IN-VIVO ANTI-PLASMODIAL ACTIVITY OF HAGENIA ABYSSINICA [FAMILY: ROSACEAE] IN PLASMODIUM BERGHEI INFECTED IN MICEHTML Full Text
IN-VIVO ANTI-PLASMODIAL ACTIVITY OF HAGENIA ABYSSINICA [FAMILY: ROSACEAE] IN PLASMODIUM BERGHEI INFECTED IN MICE
Tafere Mulaw Belete * 1 and Tefera Abula Orijino 2
Department of Pharmacology 1, College of Medicine and Health Sciences, University of Gondar, P.O. Box 197, Gondar, Ethiopia.
School of Medicine 2, College of Health Sciences, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia.
ABSTRACT: Malaria is a significant public health problem in the world, and the rising problem of drug resistance and the availability of a limited number of effective anti-malarial drugs underline how essential it is to find new anti-malarial agents. This study was undertaken to evaluate the in-vivo antimalarial activities of an extract of Hagenia abyssinica that is used traditionally to treat malaria in Ethiopia. The rodent malaria Plasmodium berghei was used to infect healthy 7-week-old Swiss albino mice weighing 24-28 gm. Each of the extracts (100 mg/kg, 200 mg/kg, and 400 mg/kg) was administered to different groups of mice. The parameters of parasitemia, survival time, body weight, and packed cell volume were measured using repository test and Peter's test. The extract significantly inhibited parasitemia and increased survival time in infected mice in a dose-dependent manner. The extract prevented loss of weight, packed cell volume. This study concluded that the extract has a promising anti-malarial activity which supports the in-vitro anti-plasmodial activity and the traditional use of the plant for treatment of malarial infection.
Antiplasmodial activity, Hagenia abyssinica, Plasmodium berghei, Medicinal plant
INTRODUCTION: Malaria is the most prevalent and lethal protozoan disease in the world infecting 216 million and killing 445,000 persons in 2017, mostly pregnant women and children under five. Most of these cases (88%) and deaths (90%) occurred in Sub-Saharan African countries. Most deaths and drug-resistant infections occur due to infection with Plasmodium falciparum, which is the most virulent Plasmodium species 1.
Even though malaria disease is preventable and curable, it is still one of the global public health issues especially Africa. Greater than 75% of the country is malarious with approximately 68% of the population living in areas at risk of malaria, making malaria the major public health issues in Ethiopia 1, 2.
Resistance to anti-malarial drugs has been developed for all anti-malarials agents, and it is a major threat to the global effort for the control of malaria. The inappropriate, ineffective, and inefficient use of anti-malarials drugs increases drugs resistance. There is broad consensus on the urgent need for new drugs developed to treat malaria, with broad therapeutic potential, good pharmacokinetic character, acceptable safety and novel mode of action, to widen the range of treatment and to prevent the development of anti-malarial drug resistance 2, 3. One approach to this is the investigation of medicinal plants and natural products. The identification of bioactive compounds from medicinal plants is a highly promising potential approach for isolating new, safe and effective lead compounds 3. In History, the most of the anti-malaria drugs have been isolated from plants or structures modification on plant lead compounds. More than 50% of all drugs in clinical use today contain certain substances of natural origin; most of these products are from plant sources 4. Previous findings of anti-malarial agents such as quinine isolated from cinchona bark extract and artemisinin found from Artemisia annua encouraged the possibility of finding new anti-malarial drugs from plant sources 4, 5.
Ethiopia, which has different kinds of medicinal plants has accumulated over generations a great number of well-known remedies Hagenia abyssinica (Family: Rosaceae), is one of them. H. abyssinica (Bruceae) J. F. Gmel is a deciduous tree with distinct male and female trees, both of which are endowed with colorful flowers. Ethnobotanical studies revealed that H. abyssinica has a wide range of therapeutic use since ancient times against malaria, fever, intestinal worms (tapeworm), stomachache, diarrhea, healing of the wound, typhoid and livestock disease 6. Several bioactive metabolites characterize the chemical composition of the Hagenia abyssinica. As active principles in this species, the phloroglucinol derivatives Including kosotoxin, protokosin, koisdine and kosin isolated. It also has volatile oil, acrid resin, tannic acid, quercetin 3-O-β-glucoside, rutin and quercetin glucuronide 7, 8. The phenolic compound, Kosin which is isolated from the flowers have comparable taenicides potency as the marketed drugs, niclosamide.
Hagenia abyssinica has a spasmolytic activity which relieves nonspecific abdominal complaints which could arise from the high secretion of acetylcholine and histamine in the gastrointestinal tract. Hagenia abyssinica has cytotoxic activity against colonic adenocarcinomas with varying growth characteristics and morphology. The hexane extracts of leaves of H. abyssinica were the most active extracts with the highest zones of inhibition against S. aureus, MRSa and P. aeruginosa 8, 9. From 1817 to1954, Hagenia abyssinica was listed in the pharmacopeias of 29 countries. To our knowledge, the stem bark extract of H. abyssinica also found to have significant in-vitro anti-malaria activity against Plasmodium falciparum 10. In the endemic area including Ethiopia where malaria prevails, traditional herbal medicines are often used for antipyretic and anti-malarial therapy. But, there is little scientific information is available to the safety and efficacy of these herbal remedies. Thus, based on ethnobotanical and in-vitro studies mentioned above the present study evaluated the in-vivo anti-malarial activities of H. abyssinica stem bark extracts of against Plasmodium berghei in mice 11, 12.
MATERIALS AND METHODS:
Collection and Preparation of the Plant Materials: The plant material (stem bark) was collected during dry season from Wegera district 780 km North-west of Addis Ababa, Central Ethiopia and identified by in the National Herbarium, Department of Biology, Addis Ababa University, where a specimen with voucher number (HA 01) was deposited. The stem was cleaned peeled and air dried under open shade at room temperature. Then the dried materials were ground to a coarse powder using mortar and pestle.
Extraction of Plant Materials: The crude extracts were prepared by cold maceration technique. The coarse powder (50 g) was macerated in one liter 80% methanol for 72 h on an orbital shaker (VWR, USA) at 120 rotations per min to facilitate the extraction process. The filtrate was separated from the mark using Whatman filter paper 18 cm diameter and 0.1 μm pore diameter, and the mark was re-macerated two times. The filtrates were dried in an oven at the temperature not exceeding 40 ℃ then further drying process was carried out in desiccators for removal of water. Finally, the dried extract was stored with the vial in a refrigerator at 4 °C until the beginning of the actual experiment 13, 14.
Experimental Animals: Swiss albino mice are weighing 24-30 gm and aged 7 weeks of both sexes were inbred at the Animal House of from University of Gondar animal house, transported within a cage and acclimatized in the laboratory for one week before the study conducted. The mice were kept at room temperature and exposed to a 12h light/ dark cycle, allowed free access to food (pellets) and water in plastic cages with a metal cover for free passage of air. This study was undertaken in line with the internationally accepted laboratory animal use and care guideline 15.
Rodent Parasites (Plasmodium berghei): Chloroquine (CQ) sensitive P. berghei, ANKA strain was used in this study. The parasites were obtained from Aklilu Lemma Institute of Pathobiology, by inoculation of uninfected mice with blood from P. berghei infected mice. The parasites were maintained by successive inoculations of mice every five days with 0.2 ml of the infected blood.
Drugs and Reagents: Chloroquine (Addis Pharmaceuticals Factory, Ethiopia), trisodium citrate (Deluxe Scientific Surgico, India), ketamine (Deluxe Scientific Surgico, India), methanol (Okhla Industrial, India), Giemsa (Science lab, USA) and normal saline (Addis Pharmaceuticals Factory, Ethiopia) were used. All reagents were analytically graded and procured from legal pharmaceutical suppliers.
Acute Toxicity Testing: A preliminary toxicity study was performed to determine the safe dose range that could be used for the studies rather than to give full toxicity data on the plant. Acute toxicity testing was done using OECD-425 guidelines. A single limit dose of 2000 mg/kg body weight of the extract was administered to the first mouse with the oral route by gavage, and then other four mice (24-28 gm each) were sequentially treated based on the result of the first mouse. Before administration of the extract, the mice were inhibited from free access to food for 3 h. Similarly, food was withheld for 1 h after the extract administration. Following administration of the extract, the mice were observed for toxicities like diarrhea, hair erection, tremors, convulsions, salivation, weight loss, lethargy, and paralysis periodically for the first four hours during the 24 h period and later followed for 14 days to assess any death 16.
Phytochemical Screening of the Crude Extract: Phytochemical screening consists of performing simple chemical tests used to analyze the presence of particular phytochemical using standard procedures according to 17, 18.
Detection of Alkaloids (Mayer’s Test): 1.36 gm of mercuric chloride and 5 gm of potassium iodide were dissolved in 60 ml and 10 ml of distilled water respectively. These two solvents were mixed and diluted to 100 ml using distilled water. To 1 ml of acidic aqueous solution of samples, a few drops of reagent were added. Formation of white or pale precipitate indicates the presence of alkaloids.
Detection of Saponins (Foam Test): A drop of sodium bicarbonate was added in a test tube containing 50 ml of an aqueous extract of the plant. The mixture was shaken and kept for 3 min. A honeycomb like froth formation shows the presence of saponins.
Detection of Tannins (Lead Acetate Test): 5 gm of the extract was stirred in 10 ml of distilled water, filtered and a few drops of 1% solution of lead acetate were added to the filtrate. Yellow or red precipitate formation indicates the presence of tannins.
Detection of Cardiac Glycosides (Keller Killian's Test): 0.5 gm crude extract was stirred in 1 ml of glacial acetic acid that contain one drop of ferric chloride solution. This was then under layer with 1 ml of concentrated sulphuric acid. Brown ring formation at the interface shows the presence of a de-oxy sugar characteristic of cardenolides.
Detection of Resins (Precipitation Test): 30 gm extracts were further extracted with 5 ml 96% ethanol. The resultant alcoholic extract was added into 20 ml of distilled water in a beaker. Precipitate shows the presence of resins.
Detection of Phenols (Ferric Chloride Test): To 1 ml of an alcoholic solution of the crude extract, 2 ml of distilled water followed by a few drops of 10% aqueous ferric chloride solution were added. Formation of blue or green color shows the presence of phenols.
Detection of Flavonoids (Shinoda’s Test): 2 mg of the extract was dissolved in 5 ml of ethanol, and to this 10 drops of diluted HCl and the small amount of magnesium were added and the solution was boiled for few minutes. The appearance of reddish pink or brown colour showed the presence of flavonoids.
Detection of Anthraquinone (Ninhydrin Test): 1 gm extract was boiled for two minutes with 5 ml of 0.5N KOH and 0.5 ml of 5% H2O2 mixture. After cooling, the suspension is filtered through the glass wool. The filtrate was treated with six drops of acetic acid, and the resulting solution was mixed with 5 ml of toluene. The upper layer is separated with a pipette and transferred to a test tube and 2 ml of 0.5N KOH was added. Infra-red color appearance in the aqueous layer indicates the presence of anthraquinone.
Detection of Terpenoid (Copper Acetate Test): 1 ml of conc. H2SO4 and 2 ml of chloroform were added to 1 mg of extract, and the appearance of reddish brown color indicated the presence of terpenoid.
In-vivo Anti-plasmodial Activity Testing:
Infecting of Mice: Donor mouse blood which is infected with the P. berghei used for inoculums preparation. After anesthetizing with ketamine (120 mg/kg) by intraperitoneal route, blood was collected from a donor mouse having parasitemia of 30 to 37% by heart puncture into a test tube having anticoagulant (0.5% trisodium citrate). Then, the infected blood was diluted with normal saline (0.9% NaCl) so that the final suspension contained about 1 × 107 infected erythrocytes in every 0.2 ml of suspension. This 0.2 ml suspension was inoculated intraperitoneally to initiate infection.
Grouping and Dosing of Mice: Twenty-five infected mice of both sexes were grouped randomly into five groups of five mice each. Group, I mice were treated with distilled water (used to dissolve the crude extract, 10 ml/kg, served as negative control), group II, III and IV mice were treated with 100, 200, and 400 mg/kg of crude extract, respectively and group (V) mice were treated with the standard drug (chloroquine,10 mg/kg, served as positive control). The safety data of the plant was obtained from acute toxicity testing as the crude extract was safe at 2 g/kg body weight in mice and this was a base for selecting the doses (100, 200 and 400 mg/kg) of the extract for the current anti-malarial effect study.
Test on Early Malaria Infection (4-Day Suppressive Test): Parasite suppressive effect of the crude extract was assessed using the method mentioned by Peters 19. Twenty-five mice were first infected intraperitoneally with the inoculum (1 × 107 P. berghei erythrocyte, 0.2 ml) on the first day (D0). After 3 h post-infection, the test groups were treated with 100, 200 and 400 mg/kg/day doses of the extract. The reference drug group was treated with chloroquine (10 mg/kg) and the control group received distilled water 0.2 ml/kg.
All the treatments were repeated for the next three days (D1 to D3). On the fifth day (D4) thin blood films were fixed with methanol, stained with 10% Giemsa at pH 7.2 for 10 min which is taken from the tail of each mouse. Average percent parasitemia and suppression was determined by using the following formula 20.
Percentage of parasitaemia = (Number of parasitized RBC) / (Total number of RBC count) × 100%
Average % suppression= (A-B) / A × 100%
Where, A = Mean parasitaemia of negative control group, B = Mean parsitaemia of extract treated group.
Prophylactic Anti-malarial Test (Repository Test): When the extract found effective in four-day suppressive test further evaluation the extract for its prophylactic effect undertaken. The prophylactic effect of the extract was assessed by applying the method described by Fidock et al. 21 Twenty-five mice of both sexes were weighed and randomly assigned into two control and three test groups Group I mice was given distilled water 0.2 ml/kg body weight. Groups II, III and IV, were given 100, 200 and 400 mg extract/kg body weight orally respectively while group V mice received 10 mg chloroquine/kg body weight orally daily for four consecutive days (D0 to D3). On the fifth day (D4), a standard inoculum (1×107 P. berghei erythrocytes, 0.2 ml) was administered intraperitoneally for each mouse. After 72 h (D7), blood smears were prepared from the tail of each mouse on a microscopic slide to determine parasitemia level.
Monitoring of Body Weight and PCV Changes: Body weight loss is one feature of rodent malaria infections. Body weight change is a crucial indicator used to measure the efficacy of the crude extracts 21. The body weight of each mouse was recorded before infection (day 0) and on (day 4) after treatment using sensitive digital balance. PCV measurement is another essential indicator to know the effectiveness of the crude extracts in preventing red blood cell hemolysis due to the parasite infection. The PCV of each mouse was recorded before infection (day 0) and on (day 4) after treatment. Blood was collected from the tail of each mouse with heparinized microhaematocrit capillary tubes. The tubes were sealed by crystal seal and placed in a microhematocrit centrifuge with the sealed ends outwards. The blood was centrifuged at 12,000 rpm.
PCV = (Volume of erythrocytes in a given volume of blood) / (Total blood volume) × 100%
Monitoring of Mean Survival Time (MST): MST is a crucial indicator that is used to evaluate the efficacy of antiplasmodial activity of the crude extracts. A crude extract that results in survival time greater than that of infected non-treated mice were considered as active 22. Mortality of the study mice was recorded daily and the number of days from the time of infection with P. berghei until the incidence of death was recorded for each mouse in both the treatment and control groups throughout the follow up period. MST for each group was calculated using the following formula:
MST = Sum of survival time of all mice in a group daily ⁄ Total number of mice in the group
Data Analysis: Data were entered, cleaned, coded, checked and analyzed by using SPSS version 20. The one-way analysis of variance (ANOVA) followed by Tukey‟s HSD posthoc test, was used to determine the significance in the comparisons of parasitemia suppression, weight change, PCV change, and survival time among the groups. Paired t-test was also used to compare some parameter between initial and final results. The results were considered significant when P<0.05.
Phyto-chemical Analysis: Preliminary phyto-chemical screening stem bark crude extract of H. abyssinica showed the presence of alkaloids, anthraquinones, terpenoids, phenolic compounds, tannins and flavonoids
TABLE 1: PHYTOCHEMICAL CONSTITUENTS OF METHANOL EXTRACT OF THE STEM BARK OF H. ABYSSINICA
|Secondary metabolite||Reagent||Test Result|
|Cardiac glycosides||Legal’s Test||-|
|Terpenoid||Copper acetate Test||+|
|Phenols||Ferric Chloride test||+|
|Tannins||Lead acetate test||+|
(+) present (-) absence
Toxicity Test: The acute toxicity study showed the crude extract did not cause mortality of mice within 24 h up to 2 g/kg. Gross behavioral and physical observation of mice also did not display signs of acute toxicity like salivation, lacrimation, hair erection, tremors, convulsions and reduction in feeding and motor activities. Based on these evidence the median lethal dose (LD50) of the extract after single oral administration in five female mice was found to be greater than 2 g/kg body weight.
Effect of the 4-Day Suppressive Test: The result of the 4-day suppressive test showed that the crude extract has significant (P<0.001) average percentage of chemosuppression against P. berghei at all dose levels (100-400 mg/kg) when compared to negative control (distil water treated) group. The dose of the crude extract at 400 mg/kg showed the highest percentage parasite suppression activity (83.33%, P<0.001), followed by 200 (79.6%, P<0.001) and 100 (65.3%, P<0.05). Significant parasitemia reduction (P<0.05) was also observed in all test groups treated with the crude extract.
But the standard drug, chloroquine (10 mg/kg), has a parasitemia suppression of 100%. All the mean survival time of the extract treated group was significant (P<0.05) when compared to distill water treated group of mice. The mean survival time increases as the dose increases which is dose-dependent effect. The standard drug, chloroquine (10 mg/kg), showed a significant effect on percentage parasite suppression and mean survival time.
TABLE 2: EFFECT OF EXTRACT ON PARASITAEMIA IN FOUR-DAY SUPPRESSIVE TEST
|Test Dose||Average % Parasitaemia||Average % Suppression||Mean Survival time ± SEM (days)|
|NC||24.0 ± 1.87||0.0||6.8 ± 0.37|
|HA100 mg/kg||8.33 ± 1.67a3||65.29 ± 1.7a3||10.0 ± 0.58a1|
|HA 200 mg/kg||5.0 ± 1.87a3||79.6 ± 2.2a3||12.4 ± 1.17a3|
|HA 400 mg/kg||4.0 ± 1.87a3||83.33 ± 4.3a3||15.2 ± 0.97a3|
|CQ 10 mg/kg||0.0a3b1c1||100 ± 0a3b1c1||28 ± 0.00a3b1c1|
Values are expressed as percentage and mean ± SEM (n=5). a as compared to NC; b as compared to HA100 mg/kg; c as compared to HA 200 mg/kg; d as compared to HA 400 mg/kg; 1 P<0.05; 2 P<0.01; 3 P<0.001. HA, Hagenia abysinica; CQ, Chloroquine; NC, Negative control; SEM, Standard error of mean.
TABLE 3: EFFECT OF EXTRACT ON BODY WEIGHT AND PACKED CELL VOLUME OF P. BERGHEI-INFECTED MICE IN 4-DAY SUPPRESSIVE TEST
|Test Dose||BW D0 ± SEM||BW D4 ± SEM||% Change||PCV D0 ± SEM||PCV D4 ± SEM|
|Dit. H2O 0.2 ml||24.4 ± 0.9||23.8 ± 0.9||-2.46b3c3d3e3||61.43 ± 0.75||51.33 ± 1.39|
|HA100 mg/kg||24.3 ± 0.7||25.5 ± 0.6||4.94||60.24 ± 1.14||51.67 ± 1.23|
|HA 200 mg/kg||25.2 ± 0.6||26.8 ± 1.1||6.34||52.13 ± 1.48||49.87 ± 1.51|
|HA 400 mg/kg||23.7 ± 0.7||25.8 ± 1.8||8.86||56.32 ± 2.03||55.75 ± 1.5|
|CQ 10 mg/kg||24.3 ± 0.6||26.5 ± 0.92||9.05||54.26 ± 3.43||56.01 ± 3.45|
|Dit. H2O 0.2 ml||24.4 ± 0.9||23.8 ± 0.9||-2.46b3c3d3e3||61.43 ± 0.75||51.33 ± 1.39|
Data expressed as a percentage and meant ± SEM (n=5). A as compared to 0.2 ml distal water; b as compared to 100 mg/kg; c as compared to 200 mg/kg; and d as compared to 400 mg/kg; e as compared to CQ 1 P<0.001; 2 P<0.01. HA, Hagenia abyssinica; CQ, Chloroquine; PCV, Packed cell volume; D0, day 0; D4, day 4; SEM, Standard error of the mean.
As presented in Table 3 the body weight of the mice was measured before and after the treatment day four. The comparison analysis showed that the crude extract treated groups prevented weight loss significantly (P<0.05) at all doses level when compared to distilled water treated control group. The crude extract treatment, in a dose-dependent manner, prevented from a significant decrease in PCV. The decrease in PCV was highest at 100 (P<0.001) followed by 200 (P>0.05) and 400 (P>0.05) as compared to chloroquine (10 mg/kg). But, there was no difference shown a change in PCV between the distilled water treated group and HA100 treated group.
Effect on Repository Test: The crude extract treatment, significantly reduced parasitemia level by 58.3%, 66.6%, and 78.3% for HA100, HA200, and HA400, respectively, as compared to the distilled water treated control group (P<0.001 in all treated groups). The inhibition by chloroquine (10 mg/kg) was significantly higher than the inhibition by all crude extract doses and the distilled water treated control group (P<0.001).
TABLE 4: PROPHYLACTIC EFFECT THE EXTRACT ON MICE INFECTED BY P. BERGHEI
|Test Dose||Average % Parasitaemia||Average % suppression||Mean Survival time ± SEM(days)|
|NC||60.0 ± 04.0||0.0||7.67 ± 0.33|
|HA100 mg/kg||25.0 ± 22.0||58.3a1||8.16 ± 0.17a1|
|HA200 mg/kg||20.0 ± 5.3a1||66.6a1||8.67 ± 0.42a1|
|HA400 mg/kg||13.0 ± 4.5a1||78.3a1||14.00 ± 0.58a1b1c1|
|CQ 10 mg/kg||0||100a1b2||20.00 ± 0.0a1b1c1d1|
|NC||60.0 ± 04.0||0.0||7.67 ± 0.33|
Data expressed as a percentage and meant ± SEM (n=5). a as compared to 0.2 ml distil water; b as compared to 100 mg/kg; c as compared to 200 mg/kg; and d as compared to 400 mg/kg; 1 P<0.001; 2 P<0.01. HA, Hagenia abyssinica; CQ, Chloroquine; PCV, Packed cell volume; D0 day 0; D4, day 4; SEM, Standard error of the mean.
DISCUSSION: The emergence of multi-drug resistant malaria-causing parasite and the absence of safe and effective malaria vaccine attracted scientists’ attention to search a new anti-malarial agent. It is interesting that candidate agents from medicinal plants utilized in traditional medicine for the management of malaria to be evaluated. The therapeutic effects of plants occur due to the presence of bioactive agents 21. The in-vivo anti-plasmodial effect of the extract displayed that the stem bark of H. abyssinica has a bioactive agent that have anti-malaria effect 5, 6, 27, 28.
Phytochemical screening of the extract showed the presence of triterpenes, tannins, alkaloids, terpenoids, flavonoids, anthraquinones, glycosides and phenols Table 1. Alkaloids which are found in this extract are known bioactive agent for their anti-malarial effect in other plants including cinchona bark (Cinchona officinalis) from which the most effective anti-malarial agent quinine was discovered 21, 22. The crude extract also contains phenols which are well known for its anti-oxidant and free radical scavenging activity that can prevent oxidative damage induced by the parasite. Anthraquinones & flavonoids were identified as active anti-malarial agent and the later classes of agents supposed to act by blocking fatty acid synthesis of the parasite 23. Also, to these activities, the crude extract may also have another crucial effect on the hosts, such as acting as anti-inflammatory, analgesics, anti-oxidant, and antipyretics or as immune stimulatory activity. The crude extract may act either of the above singly or synergistically by different mechanisms suggested or via another unknown mechanism 18, 23.
Hydroalcoholic (80% methanol) solvent was used in the extraction process because it extracts polar and moderately polar bioactive constitutes (s) 24. The stem bark was prepared as a decoction to in a similar way of traditional preparation by traditional practitioners. Young mice were also used to prevent the effect of anemia in old mice and some physiological changes due to aging which may affect the treatment outcome. The in-vivo model was used since it is suitable to know the pro-drug effect and the effect of the immune system in the removal of the parasite. A rodent malaria model was used in predicting the effect due to their similar disease features to those of plasmodial infection in a human, when infected with P. berghei and also several anti-malarial drugs such as quinine mefloquine, halofantrine, and artemisinin derivative were found using this model 25. The 4-day suppressive method is a standard test which commonly utilized to evaluate the anti-malarial effect of the candidate agent on early infections, and repository test which is frequently used to evaluate the prophylaxis effect of the candidate agent. In both evaluations method determination of percentage parasitemia suppression is the most reliable parameter in the anti-malarial drug discovery 22, 26.
The results showed that percentage of parasite suppression in the repository test and 4-day suppressive test significantly reduced in extract-treated groups, when compared to mice in the negative control group with 83%, and 78.3% respectively. This shows that the candidate agent has the anti-plasmodial effect. An in-vivo anti-malarial impact of a crude extract categorized as moderate, good, and very good, if the crude extract displayed percentage parasitemia suppression equal to or greater than 50% at a dose of 500, 250, and 100 mg/kg b.w per day, respectively 27. Based on this category, the extract has an excellent anti-malarial effect.
PCV was measured to assess the effectiveness of the candidate agent in preventing hemolysis because P. berghei infected mice suffer from anemia due to the destruction of infected erythrocytes, dyserythropoiesis, and erythropoietic suppression. In this study, the extracts prevented reduction of PCV in extract administered as compared with that in the negative control mice. This shows the extract prevented anemic conditions that were manifested negative control mice due to severe infection. This may be due to decreased parasitemia level in the in mice treated with the extracts in a dose-dependent manner. In the untreated mice, the parasitemia level increased and resulting in more red blood cells destruction and marked decrease of hematocrit PCV 28.
The life-prolonging potential of the extract in malaria-infected mice could be seen from its prolongation of the mean survival time. According to Peters the mean survival time of mice is considered as evidence for the anti-malarial activity of the plant extract. A plant extract that results in survival time greater than that of infected non-treated mice were considered as bioactive. In this study, there was lower parasitemia level in the extract administered group and longer survival time of the mice at the highest doses of the extract indicating strongly associated with a dose-dependent pharmacological activity in other words the extract suppressed the growth of P. berghei, and this was in agreement with another in-vivo anti-malarial test 29.
These findings were further supported by measuring Body weight change in mice which is an important parameter that measures the antiplasmodial activity of plant extracts. Because loss of body weight is one of the manifestations of P. berghei-infected mice, possibly due to the appetite suppressant and the disordered metabolic activity effect of the parasite 30. All Mice treated with the extract showed an increased in body weight gain when compared to the negative control group, hence this might indicate that the anti-malarial effect of the extract. The present study on body weight is in agreement with that of Dicasso 31.
CONCLUSION: This study has shown the efficacy of the stem bark extract of H. abyssinica supporting its traditional usage to malaria. The results also indicated that the extract has wide safety of margin making it a potential source for isolation of safer and cost-effective alternative drug with a new mode of action in the prophylaxis and treatment of malaria. Therefore, further studies on the extract could be carried out to isolate and characterize the active principle from this plant.
ACKNOWLEDGEMENT: We would like to acknowledge the School of Pharmacy (University of Gondar) for providing necessary materials and resources.
COMPETING INTEREST: The authors declare that they have no competing interests
ETHICS APPROVAL: The Ethical clearance to experiment on mice was obtained from the Institutional Ethics Review Board of College of health science, the University of Gondar with registration number UOG/18/363/07. The mice were handled by national guidelines for handling laboratory animals.
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How to cite this article:
Belete TM and Orijino TA: In-vivo anti-plasmodial activity of Hagenia abyssinica [Family: Rosaceae] in Plasmodium berghei infected in mice. Int J Pharmacognosy 2019; 6(2): 66-74. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.6(2).66-74.
This Journal licensed under a Creative Commons Attribution-Non-commercial-Share Alike 3.0 Unported License.
T. M. Belete * and T. A. Orijino
Department of Pharmacology, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia.
29 January 2019
25 February 2019
27 February 2019
28 February 2019