Antimalarial Activity of Cocos nucifera Husk Fibre: Further Studies

In this study, the antimalarial and toxicity potentials of husk fibre extracts of five Nigerian varieties of Cocos nucifera were evaluated in vitro. The only active extract fraction, West African Tall (WAT) ethyl acetate extract fraction, was then evaluated for its phytochemical constituents, antimalarial and toxicity potentials at varying doses (31.25–500 mg/kg body weight) using various organ function indices. The results revealed that WAT ethyl acetate extract fraction (WATEAEF) contained alkaloids, tannins, and flavonoids and was active against Plasmodium falciparum W2 strain maintained in continuous culture, with a selectivity index of 30.3. The same extract fraction was active in vivo against Plasmodium berghei NK65, causing more than 50% reduction in parasitaemia on days 4 and 6 after inoculation at various doses administered. WATEAEF did not significantly alter (P > 0.05) function indices of the liver and cardiovascular system at all doses administered but significantly increased (P < 0.05) plasma creatinine concentration at 250 and 500 mg/Kg body weight compared to controls. The results of this study suggest that WATEAEF possesses antimalarial activity and may not adversely affect normal liver function nor predispose subjects to cardiovascular diseases but may impair normal kidney function at higher doses. Further studies are underway to isolate the active principles.


Introduction
Malaria is one of the most dreaded human parasitic diseases in the tropics and subtropics, especially in Africa where 81% of cases and 91% of deaths have been estimated to occur, with children under five years of age and pregnant women being most severely affected [1]. Nigeria accounts for a quarter of all malaria cases in Africa [2], mostly caused by Plasmodium falciparum [3], with an estimated 100 million malaria cases and over 300,000 deaths per year [4]. In addition to its direct health impact, malaria imposes a huge economic burden on afflicted individuals and nations, through high healthcare cost, missed days at work, and reduced economic output and productivity [5].
The continuous spread of P. falciparum resistance to antimalarial drugs poses a serious threat to malaria control programs. This, in addition to the high cost of the potent antimalarial drugs, has left the poor masses to be heavily reliant on traditional herbal medicines, which are often affordable and available [3]. Thus, the use of plant remedies has steadily increased worldwide in recent years, as well as the search for new phytochemicals that could be developed as 2 Evidence-Based Complementary and Alternative Medicine useful drugs for the treatment of malaria and other infectious diseases [6]. Cocos nucifera husk fibre and white flesh are used in folk medicine for the treatment of malaria [3,7]. Our recent in vitro studies have authenticated the acclaimed antimalarial action of the husk fibre extract of Cocos nucifera [8]. The present study was, therefore, set out to evaluate the antimalarial and toxicity potentials of the husk fibre extracts of five Nigerian varieties of Cocos nucifera in vitro and the most active in vivo, with the aim of identifying the most potent variety for rational antimalarial drug design.

Chemicals.
Absolute n-hexane, ethyl acetate, methanol, and ethanol were obtained from Sigma-Aldrich Laborchemikalien GmbH, Germany. Giemsa stain was obtained from Anosantec Laboratory, UK. Sodium chloride and glucose were both obtained from British Drug House Chemical limited, Poole, England. Sodium citrate was obtained from Merck, Darmstadt, Germany. Disodium hydrogen phosphate and potassium dihydrogen phosphate were obtained from Kermel Chemicals, China. Immersion oil was obtained from Panzonar laboratory Supplies, Button road, Canada. RPMI 1640 medium, sodium bicarbonate, L-glutamine, D-sorbitol, and HEPES were obtained from Sigma (St. Louis, MO, USA). Other reagents were of analytical grade and were prepared with injection water.

Preparation of Extracts.
The extracts were prepared according to the method of Adebayo et al. [9]. The fresh samples were allowed to dry under shade at room temperature and then pulverized into powder. Four hundred and fifty grams (450 g) of the powder of each variety was successively extracted with 5000 mL each of n-hexane, ethyl acetate, and methanol for 72 h per solvent in a tightly stoppered glass container. After each solvent extraction, the content was filtered with Whatman filter paper no. 1. The filtrates were then concentrated under pressure using rotary evaporator at 40 ∘ C, thereby generating the crude extracts.

Qualitative Phytochemical Screening.
Phytochemical screening was carried out for the ethyl acetate extract fraction using standard procedures described by Sofowora [10] to evaluate the presence of tannins, anthraquinones, alkaloids, terpenes, saponins, flavonoids, phenols, steroids, and cardiac glycoside.

In Vitro
]-Hypoxanthine Incorporation Assay. The in vitro tests were performed with blood parasites of P. falciparum W2 clone, which are chloroquine resistant, kept in continuous culture at 37 ∘ C in human erythrocytes, using the candle jar method as described by Trager and Jensen [11]. The antiplasmodial effects of the extract fractions were measured through inhibition of parasite growth, by the [ 3 H]-hypoxanthine incorporation assay, as described by Desjardins et al. [12] and modified by Zalis et al. [13]. For the test, a stock solution of each extract fraction was diluted in complete culture medium without hypoxanthine (RPMI 1640 containing 10% human serum, 2% glutamine, and 75% NaHCO 3 ). Blood stage parasites in the ring form obtained in sorbitol-synchronized blood (180 L/well) were cultured in 96-well culture plates at 1% parasitemia and 1% hematocrit and then incubated with the extracts. Controls without extracts or with chloroquine, used as the reference antimalarial drug, were run in parallel. After a 24 h incubation period, 20 L of medium containing [ 3 H]hypoxanthine (0.5 Ci/well) was added to each well, followed by incubation for 18 h at 37 ∘ C. The plates were frozen and thawed, and the cells were harvested (Tomtec 96-Harvester; Tomtec Inc., Handem, CT, USA) on prewet glass-fiber filters (Wallac Ou, Turku, Finland), which were placed in sample bags (Wallac) and immersed in scintillation fluid (OptiPhase Super Mix, Wallac). Radioactive emission was counted in a 1450 Microbeta reader (Wallac).
The half-maximal inhibitory concentrations (IC 50 ) as compared to the drug-free controls were estimated by using curve-fitting software (Microcal Origin Software 8.0, Inc.). Each experiment was repeated at least two times. The microtitre plates used for the radioactive assay were kept in a designated place and are meant to be there for a minimum of eighty years.

Histidine-Rich Protein 2 (HRP2)
Assay. This was performed according to the method of Noedl et al. [14]. The samples from the continuous cultures were washed and resuspended with RPMI 1640 (with 10% human serum) and uninfected erythrocytes to obtain 0.05% parasitemia and 1.5% hematocrit. For the test, a stock solution of each extract fraction was diluted in complete culture medium without hypoxanthine (RPMI 1640 containing 10% human serum, 2% glutamine, and 75% NaHCO 3 ). Serial twofold dilutions (seven concentrations and one drug-free control) of the drugs (20 L/well) were dispensed into standard 96-well microtitre plates (Costar 3599 plates), and 180 L of CMM was added to each well. The plates were then incubated for 72 h at 37.5 ∘ C. They were subsequently frozen-thawed twice to obtain complete hemolysis. 100 L each of the hemolyzed culture samples was transferred to the ELISA plates, which are precoated with monoclonal antibodies against P. falciparum HRP2 (capture antibody of the immunoglobulin M class; code CPF4), and the plates were incubated at room temperature for 1 h. Subsequently, the plates were washed four times with the washing solution provided with the test kit, and 100 L of the diluted antibody conjugate (an indicator antibody of the immunoglobulin G1 isotype; code CPF6) was added to each well. After incubation for an additional 1 h, the plates were washed four times, and 100 L of diluted (1 : 20) chromogen (tetramethylbenzidine) was added to each well. The plates were then incubated for another 15 min in the dark, and 50 L of the stop solution was added. Spectrophotometric analysis was performed with an ELISA plate reader (Spectra-MAX 340 Microplate spectrophotometer; Molecular Devices, Sunnyvale, CA, USA) at 450 nm.

Cytotoxicity Test Using Cultures of Hepatoma Cell Line.
Hep G2 A16 hepatoma cells were kept at 37 ∘ C in RPMI supplemented with 5% fetal calf serum (complete medium), in a 5% CO 2 environment. Cells from confluent monolayers were trypsinized, washed, counted, resuspended in complete medium, distributed in 96-well microtiter plates (4 × 10 4 cells/well), and then incubated for another 18 h at 37 ∘ C. The extract fractions prepared as stock solutions in DMSO were diluted in incomplete RPMI without fetal calf serum. After 24 h incubation at 37 ∘ C, 20 L of MTT solution (5 mg/mL in RPMI 1640 without phenol red) was added to each well [15]. After 3 h incubation at 37 ∘ C, the supernatant was removed, and 100 L of dimethyl sulphoxide was added to each well. The culture plates were read using a spectrophotometer with a 570 nm filter and a background of 630 nm. The minimum lethal dose (MLD) that killed 50% of the cells was determined as reported by do Ceu de Madureira et al. [16]; each assay was performed two times at least. Based on the values of cytotoxicity and antimalarial activity the selectivity index of activity, (SI) was calculated using the formula SI = MLD 50 /IC 50 .

In Vivo Studies
The animals were housed in standard plastic cages and acclimatized for a period of two weeks. They were maintained under standard conditions (12 h light and 12 h dark cycle) and had access to chow (Bendel Feeds, Ewu, Edo State, Nigeria) and clean tap water ad libitum.

4-Day Suppressive Test in Animal
Model. The antimalarial tests were performed using P. berghei NK65 strain, maintained by serial weekly passages of infected blood in mice. Tests were performed as described by Peters [17] with some modifications [18]. Briefly, thirty-five mice inoculated by intraperitoneal route with 1 × 10 5 infected red blood cells were kept together for 2 to 16 h, divided randomly in seven groups of 5 mice per cage, and then treated daily by oral route for 3 consecutive days with various doses of the active extract fraction as follows. All solutions were freshly prepared before administration. The extract fraction was then dissolved in 5% DMSO and then diluted so that each mouse received 200 L. At several days after parasite inoculation, blood smears were prepared from each mouse tail, methanol-fixed, stained with Giemsa, and then microscopically examined by counting parasitemia in up to 6000 erythrocytes. Inhibition of parasite growth in the drug-treated groups was calculated in relation to the nontreated control mice. The cumulative mortality of the animals was daily monitored up to day 30 after inoculation.

Toxicity Test in Animal Model
Animal Grouping and Extract Administration. Thirty adult Swiss mice were randomly divided into six groups of five mice each and daily administered 200 L of the various doses of most active extract fraction (WAT ethyl acetate extract fraction) dissolved in 5% DMSO by the oral route for 7 days as follows. Sample Collection and Preparation. At the end of the 7day experimental period, the mice were sacrificed by slight diethyl ether anaesthesia, and venous blood was collected into EDTA bottle to prevent clotting. The EDTA blood sample was centrifuged at 3000 rpm for 5 min and the plasma pipetted out. This was stored frozen until needed for analysis.

Biochemical Assays.
The protein content of the plasma was determined using the Biuret method as reported by Gornall et al. [19]. The plasma creatinine and urea concentrations were determined by the methods of Bartels and Bohmer [20] and Veniamin and Vakirtzi-Lemonias [21], respectively. Bilirubin concentration in the plasma was determined using the method described by Winsten and Cehely [22]. The procedure described by Doumas et al. [23] was used for the determination of plasma albumin concentration of the mice. The determination of plasma globulin concentration was done using the method described by Tietz [24] by subtracting the concentrations of liver and plasma albumin from the total liver and plasma protein concentrations, respectively. Alkaline phosphatase activity was determined by the method of Wright et al. [25]. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined by monitoring the concentrations of -keto acid hydrazones formed with 2, 4-dinitrophenyl hydrazine based on the procedure of Reitman and Frankel [26]. The method described by Wroblewski and La due [27], with slight modification, was used for the determination of lactate dehydrogenase activity. The method described by Bradley et al. [28], with slight modification, was used for the determination of glutamate dehydrogenase activity.

Statistical
Analysis. Data were analyzed using Duncan multiple range test following one-way analysis of variance (ANOVA) using SPSS 16.0 computer software package (SPSS Inc., Chicago, IL, USA). Differences at < 0.05 were considered significant.

Results
The in vitro evaluation of the extract fractions for antiplasmodial activities revealed that only the ethyl acetate fraction of the husk fibre extract of WAT was active against Plasmodium falciparum W2 strain maintained in continuous culture (IC 50 = 10.94 g/mL), with a selectivity index of 30.3 (Table 1). Only extracts with IC 50 less than 25 g/mL are generally considered active [18]. The phytochemicals present in the WAT ethyl acetate extract fraction were found to be alkaloids, tannins, and flavonoids (   of WAT ethyl acetate extract fraction, at all doses investigated, did not significantly ( > 0.05) alter the plasma total and conjugated bilirubin concentrations (Table 5), as well as plasma albumin and globulin compared to controls ( Table 6). All doses of the extract fraction administered caused no significant alteration ( > 0.05) in plasma total cholesterol concentration, HDL cholesterol concentration, and atherogenic index compared to controls (Table 7). However, the extract fraction significantly increased ( < 0.05) the plasma triglyceride concentration at the dose of 31.25 mg/kg body weight compared to control. Moreover, WAT ethyl acetate extract fraction, at all doses administered, did not significantly alter ( > 0.05) plasma alkaline phosphatase and alanine aminotransferase activities but significantly reduced ( < 0.05) plasma aspartate aminotransferase activity compared to controls (Table 8). However, plasma lactate dehydrogenase activity was increased significantly ( < 0.05) at the dose of 250 mg/kg body weight and glutamate dehydrogenase activity at the doses of 62.5 and 250 mg/Kg body weight compared to controls (Table 9).

Discussion
Cocos nucifera is abundant in Nigeria and other parts of West Africa [29]. The plant husk fibre has been reported to be used in the middle belt region of Nigeria as an antimalarial remedy, which has also been authenticated through in vitro studies [8]. The results of this study lend credence to our earlier report on the antiplasmodial activity of the widely grown tall variety of Cocos nucifera [8] and also established the in vivo antimalarial activity of the active extract fraction, WAT ethyl acetate extract fraction. Phytochemical screening of WAT ethyl acetate extract fraction in this study revealed the presence of flavonoids, tannins, and alkaloids (Table 2). This result is similar to the report of Silva et al. [30] on the phytochemicals present in the ethyl acetate extract of Cocos nucifera (Olho de Cravo variety) husk fibre. Moreover, tannins and polyphenolic compounds such as flavonoids, of which catechins are the most prominent, have been reported to be present in abundance in the coconut husk fibre and are responsible for its antibacterial, antiviral, antileishmanial, antinociceptive and, free radical scavenging activities [31,32]. Flavonoids and tannins present, which are phenolic compounds, act primarily as antioxidants or free radical scavengers, and may alleviate the oxidative stress associated with malaria, which plays an important role in the development of anaemia in malaria [33]. The concentrations of total protein, bilirubin, and albumin in blood can be used to ascertain different types of liver damage [34]. Bilirubin is an important product of haemoglobin catabolism with biological and diagnostic values [35]. Absence of a significant ( > 0.05) change in both total and conjugated bilirubin suggests that the extract 6 Evidence-Based Complementary and Alternative Medicine  fraction, at the doses administered in this study, is not capable of causing haemolysis and impairment of the secretion of conjugated bilirubin into the bile duct in the liver [36]. Albumin is synthesized in the liver. Thus, the lack of alteration in plasma albumin concentration suggests that the synthetic function of the liver has not been compromised, at all doses of the extract fraction administered [37]. Globulins are heterogeneous complex mixture of protein molecules with diverse functions in the body [38]. Also, there was no significant change in the globulin concentrations in plasma caused by the extract fraction at the doses administered in this study. Thus, the roles of these plasma proteins in the transportation of nutrients, defense mechanism, coagulation processes, maintenance of blood osmotic pressure, and buffering capacity of the blood may not be affected by the extract fraction [38].
Serum urea and creatinine concentrations are used for the assessment of renal sufficiency. Higher than normal levels serum urea and creatinine are indicators of deficiency in renal function [39]. Moreover, increase in serum urea level may also be due to the increase in protein catabolism [40] while the decreased urea level may be attributed to impairment of the urea cycle leading to reduced production of urea [9]. The extract fraction, at all doses administered, showed no significant alteration in the plasma urea concentration, suggesting that the urea cycle and protein catabolism were not adversely affected by the extract fraction. However, the increase in plasma creatinine concentration at the doses of 250 and 500 mg/Kg body weight suggests that the extract fraction may impair renal function at higher doses.
In recent times, there has been an increase in the prevalence of coronary heart disease (CHD) and CHDrelated deaths possibly due to the mismanagement of the risk factors that predispose to this disorder [41]. The major identified risk factors are elevated serum LDL-cholesterol concentration, reduced serum HDL-cholesterol concentration, and high blood pressure [42,43]. Studies have shown that lowering levels of serum cholesterol decreases the incidence of coronary heart diseases [44]. The atherogenic index (total cholesterol/HDL-cholesterol) is a reliable and strong indicator of cardiovascular diseases. Myocardial infarction increases considerably when the ratio is higher than 5 [45,46]. All the values for atherogenic index observed in this study were less than 3 and were not significantly different ( > 0.05) from control, suggesting that WAT ethyl acetate extract fraction may not predispose subjects to coronary heart disease, thereby not giving rise to further cardiovascular complications during malaria treatment.
The measurement of the activities of enzymes in tissues and body fluids plays a paramount and well-known role in disease investigation and diagnosis [47]. These enzymes, such as phosphatases, dehydrogenases, and transferases, get into the blood through leakage from disrupted cell membranes in damaged tissues [48,49]. Alkaline phosphatase (ALP) has been reported to be a marker enzyme for plasma membrane and endoplasmic reticulum [50]. ALT activity in the plasma is a more specific indicator of liver damage affecting cell integrity [51]. ALP and ALT activities, at all doses of the extract fraction administered, were not significantly changed in the plasma, suggesting that the extract fraction may not interfere with plasma membrane integrity and other metabolic activities mediated by ALP in the liver [52]. Lack of change in plasma ALP activity also suggests that the extract fraction does to cause hepatobiliary obstruction [51]. The reduction in plasma AST activity, at all doses of the extract fraction administered in this study, suggests inhibition of the enzyme in situ rather than damage to the liver or heart. Generally, plasma lactate dehydrogenase activity was not affected  by the extract fraction, except at the dose of 250 mg/kg body weight, suggesting that the extract fraction not predispose subjects to myocardial infarction and haemolysis [51].
In conclusion, the results of this study suggest the husk fibre of the West African Tall variety of Cocos nucifera as a potential source for novel antimalarial drug, pinpointing its ethyl acetate extract fraction as being responsible for its antimalarial activity. This active extract fraction may not possess hepatotoxicity potential nor predispose subjects to cardiovascular diseases. However, it may impair normal kidney function at higher doses. Further studies on biofractionation are underway to isolate the active compound(s) from the active extract fraction, with the aim of getting rid of the nephrotoxicity of the extract fraction at higher doses and reducing the expensive cost of the treatment of the disease, especially amidst the poor populace of the country.