Introduction: This study assessed the toxicity and antiplasmodial profile of the ethanolic leaf extract of Chromolaena odorata on Plasmodium berghei-infected mice.
Methods: The extract was screen qualitatively and quantitatively for phytochemical constituents. Adult Swiss albino mice (20-23 g) of n = 5/group were inoculated with Plasmodium berghei intraperitoneally and were orally treated with the extract (250, 500 and 1000 mg/kg) and CQ (10 mg/kg) (Standard) daily, respectively. In the sub-acute toxicity study, mice (n = 5/group) were treated with the extract (250, 500 and 1000 mg/kg) daily for 28 days, respectively. After treatments, blood samples were collected and examined for percentage parasitemia, inhibition and biochemical parameters.
Results: The extract contains flavonoids, alkaloids, steroids, tannins, terpernoids, glycosides and saponins. It has high flavonoids (9.04 mg) quantity and low steroids (0.41 mg) quantity. The acute toxicity study of the extract showed no mortality with 3162 mg/kg as the lethal dose 50. The extract exhibited significant (p < 0.05) curative, suppressive and prophylactic antiplasmodial activities in a dose-related fashion when compared to parasitized control. Curatively, the extract (250, 500 and 1000 mg/kg) produced 84.81%, 90.70% and 95.63% parasitemia inhibitions, respectively while CQ produced 94.31% parasitemia inhibition. MST was increased by the extract in a dose-dependent fashion when compared to parasitized control. The altered haematological parameters were restored by the extract in a dose-dependent fashion when compared to parasitized control. The acute toxicity study of the extract (250, 500 and 1000 mg/kg) significantly impaired renal and liver function biomarkers in a dose-related fashion when compare to normal control at p < 0.05, p < 0.01 and p < 0.001, respectively.
Conclusion: Chromolaena odorata leaf extract has promising antiplasmodial activity.
Chromolaena odorata, Leaf, Extract, Toxicity, Antiplasmodial, Mice
Malaria is one of the serious infectious diseases of primary health concern. It is highly poses a significant socioeconomic burden on most African and Asian countries were its incidence is very high [1,2]. It affects nearly half of the world's population and causes deaths yearly, especially, among children in Africa . Malaria is managed through vector control methods and drugs for both treatment and prevention. The use of artemisinin-based combination therapies (ACTs) has contributed to substantial and appreciable decline in malaria-related deaths; but the emergence of drug resistance threatens the use of ACTs . Also, the cost and toxicities caused by some antimalarial drugs are significant challenges in malaria treatment . In developing nations with high malaria burden the aforementioned challenges created decisions for the alternative use of materials of plant origin as remedy for malaria .
There are information on age-long folkloric uses of plants as rich sources of medicines. It is recorded that over 80% of the world population rely medicines from plants. The past decade the global use of herbal medicines has significant increase due to proven efficacy and safety . Most communities in different localities in the world have various folkloric knowledge on medicinal plants, their utilization, management and conservation . Plant products and secondary metabolites are alternative agents for malarial treatment. Medicinal substances of plant origin have been used to treat malaria for thousands of years by humans indifferent parts of the world. Quinine the first antimalarial drug was isolated from Cinchona (Rubiaceae) species, while artemisinin was extracted from Artemisia annua .
Chromolaena odorata (L)(C. odorata)is one of herbs that belongs to Asteraceae, sunflower family . It is an ornamental plant considered to be one of the most invasive environmental weeds of roadsides, wastelands and other exposed areas in the world . It is a flowering shrub that is native to Central and North America, and was later introduced to Africa, Asia, and Australia . C. odorata is known by other names such as baby tea, bitter bush, Armstrong’s weed, butterfly weed, devil weed, king weed, Christmas bush, Siam weed, eupatorium, paraffin weed and turpentine weed . It contains bioactive compounds such as steroids, flavonoids, alkaloids, tannins and saponins, which can act singly or synergistically to exert different biological effects . In sub-Saharan Africa and it is use for the treatment of different ailments and disease conditions such as diabetes, inflammation, wounds and fever . In Benin and Ghana, infusion from fresh leaves of C. odorata is used to treat malaria . It has been shown to have an in-vitro antiplasmodial activity  with limited in-vivo antiplasmodial studies. This study as certain the toxicity and antiplasmodial profile of the ethanolic leaf extract of C. odorata on Plasmodium berghei infected mice.
C. odorata was obtained from Ignatius Ajuru University, Rumuolumeni Obio/Akpor Local Government Area, Rivers State, Nigeria (Latitude of 4.49’4” and longitude of 6.51’24) C. odorata was deposited in Rivers State University Botanical garden where it was identified by a plant taxonomist.
The ethanolic extract of C. odorata was prepared as described by Bligh and Dyer, 1959  with slight modification. The fresh leaves of C. odorata was washed in clean water and blended using porcelain and electric blender. The leaves were weighed before grinding. The blended paste of C. odorata was macerated in ethanol solvent (100%) and allowed to stand overnight after which, the slurry was filtered using Whitman filter paper. The filtrate was the concentrated in a rotary evaporator (45 °C) and the residue was dried under reduced pressure to determine the dry weight of the residue and the percentage extraction yield was calculated. The extract was properly stored in an air tight container in a refrigerator until needed.
Phytochemical analysis of the extract of C. odorata was performed using standard procedures as reported by Trease and Evans (1989)  and Harborne (1998) . The extract was examined for alkaloids, anthraquinones, saponins, flavonoids, cardiac glycosides, tannins, steroids and terpernoids.
The median lethal dose (LD50) of the extract was determined using the method described by Alaribe, et al. . Sixteen mice (25 g-28 g) of n = 4/group were used. The mice were subjected to 24 hours starvation before the oral administration of the extracts (1000, 2000, 3000 and 5000) dissolved in Tween-80 (20%), respectively. The control received only Tween-80 (20%). Physical observations such as degree of restiveness, aggressions and calmness were observed. The mice were also observed for toxicity and fatalities within 72 hours. The LD50 was calculated using the modified formula reported by Enegide, et al.  LD50 = √ ab
a = least tolerable dose;
b = maximum tolerable dose.
Twenty five Swiss albino mice were grouped into 5 of n = 5/group and were treated with normal control (Normal saline 0.2 mL), solvent control [Tween 80 (20%)], extract (250 mg/kg, 500 mg/kg and 1000 mg/kg), respectively for 28 days. Thereafter, blood samples were collected and evaluated for renal and liver biochemical markers.
Ethical approval was granted the research and ethics committee of the Department of Biology, Faculty of Natural and Applied Sciences, Ignatius Ajuru University of Education.
Donor mice parasitized with P. berghei (NK65) were obtained from the Faculty of Basic Clinical Science, University of Port Harcourt, Rivers State, Nigeria. Adult mice of both sexes (25 to 28 g) were used were obtained from the aforementioned school. They were housed in wooden/wire gauze cages and acclimated for 2 weeks with access to water freely before use. The mouse were handled according to United States of America guidelines for the use of laboratory animals (2003). The mice were inoculated intraperitoneally with blood samples (0.2 mL) containing P. berghei (1x107) collected from the donor mice through cardiac puncture.
It was determined using the protocol explained by Ryley and Peters . Thirty albino mice (25-28g) of 6 groups of n = 5/group were parasitized with P. berghei (1x107) and allowed for 3 days. The groups which served as normal control and parasitized control were treated with the solvent [Tween 80 (20%)], while the standard control was treated with CQ (10 mg/kg) for 3 days. Other parasitized groups were treated with the C. odorata leaf extract (250 mg/kg, 500 mg/kg and 1000 mg/kg), respectively for 3 day. During treatment, on day 1, 3, and 5, tails blood samples were collected from the mice on slides and thin blood smears produced. The blood smear were air dried, washed and stained. Percentage of parasitemia was determined by counting the parasitized erythrocytes out of 200 erythrocytes in random fields of the microscope. Percentage parasitemia and inhibitions were calculated using the formula below.
PP = Percentage parasitaemia,
PRBC = parasitized red blood cells, and
RBC = red blood cells
APP: Average percentage parasitemia
APPC: Average percentage parasitemia in control group
APPT: Average percentage parasitemia in test group.
It was evaluated using the method reported by Knight and Peters . Twenty five Swiss albino mice were grouped into 5 of n = 5/group and inoculated with P. berghei (1x107). After 2 hr, the mice were treated with the C. odorata leaf extract 250 mg/kg, 500 mg/kg and 1000 mg/kg, respectively for 3 day. The parasitized and the standard controls were treated with normal saline (0.2 ml) and CQ (10 mg/kg) for 3 days, respectively. On day 4, blood smears were produced on slides, air dried, and washed. The slides were stained and examined for percentage of parasitemia and inhibitions as stated above.
It was performed as reported by Peters . Twenty five Swiss albino mice were grouped into 5 of n = 5/group and were treated with the C. odorata extract (250 mg/kg, 500 mg/kg and 1000 mg/kg), respectively for 3 day. The parasitized control and the standard control were treated with normal saline (0.2 ml) and CQ (10 mg/kg) for 3 days, respectively. On day 4, the mice were inoculated with P. berghei (1x107). After, 24 hours, tail blood smears were produced on slides, air dried, and washed. The slides were stained and examined for Percentage parasitemia and inhibitions as stated above.
In the curative study, from the inception of infection until death, mortality of each mouse was monitored and recorded in days. Mean survival time (MST) of mice in each group was determined as shown below.
Samples of blood collected from the curative mice group were evaluated for hemoglobin (Hb), packed cell volume (PCV), neutrophils (NEU), red blood cells (RBC), lymphocytes (LYM) monocytes (MON), white blood cells (WBC), Aspartate aminotransferase (AST), alkaline phosphatase, alanine aminotransferase (ALT), Creatinine (CR), urea (UR), total bilirubin (TBL) and total protein (TP) using laboratory reagents according to the manufacturer specifications.
Results as mean ± standard error of mean (SEM). Analysis of variance (ANOVA) and Tukey’s tests were used for data analysis with the aid of Graph Pad Prism (Version 5.0, Graph Pad Software Inc., La Jolla, California, U.S.A). P-values < 0.05, < 0.01 < 0.001 were used as significance.
The qualitative screening of C. odorata leaf extract shows that it contains alkaloids, cyanogenic glycosides, flavonoids, saponins, steroids, tannins and terpernoids (Table 1). The quantitative screening of C. odorata leaf extract shows that alkaloids (6.23 mg), flavonoids (9.04 mg), and alkaloids (6.23 mg) were present in high quantities whereas saponins (1.24 mg) and steroids (0.41 mg) were present in low quantities (Table 2).
Table 1: Qualitative phytochemical composition of Chromolaena odorata leaf extract. View Table 1
In acute toxicity study the C. odorata extract (1000, 2000, 3000, 4000 and 5000 mg/kg) did not produce mortality, but at 5000 mg/kg dose notable physical changes occurred, which include lethargy, decreased locomotor activity, diarrhoea and pilo erection (Table 3). In the sub-acute toxicity study, C. odorata extract (250, 500 and 100 mg/kg) administered for 28 days significantly increased serum AST, ALT, ALT, TB, Creatinine, urea and uric acid levels in a dose-related fashion at p < 0.05, p < 0.01, and p < 0.001, respectively when compared to normal control (Table 4 and Table 5). On the other hand, C. odorata extract (250, 500 and 100 mg/kg) administered for 28 days significantly decreased total protein in a dose-related fashion when compared to normal control (Table 4 and Table 5).
Table 2: Quantitative phytochemical composition of Chromolaena odorata leaf extract. View Table 2
Table 3: Acute toxicity evaluation of Chromolaena odorata leaf extract in mice. View Table 3
Table 4: Effect of Chromolaena odorata extract on liver biomarkers of mice. View Table 4
Table 5: Effect of Chromolaena odorata extract on renal biomarkers of mice. View Table 5
In the curative study, C. odorata leaf extract produced dose-related decreases in percentage parasitemia in day 1, 2 and 3 with significant difference at p < 0.05 when compared to parasitized control (Table 6). The decreased percentage parasitemia produced by C. odorata extract at 1000 mg/kg was not different from CQ. C. odorata extract produced 84.81%, 90.70% and 95.63% inhibitions at 250, 500 and 1000 mg/kg, respectively while CQ produced 94.31%parasitamia inhibition (Table 6).
Table 6: Curative effect of Chromolena odorata leaf extract on parasitized mice. View Table 6
In the suppressive and curative antiplasmodial studies, C. odorata extract in a dose-dependent fashion significantly decreased percentage parasitemia at p < 0.05 when compared to parasitized control. No significant difference in percentage parasitemia was observed in C. odorata extract (1000 mg/kg) when compared to CQ (Table 7). In the prophylactic study, the following inhibitions; 88.44% and 94.14% 96.14% were produced by C. odorata extract (250, 500 and 1000 mg/kg), respectively whereas CQ produced 98.85% parasitemia inhibition (Table 7).
Table 7: Suppressive and prophylactic antiplasmodial activities of Chromolena odorata leaf extract on parasitized mice. View Table 7
P.berghei-infected mice showed decreased PCV, HB, RBCs and increased WBCs, MON and LYM when compared to normal control at p < 0.05 (Table 8). C. odorata extract (250, 500 and 100 mg/kg) increased PCV, HB, WBCs and decreased WBCs, MON and LYP when compared to the parasitized control at p < 0.05. C. odorata extract (1000 mg/kg) increased PCV, HB,WBCs and decreased WBCs, MON and LYM when compared to CQ at p < 0.05 (Table 8).
Table 8: Effect of Chromolaena odorata extract on the haematological parameters of parasitized mice. View Table 8
The use of medicinal plants in the treatment of ailments is part of the culture of the indigenous people of African. From time immemorial, humans have relied on plants as sources of medicines to treat diseases and provide for different health needs . For decades, plants have provide medicines for the malaria treatment with the discovery of two major antimalarial drugs- quinine and artemisinin, which are used worldwide. Thus, medicinal plants have potential as new sources of antimalarial drugs . The extraction of bioactive compounds from medicinal plants based on ethno medical data or on traditional use is a promising step toward the development of new antimalarial drugs . This study assessed the toxicity and antiplasmodial profile of the ethanolic leaf extract of C. odorata on Plasmodium berghei-infected mice. In-vivo model was used as compared with an in-vitro model to account for a prodrug effect and the involvement of the immune system in the eradication of infection . The primate model provide a better prediction for the assessment of potential antimalarial agents, but the rodent was used in this study because it is the first step used to screen most in-vivo antimalarial activities of test compounds . The rodent model has been also validated through the discovery of several conventional antimalarial agents. P. berghei was used in this study since it is frequently used for the assessment of potential antimalarial agents in rodent model and has higher accessibility. Due to the sensitivity and significant suppression of P. berghei by CQ, it was used as the standard . The 4-day suppressive test, which evaluates the activity of a test compound on early malaria infection, and Rane’s test, which estimates the curative ability of a test compound on established infection, are the two adopted methods frequently used for the screening of potential antimalarial agents . In the qualitative screening the extract was found to harbour notable phytochemicals of paramount importance, which include alkaloids, saponins, tannins, flavonoids, terpenoids and steroids. Similarly, Igboh, et al.  reported the presence of the aforementioned phytochemicals in C. Odorata leaf extract. Quantitatively the extract contains higher quantities of flavonioids and alkaloids with low quantities of saponins and steroids. No mortality was observed in the acute toxicity evaluation of C. odorata extract which shows it may be safe. But physical observations such as piloerection, lethargy, diarrhoea and reduced locomotor activity were observed at the highest dose of the extract (5000 mg/kg). The sub-acute toxicity assessment of C. odorata for 26 days shows altered levels of renal and liver function biomarkers. This shows that long-term use of the extract may impair renal and liver function. In the 4 day suppressive study, the extract exhibited notable suppressive activity against P. Berghei in a dose- dependent fashion, which was at par with CQ at 1000 mg/kg of the extract. The extract also showed pronounced curative and prophylactic activities in a dose-related fashion, which were similar to CQ at the highest dose of the extract. Similarly, funmilayo, et al.  reported the antiplasmodial activity of the methanolic leaf extract of C. odorata on P. berghei-infected mice. In-vivo antiplasmodial activity can be classified as moderate, good and very good if an extract displayed a respective percent parasite suppression equal to or greater than 50% . C. odorata produced 84.81 % parasitemia suppression at the least dose, which indicates a very good antiplasmodial activity. MST of the mice were prolonged by the extract in the curative study, maximum prolongation of MST was observed at the highest dose of the extract, which was similar to effect produced by CQ. This further add credence to the suppression of P. berghei, by the C. odorata extract resulting in decreased overall pathologic effect of the parasite on the mice. On the haematological parameters, anaemic condition was observed in the parasitized mice marked by low PCV, RBCs and HB levels. The observed anaemic conditions in parasitized mice was curtailed by C. odorata extract in a dose-related fashion. Studies associated malaria-related anaemia to the destruction of infected RBCs and erythropoietic suppression by parasites , thus the C. odorata extract might have curtailed P. berghei-induced anaemia by inhibiting the aforementioned mechanism. The antiplasmodial activity of C. odorata extract can be attributed to its phytochemical constituents. Phytochemicals have been associated with antiplasmodial activity through different possible mechanisms. These mechanisms include endoperoxidation by terpenes, disruption of the ability of the parasite to detoxify heme into nontoxic malaria pigment by alkaloids, blocking protein synthesis by alkaloids and chelation of nucleic acid base pairing by flavonoids. It also include the immunomodulatory effects of steroids and flavonoids, and the free radical scavenging effects of tannins .
The observations in this study showed that C. odorata leaf extract may be a source for the development of new plant-based antimalarial agents which support it folklore use for the treatment of malaria. This study suggest further studies on the fractionation, isolation and characterization of the active principle responsible for the observed antiplasmodial activity.