Antimalarial and Haematological Effects of the Methanolic Leaf Extract of Gongronema latifolium on Mice Infected with Plasmodium berghei

MATERIALS AND METHODS

Study area: The study was carried out at the Department of the Pharmacology and Toxicology, Animal Science Sub-Laboratory, University of Nigeria Nsukka, from July, 2018 to September, 2019.

Chemical and test reagents: Chemicals and test reagents used include artemether/lumefantrine (20 mg/120 mg) (Artelum^®, May and Baker, Nig), Blood samples, methanol, (Fischer Scientific and Chemical Company, Germany). Plant extract, Distilled water (Energy and Resources Centre, UNN), Leishman stain (Sigma Chemical Company Ltd., USA) Phosphate buffer. Other reagents were contained in the commercial kits, products of Randox, QCA, USA and Biosystem Reagents and Instruments, Spain. All reagents were of analytical grade.

Equipment/Instruments: The following instruments were used for the experiment: Adjustable micropipette (PERFECT, USA), Chemical balance (Gallenkamp, England), Digital photo colorimeter (E1, 312 Model, Japan), Incubator (Gallenkamp, England), Microscope slides (UNESCOPE, USA), pH meter (Pye, Unicam 293, England), Refrigerator (Kelvinator, Germany) and Spectrophotometer (NOVASPEC LKB Biochrom, model 4049, Germany, Whatman qualitative filter paper, gloves, EDTA bottles, bijou bottles, cages, syringes and needles.

Collection of the experimental plant: Fresh leaves of G. latifolium were collected from Ogige Market near the University of Nigeria, Nsukka in July, 2018. The plant was identified by Mr. Felix Nwafor, a taxonomist in the Department of Pharmacognosy and Environmental Medicine, University of Nigeria, Nsukka. A voucher specimen was preserved at the herbarium of the Pharmacognosy Department, University of Nigeria, Nsukka, Nigeria.

Animals: Adult male and female Swiss albino mice (50) weighing between 18-25 g were obtained from the Animal-House of the Department of Pharmacology and Toxicology, University of Nigeria, Nsukka, Nigeria. The animals were observed under 12 light/12 hrs dark cycle in metabolic cages in a well ventilated (average room temperature of 28 ) rodent cubicle. They were fed with a mice pellet diet and given free access to clean drinking water. The animals were kept in well-disinfected rooms (to avoid any form of parasite infections) and allowed to acclimatize for two weeks before the start of the experiment. All the animals were handled according to the regulations of the National Code of Conduct for animal research ethics and Use of Laboratory Animals. And also the approval of the ethical committee of the Faculty of Pharmaceutical Science of the University of Nigeria on the use of animal subjects with certificate numbers FPSRE/UNN.

Rodent parasite: Artemether/lumefantrine sensitive P. berghei (NK65 Strain) employed in the study was obtained from Nigeria Institute of Medical Research (NMR1), Lagos.

Crude extraction of collected leaves: The maceration protocol as described by Maina et al.⁷ saw 1 kg of G. latifolium powdered dry leaves extracted with 95% methanol. The extract was present in the refrigerator till it was needed for the study.

Phytochemical analysis: The whole methanolic extract was subjected to phytochemical investigation, using the method described by Altemimi et al.⁸. The test was carried out to determine the presence or absence of certain pharmacological active principles in the plant.

Pharmacological testing
Acute toxicity test: The methanol extract was administered in normal saline. The test was carried out in two stages as described by Rispin et al.⁹ using a total of 12 mice of weight 15-32 g.

Blood collection: Blood collection was through the retro-orbital route and the blood sample was placed in an EDTA bottle for haematological analysis.

Mice parasite: Artemether/lumefantrine sensitive P. berghei (NK65 Strain) was obtained here with kind permission from malaria-infected mice at Nigeria National Medical Research Institute (NMRI) Lagos, Nigeria. Ten drops of the parasitized blood were obtained with the aid of a capillary tube through the ocular region of the mice, diluted with 1 mL of normal saline. Thereafter, 0.2 mL of the diluted parasitized blood was used to passage experimental animals (mice) for the study.

Infection of mice with P. berghei and administration of leaf extracts: The 25 (5 mice per group) healthy mice were divided into 5 groups each for the suppressive and curative tests, respectively as follows:

The route of administration (treatment) was via the oral route with the aid of an oral intubation tube.

Antiplasmodial activity
Four day suppressive test (Peters 4 days suppressive test): The standard 4 days suppressive test was employed. This test is the most extensively used preliminary test in which the effectiveness of the extract is evaluated via comparison of blood parasitemia (in extract-treated and untreated mice^10-12. Treatment was started 3 hrs after. Infection on day 0 and continued daily for 4 days (i.e., from day 0-3) on the 5th day (day 4), thin smears of blood were prepared from the tail of each mouse placed on microscopic slides fixed in methanol and Leishman stained (10%) at PH 7.2 for 15 min. Parasitemia was determined via counting the number of parasitized RBCs in ten random microscopic fields of at least 1000 erythrocytes. Then average percent parasitemia and percent suppression were calculated by using the following formula:

Evaluation of established infection (Rane’s test): The curative effect of the extract was evaluated following the method described by Kapishnikov et al.¹³. Infected RBCs were injected into mice intraperitoneally on day 0. About 72 hrs later, mice were randomly distributed into groups and dosed once daily for 5 consecutive days. Leishman stained thin blood film was prepared from the tail number of each mouse daily for 5 days to monitor parasitemia. The animal used was the same as in the suppressive model.

Haematological studies
Blood collection: Blood collection was through the retro-orbital route. The sample was collected with the help of a 5 mL syringe attached to needle (21 SWG) plain capped bottles containing Ethylene Diamine Tetraacetate (EDTA). The samples were used for the estimation of the different variables.

Determination of the packed cell volume, haemoglobin concentration, red blood cell count and white blood cell count was done.

Measurement of packed cell volume (PCV): The PCV was considered to predict the efficacy of the extract against hemolysis due to the rising parasite level associated with malaria. Heparin EDTA coated capillary tubes were employed for blood collection from the tail of each mouse. Blood filled tubes were sealed with sealing plasticine and then placed in a microhematocrit centrifuge with the sealed end outwards centrifugation for 5 min at 300 rpm. Finally, tubes were removed from the centrifuge and PCV was evaluated via a standard microhematocrit reader. The PCV, a measure of the proportion of RBCs to plasma, was measured before inoculation with the parasite and at day 4.

Hemoglobin concentration (g dL^–1): The Sahli haemoglobinometer (Dolphin Equipment Corporation Company, Pelham, New York, USA) Sahli haemoglobin metre was are used for the determination of HB concentration and calculations done according to the methods of Agrawal et al.¹⁴.

Up to the ten mark of the Sahli tube was 0.1N HCl placed. With the Sahli blood pipette, 20 μL of blood was placed into the Sahli tube and was sucked up and down. The mixture was allowed to stand for 5 min for the formation of acid haematin. The dark mixture formed was diluted gradually with distilled water till the colour when compared with that in the haemoglobin metre is slightly darker than the standard. The volumes of the noted colour change were taken and the average of the slightly darker and paler were compared and was ensured that the variation is not more than ±5 for the average value.

Red blood cell count (RBC count): A haemocytometer counting chamber procedure according to Khan et al.¹⁵ was used in the determination of RBC count. Using the dilution pipette with the mixer from the hemacytometer kit, blood was drawn up to the 0.5 mark. Continuing to hold the pipette as horizontal as possible, the Ringer's solution diluent was drawn up to the 101 mark (Dilution of 1-200).

The tip of the pipette was sealed with the finger and shaken well to mix.

Half of the content of the pipette was emptied into a waste container and a small amount of the diluted blood was placed into one chamber of the hemacytometer to just fill the chamber of the hemacytometer. The preparation was allowed to sit for a minute (for cells to settle).

The RBCs/cmm was calculated by adding the cells in the 5 groups and multiplying by 10,000 (i.e., add four zeros)¹⁶.

Termination of the experiment: The animal in the 1000 mg kg^–1 group died, hence, the LD₅₀ of the extract of G. latifolium is 1000 mg kg^–1 following Lorke’s assumption⁹.

Statistical analysis: For the statistical analysis a 2019 SPSS package was used, ANOVA mean difference at 0.05 and 0.01 confidence limits were determined. Standard deviation and difference between and within groups were also analyzed.

RESULTS

Percentage yield of extract: The yield obtained from the methanol extract of G. latifolium is 0.151 and the percentage yield is 15.1%.

Acute toxicity test: Death was recorded in 1000 mg kg^–1 dose therefore, a geometric mean of the dose where the death occurred and the dose preceding the recorded death was calculated and the LD₅₀ was found to be 0. 9 g kg^–1.

Phytochemical analysis: A phytochemical test was carried out on the whole methanolic extract and shows a result of the quantitative analysis of the different phytochemicals present. G. latifolium leaves and very high concentrations of proteins, high concentrations of saponins, carbohydrates, glycosides, the moderate concentration of alkaloids, terpenoids, steroids, a small concentration of resins and flavonoids and absence of reducing sugars, tannins, acid-soluble compounds and fat and oils.

Result of statistical analysis: The result showed that the methanol extract of G. latifolium has a very high dose-dependent significant antispasmodic effect on albino mice infected with Plasmodium berghei amongst the 100, 200 and 400 mg kg^–1 dose when compared with the negative control group.

Anti-malarial activity
Four days suppressive test: The results of the study showed that G. latifolium crude extract displayed a significant decrease in mean parasite count. It is of note that the extract dose-dependently suppressed the malaria parasite significantly at all tested doses (100, 200 and 400 mg kg^–1) (p<0.05) (Table 1).

This effect was much higher than the standard drug artemether/lumefantrine and 5 mL kg^–1 of distilled water.

Anti-plasmodial activity: Mean parasitemia inhibition of Plasmodium berghei infected mice on the 5th day, for the 4 days curative test.

The result showed that the methanol extract of G. latifolium has a very high dose-dependent significant antispasmodic effect on albino mice infected with plasmodium (Berghei berghei ) amongst the 100, 200 and 400 mg kg^–1 dose when compared with the negative control group.

Effect of the G. latifolium extract on haemato parameters: The mean different value analysis of the initial and final correct test results of the various haematological parameters of the plasmodium infected mice treated with Gongronema latifolium (curative).

With the PCV, the highest dose, 400 mg kg^–1 and the standard drug have the highest effect (Table 2). At the haemoglobin concentration (Hb), the 200 and 400 mg kg^–1 doses and the standard has a statistical significance of 0.000 for each. In RBC count all the doses and the standard has the significance of 0.000 except the 100 mg kg^–1 which has 0.005. The mean packed cell volume has a level of increase though not significant, was insignificantly increased in all test groups and also with the standard drug group (p<0.05). The same pattern of the result occurred with the mean haemoglobin concentration, mean red blood cell concentration.

Peter’s 4 days test analysis showing the effect of varying doses of methanol extract of G. latifolium on mice induced with Plasmodium berghei.

The result showed a dose-dependent antiplasmodic effect across the doses when compared with the negative control (Table 3).

DISCUSSION

This study investigated the in vivo antimalarial and haematological effects of methanolic extracts of G. latifolium on albino mice, chosen as it takes into full account the prodrug effect and the immune system impact in malaria eradication¹⁷.

Plasmodium berghei and at times P. vivax can be utilized in an experimental model of malaria infection causing symptoms of pathology which mimics symptoms produced by human malaria^18,19. This rodent malaria is dissimilar to the human Plasmodium parasite, yet it is the beginning step in screening most in vivo antimalarial activities of test compounds and also the old and current antimalarials (chloroquine, artemisinin and its congeners) were discovered through this method. Peter’s 4 days suppressive procedure test which evaluates activities of (potential) antimalarial agents on early malaria infection and the curative model followed the protocols of Rane’s estimates the curative ability of test compound on established infection which is the two widely established models of antimalarial activity. Both methods determined the inhibition in parasitemia and are reliable parameters¹¹.

Methanol extract of G. latifolium has a high antiplasmodial effect (10.00±0.0) compared with the negative control (28.400). The different dosing groups 100 mg kg^–1 (10.00±0.316), 200 mg kg^–1 (4.0±509) and 400 mg kg^–1 (1.00±0.447) (Peter’s 4 days suppressive test) revealed the antiplasmodial effects of the extract with its ability in reducing parasitemia of P. berghei infected mice in a dose-dependent fashion indicating the presence of active compounds against the malaria parasite. This could be attributed to the contents of alkaloids, terpenoids, flavonoids²⁰. Although the active malarial compound in G. latifolium has not been elucidated, its antimalarial properties could be due to secondary metabolites (alkaloids, terpenoids, phenolic compounds etc.) in the plant. These groups of compounds may produce antiplasmodial effects in collaboration or singly^21,22. Alkaloids as exemplified by quinine are noted for antimalarial activity.

Survival time, particularly at the highest dose, prolonged by the test extract was associated with a decline in escalated parasitemia level, suggesting antiplasmodial activity.

The daily mean parasitemia count for the respective groups in the curative test also indicated a dose-dependent decrease in parasitemia and a dose-dependent percentage increase in the curative test or ability.

Anaemia reduction in body weight and a reduction in body temperature are features of malaria in mice. Haematological changes are some o the most common complications in malaria pathogenesis in malaria pathogens with infected patients having lower white blood cells platelets, red blood cells eosinophils²³. In the haemato parameter determined PCV was calculated to estimate G. latifolium’s ability in averting hemolysis due to rising parasitemia level. Here, anaemia in mice could be attributed to the clearance or damage of infected Red Blood Cells (RBCs) coupled with clearance of uninfected RBCs and inhibition of erythropoiesis by malaria parasite²⁴. In the suppressive test, there was a reversal of mean RBC concentration by the extract. As a result, the analysis of packed cell volume which evaluates the efficacy of test extract in preventing hemolysis was done.

The extract in the 4 days suppressive test (100, 200 and 400 mg kg^–1), substantially prevented PCV reduction. This reversal of PCV reduction by the test extract is associated with its ability in reducing parasitemia of infected mice hence, indicating a dose-dependent haematological effect for all the blood parameters measured in line with the findings of Matur and Egwuonwu²⁴ on the moderate to high efficacy of the antimalarial activities of G. latifolium. It is strongly recommended that this effect be further established by further research to establish its anti-malarial effects as it has been established for plants such as Calpurnia aurea, Croton macrostachyus, Asparagus africanus, Withania somnifera, Dodonaea angustifolia and Phytolacca dodecandra.

CONCLUSION

Gongronema latifolium has proven to have an appreciable effect on the blood parameters as studies in this research indicates. It has also proven a somewhat advance able study result on the effect on malaria parasites which was shown by its suppressive effect and confirmation by its curative effects. The research study revealed that the extract has promising antiplasmodial activity against artemisinin/lumefantrine sensitive P. berghei in a dose-dependent manner.

SIGNIFICANCE STATEMENT

This study discovers the antiplasmodial properties in Gongronema latifolium that can be beneficial to patients on other antimalarials where resistance may occur especially in Plasmodium berghei infections. Thus, a new theory in malaria therapy may have emerged.