Introduction
The most dangerous form of malaria is caused by
Plasmodium falciparum (
P. falciparum) parasites which present serious health problems worldwide. For the year 2017, there were approximately 219 million cases of malaria worldwide, compared to 217 million cases in the previous year. There were approximately 435,000 deaths from malaria globally in 2017, compared to 451,000 estimated deaths in 2016. The highest disease burden is centered in Sub-Saharan Africa, with children less than 5 years of age being the most affected group (WHO
2018). To date, the most effective ways to control these parasitic illnesses have been the use of drugs to treat the diseases and insecticides to control their transmission. Resistance to chloroquine, and lately to artemisinin derivatives and insecticides, gives fresh impetus to the search for new agents (Achan et al.
2018).
This search has led scientists to examine microorganisms, animal fur and plants (Higginbotham et al.
2014; Martínez-Luis et al.
2012; Durant et al.
2014: Isaka et al.
2018; Schorderet Weber et al.
2019; Whitman et al.
2019) for signs of activity against various diseases. In Panama we can find one of the planet’s most uniquely biodiverse environments, rich in plant and animal species potentially useful for alternative medicine studies. As part of their conservation efforts, The National Association for the Conservation of Nature (ANCON) protects a private natural reserve in Punta Patiño, Darién. This area is characterized by large extensions of coconut palm plantations of
Cocos nucifera (
C. nucifera).
The coconut tree has traditionally been used to treat several human diseases such as arthritis and asthma; and symptoms such as diarrhea and fevers; as a diuretic, and to treat malaria, among others (Lima et al.
2015). Several studies have described the traditional use of
C. nucifera husk, water and inflorescence on mice and rats to address several pathologies (Naskar et al.
2011; Rinaldi et al.
2009; Renjith et al.
2013; Alviano et al.
2004; Omoboyowa et al.
2016). One study describes the use of
C. nucifera leaf extracts as reducing amyloid-β
(1-42) aggregation in transgenic
Caenorhabditis elegans (Manalo et al.
2017). Another study describes the safety of
C. nucifera leaf extracts in mice, however, it does not evaluate the effectiveness of these extracts on any pathology (Paul et al.
2012). Because we have previously reported the in vitro antiplasmodial activity of the water extract of leaves (Tayler et al.
2019), and because to our knowledge there are no rigorous studies which describe the use of the leaves of
C. nucifera against
P. falciparum, we decided to test its in vivo potential as an antimalarial.
Our work focused on evaluating the inoculation route, acute toxicity, hematological parameters and antiplasmodial activity exerted by the aqueous extract of C. nucifera leaves from the Punta Patiño Private Natural Reserve in mice infected with Plasmodium berghei parasites.
Discussion
We carried out experiments to test the antimalarial activity of the extract from the leaves of C. nucifera on mice infected with P. berghei parasites, examining the routes of administration, the acute toxicity and different assays to determine its antiplasmodial effects.
The IM-treated mice were less susceptible to weight loss when compared with the other two routes of administration, oral and subcutaneous. Additionally, there was a greater antiplasmodial effect in these animals when compared to those treated subcutaneously and intragastrically. The latter administration of the extract involves the drug being subjected to the stomach acids which may cause it to be degraded (Levine
1970). Besides, using a gavage administration probably affected the animals’ ability to eat properly, resulting in weight loss, while subcutaneous administration may hinder the extract from reaching circulation and may not act against the parasites since the rate of absorption in this route is lower than intramuscular administration (Simmons and Brick
1970).
Our results for the acute toxicity assay showed that the extract presented signs of toxicity at the maximum concentration used (2000 mg/kg/day) when administered intramuscularly. In the group treated with the extract, two of the five mice died, with deaths beginning on the 6th day, suggesting that the dose at which half of the population dies (LD
50) lies slightly beyond 2000 mg/kg/day, since no animals died at 1000 mg/kg/day. Blood analysis of hematocrit and hemoglobin levels in all groups remained generally within the normal values up until day 8, when the three concentrations used apparently caused the hemoglobin to fall below the lower normal limit. This trend continued until day 16th suggesting that the extract negatively affects the integrity of red blood cells if used for periods longer than 8 days. Further experiments should monitor these levels more closely to determine the effects on hematocrit and hemoglobin parameters of the mice on a daily basis. Additionally, mean cell volume (MCV) parameters were analyzed in all groups. MCV is a measure of the size of red blood cells and low values (normal reference range is typically 42.3–55.2 fl/red cell) are indicative of microcytic anemia (Dasgupta
2015).This parameter was found to be normal after inoculating the extract in all treated groups, indicating that, while the extract affects hemoglobin and hematocrit levels after the first 8 days, the size of the RBCs remains unaffected for extended periods of treatment.
At concentrations of 500 and 750 mg/kg/day, the leaf extract was able to exert a parasite chemosuppression of 51% and 54%, respectively. The slightly higher activity achieved at 750 mg/kg/day suggests that this extract has a moderate suppressive activity (Deharo et al.
2001). Tests using concentrations between 750 and 1000 mg/kg/day should be explored to improve chemosuppression, but avoiding the slight toxicity found in the mice at 1000 mg/kg/day.
The extract does not show any curative effect when tested at concentrations of 500 and 750 mg/kg/day on P. berghei—infected mice. Nonetheless, in the prophylactic test, the extract at 500 and 750 mg/kg/day showed a dose-dependent activity against the parasite with P. falciparum inhibitory percentages of 28.60% and 44.91%, respectively. For a comparison with a classic anti-malarial drug, the use of chloroquine before infection was 83.56% efficient in stopping the infection.
Different products from the coconut plant have been studied extensively for their medicinal properties (Roopan
2016). Research has demonstrated the presence of metabolites such as polyphenols, glycosides, steroids, alkaloids, terpenoids, and others which could be responsible for those activities (Renjith et al.
2013; Manalo et al.
2017; Oliveira et al.
2009; Singla et al.
2011; Soumya et al.
2014). More specifically, the husk extracts of
C. nucifera have shown to have antimalarial properties when tested in vitro and in vivo (Adebayo et al.
2012; Adebayo et al.
2013; Balogun et al.
2014; Angeles et al.
2005).
Previous research by our group (Tayler et al.
2019) demonstrated the presence of flavan-3-ols (epicatechin), flavones (catechin derivatives, isoorientin, apigenin, vitexin, isovitexin, and luteolin) in the leaves of
C. nucifera from Punta Patiño, Panama, and the in vitro antiplasmodial activity of a water decoction of these leaves was described.
Flavonoids are phenolic compounds and one of the most common secondary metabolites found in plants. Flavonoids such as acacetin, apigenin, baicalein, chrysin, genistein, kaempferol, luteolin, among many others have been identified to possess antimalarial activity (Lehane and Saliba
2008). Additionally, compounds from the catechin family have been involved in antimalarial action (Sannella et al.
2007).
Fatty acid synthesis is of utmost importance in the development of malarial parasites. They are needed for parasite membrane and lipid body biogenesis (Palacpac et al.
2004) and they are used for the anchorage of parasite membrane proteins through glycosylphosphatidylinositol moieties (Gilson et al.
2006). There are three important enzymes involved in the fatty acid biosynthesis of
P. falciparum, namely 3-hydroxyacyl-[acyl-carrier-protein] dehydratase (FabZ), 3-oxoacyl-[acyl-carrier-protein] reductase (FabG), and Enoyl-[acyl-carrier-protein] reductase [NADH] (FabI) (Tasdemir et al.
2006). In a study from Tasdemir et al. (
2006) it was demonstrated that the flavonoids belonging to the gallic acid esters of catechins were the most active compounds against the parasite. They performed kinetic analyses using luteolin and (-)-catechin gallate as model compounds and revealed that FabZ was inhibited competitively. FabG was inhibited in a noncompetitive manner, whereas both compounds behaved as tight-binding noncompetitive inhibitors of FabI. Additionally, these polyphenols showed in vitro activity against chloroquine-sensitive (NF54) and -resistant (K1)
P. falciparum strains in the low to submicromolar range (Tasdemir et al.
2006). Furthermore, in the presence of catechins from green tea and other important plant polyphenols, the inhibitory effect of triclosan binding to FabI from
P. falciparum was potentiated (Sharma et al.
2007). The heightened binding of triclosan because of the high affinity of catechins was furthermore explained by molecular modeling studies based on flavonoids luteolin, quercetin, fistein, (-)-catechin gallate, and others (Banerjee et al.
2008). Modelling also correlated with the activity of luteolin against chloroquine-sensitive and chloroquine-resistant strains of
P. falciparum, following in silico studies where it was shown that luteolin had good binding affinity against dihydrooroate dehydrogenase (PfDHODH) and plasma membrane P-type cation translocating ATPase (PfATP4), two enzymes needed for the survival of the parasite. In vitro testing confirmed the finding showing low micromolar activity againt
P. falciparum. The flavonoids were found in the juice of Citrus species used traditionally for the treatment of malaria-associated fevers (Gogoi et al.
2019).
Several studies describe how catechin-related compounds such as epigallocatechin-3-gallate (EGCG) and epicatechin gallate (ECG) show moderate antiplasmodial effects. In a study by Sannella et al. (
2007), a crude extract, as well as EGCG and ECG from green tea leaves, were found to strongly inhibit the growth of
P. falciparum parasites in vitro. Additionally, they found that these catechins were able to potentiate the effect of artemisinin when used in combination against malaria parasites (Sannella et al.
2007).
The presence of flavan-3-ols (epicatechin), flavones (catechin derivatives, isoorientin and luteolin, among many others) could help explain the parasitemia reduction of P. berghei-infected mice tested in this study. It is clear, from the chemosupression percentages of 51% and 54% in a 4-day suppressive assay, in addition to a moderate prophylactic actity, that our crude extract from C. nucifera possesses antimalarial properties. Acute toxicity assays, however, showed a slight harming effect of this extract on hematocrit and hemoglobin parameters for the group which showed 54% chemosuppression. Even with this finding, the extract from C. nucifera leaves is a candidate for further analysis using a bioassay-guided isolation of compounds and/or fractions selected from the whole decoction. In addition, it would be valuable to determine the combinatorial effect this extract, and/or its compounds, could have on malaria parasites when paired with standard drugs. These approaches could result in new candidates and/or structures for drug development against human malaria.
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