drug with significant increase in the parasitemia count. The extract exhibited anti-malaria properties and standard drug drastically reduce the level Plasmodium berghei as recorded from the percentage inhibition in the graded doses of the treated plant (52 %, 61%, 47% and 37%) revealed in Table 5 and Figure 2. The surviving days showed average survival for the treated groups in comparison to the 10 mg/kg Chloroquine and negative control (