A large amount of mature and fresh ripe fruits of A. indica was harvested from Abakaliki, Ebonyi State, Nigeria. Fresh ripe fruits of A. indica were collected from the Abakaliki area of Nigeria. The analysis was carried out by GC-MS and 4 compounds were identified. Most of the compounds that were identified are presented in Table 1. Different fatty acid methyl esters of the A. indica fruit juice were identified using the National Institute of Standards and Technology (NIST) Database. The major fatty acid identified were dodecanoic acid (4.37%), oleic acid (14.19%), 13-octadecenal (17.05%) and 15-tetracosanoic acid methyl ester (47.13%). Fatty acids such as oleic (18:1), palmitic (16:0), linoleic (18:2), and stearic (18:0), were reported by Castro [38] and Duarte [39] in propolis samples. A total of 10 compounds were identified by [40] from the propolis collected from the Tamilnadu region of India, using GC-MS to show the presence of fatty acids. Among 10 compounds the major fatty acid present were as 9-octadecenoic acid (3.2%), decanoic acid (2.12%) 9,12-hexadecanoic acid (1.29%), octadecadienoic acid methyl ester (0.49%) and alcohols such as 1-tetradecanol (0.89%), octadecanol (0.69%), 1-dotricontanol (0.48%) and 2,3-epoxy-5,8-hectadecadien-1-ol (0.6%) [40, 41]. The synthetic mixes recognized in the ethanol concentration of the bark of Azadirachta indica were introduced [42]. GC-MS investigation uncovered the presence of Decosanoic corrosive, 22-trimethyl siloxy)- methyl ester, (1-Benzenesulfonyl-1H–pyrrol–3-yl) acidic corrosive, methyl ester, 1-H-pyrrole-3-propanoic corrosive, 2-(methoxycarbonyl-4-(2-ethoxy-2-oxoethyl)- 5 methyl ethyl ester, Propanoic acid,2 hydroxy-2-methyl ethyl ester, and 3,5-pyridine dicarboxylic corrosive, 2-4-6-trimethyl-diethyl ester. The antimicrobial action of leaves from Psidium guajava and A. indica was broken down by GCMS. The outcome demonstrated that 47 bioactive phytochemical mixes were recognized in the ethanol part of A. indica [42]. Biney revealed that the presence of methyl or ethyl esters of unsaturated fats can likewise be considered as qualities of the A. indica plant [43]. From this outcome, it very well may be reasoned that every one of these constituents is of pharmacological significance.
Vital metabolic activities take place in the apicoplast of the plasmodium parasite. Some of the processes include the biosynthesis of isoprene, haem, and fatty acids. Meanwhile, the pathway for fatty acid biosynthesis in the apicoplast of Plasmodium is different from the fatty acid biosynthesis pathway in humans and higher eukaryotes. Humans and eukaryotes normally use a Type I fatty acid synthase (FAS I) system, where each fatty acid biosynthetic step is catalyzed by a single protein with multiple domains. On the contrary, the apicoplast has a Type II fatty acid synthase (FAS II) system with each fatty acid biosynthetic pathway carried out by a discrete enzyme encoded by a different gene [44]. This type II FAS system is absent in humans but is common in plasmodium, bacteria, and algae [45]. Interference with the plasmodial type II FAS system can serve as a target of drug action to destroy the parasite without harming the human host. In the parasite, fatty acid biosynthesis is critical for cell membrane formation, an important source of energy, essential in signal transduction, protein acylation, growth, differentiation, and homeostasis in P. falciparum. Lipid biosynthesis is elevated during the erythrocytic phases of the parasite [45] because when the parasite is invading a host, it needs to protect itself by creating a so-called parasitophorous vacuole, in part as a protection from the immune system of the host. In this process, the parasite needs to make its fatty acids de novo to form and expand its membrane. In P. falciparum the principal membrane fatty acids are decanoic acid (10:0), lauric acid (12:0), and myristic acid (14:0).
Earlier work in 1992 by Kumaratilake and collaborators reports on the antimalarial properties of n-3 and n-6 polyunsaturated fatty acids, where acids such as 22:6 (n-3) and 20:5 (n-3) were the best in the studied series for in vitro killing of intra-erythrocytic forms of P. falciparum [46]. It was also reported that docosahexaenoic acid [22:6 (n-3)] was the best in the studied series of fatty acids in killing P. falciparum (> 90% death) at concentrations of 20–40 µg/ml [46]. The methyl esters of the fatty acids were reported to be as potent as the free acids in killing the parasite. Later work in 1995 by Krugliak and collaborators reported on the antiplasmodial effect of a series of C18 fatty acids against the FCR3 strain of P. falciparum, and these fatty acids displayed some inhibitory activity (≤ 200 µg/ml) against both the intact infected cells and the free parasites [47]. In this particular work, oleic acid (9–18:1) was the most inhibitory fatty acid with an IC50 of 23 µg/ml, and linoleic acid (9,12–18:2) displayed an IC50 of 76 µg/ml. In 2005, a naturally occurring C18 fatty acid, named scleropyric acid, was isolated from the twigs of Scleropyrum wallichianum, and also shown to display good antiplasmodial activity (IC50 = 7.2 µg/mL) against a K1 multidrug-resistant strain of P. falciparum [48].
Despite all current measures to curtail the spread of malaria infection, the disease is still ravaging the human population, particularly in malaria-endemic regions of the world. The economic burden continues to soar in addition to its comorbidities [49–51]. Current efforts in malaria research are towards the development of medications with less economic cost and more efficacy in combating this menace [52, 53]. Over the years, studies on the medicinal potentials of Azadirachta indica have predominantly focused on some parts of the plant such as the seeds, leaves, bark, roots etc. Some studies have developed extracts from the leaves, stem bark, and roots to treat several human diseases and ailments [54–57]. However, fruit juice has since been neglected and has never been exploited as a useful source of nutraceuticals that can be used in the treatment of diseases. Igwenyi revealed the biochemical composition and nutritional potentials of the fruit juice of Azadirachta indica and suggested that more investigations should be done to explore possible therapeutic potentials of the fruit juice [33]. Interestingly, this present study evaluated the potential of the fruit juice of Azadirachta indica in clearing malaria parasites in addition to replenishing blood levels usually depleted due to malaria pathogenesis.
As expected, our result revealed a high level of parasitemia in animals infected with Plasmodium berhei (NK 65). Treatment with Artesunate which is a standard drug for malaria studies and treatment led to a significant reduction in the levels of the parasites in the treated animals. Both World Health Organization and Malaria Consortium have since recommended the use of Artemisinin Combination Therapy as a standard drug for clinical treatment of malaria infection particularly in the malaria endemic regions of the world [6, 58]. Interestingly, treatment of the infected animals with fruit juice of Azadirachta indica as shown in Fig. 2 led to a significant reduction (p˂0.05) in the levels of Plasmodium berhei (NK 65). More interestingly, there were no significant differences (p > 0.05) between the anti-parasitic effect of the standard drug treatment and the treatment with fruit juice of Azadirachta indica on all the days of treatment. Fruit juice of Azadirachta indica is yet to be reported to have anti-plasmodial activity, hence this study has shown a novel therapeutic potential of Azadirachta indica fruit juice. Additionally, the use of the fruit juice of Azadirachta indica as medication for malaria infection offers some metabolic advantage over conventional drugs particularly as it relates to drug metabolism in the liver. Drug as xenobiotic increases metabolic burden on the liver [59, 60]whereas juice is barely metabolized in the liver due to its organic nature and high nutritional content.
Furthermore, one of the hallmarks of the pathogenesis of malaria infection is the destruction of the structural integrity of the Red Blood Cells (RBC) leading to the rupture of the membrane and further release of haemoglobin in the blood [61–63]. The haemoglobin is further broken down to its primary structure of the amino-acid sequence. This is incorporated by the parasite for the building of its proteins while the heam is processed to bilirubin for excretion [64, 65]. We confirmed this hypothesis in Fig. 3 of our result. Plasmodium berhei (NK 65) infection led to a significant reduction in the levels of haemoglobin in the infected and untreated group. Conversely, treatment with Artesunate led to a significant elevation (p˂0.05) in the mean values of haemoglobin in the animals. Moreover, treatment with fruit juice elevated the mean values of haemoglobin in the animals to values comparable to that of the standard malaria drug. Perhaps, this may partly be due reduction in parasitic load in the animals and partly due to the repair of the cell membrane of the damaged RBC. The fruit juice may contain bioactive components that could be helpful in membrane repair and resistance to oxidative processes as previously suggested by Igwenyi [33].
We further examined the effects of the juice on Packed Cell Volume (PCV) (Fig. 4). PCV indicates the percentage of RBC in circulation, thus the volume occupied by the RBCs in a particular volume of blood. A decrease in PCV indicates RBC loss perhaps due to cell destruction, general blood loss, and/or failure of hemopoietic cells in the bone marrow [66–69]. As expected, infection with Plasmodium berhei (NK 65) caused a significant decrease in the mean value of PCV in infected and untreated animals. It is a known fact that Plasmodium berhei (NK 65) thrives in the blood stage of malaria pathogenesis by invading and destroying the RBC [5, 18]. Hence, the PCV in the infected and untreated animals may have decreased due to RBC destruction which is consistent with studies of others. Interestingly, treatment with the fruit juice significantly increased (p˂0.05) the PCV to a mean value comparable to the standard drug group. In clinical treatment of malaria infection particularly in pregnant women, blood tonics are usually prescribed in addition to the antimalarial drug [19, 70–72]. This is to replenish the lost RBC due to malaria infection whereas the critical antimalarial component clears the parasites. Thus, we may have identified yet another novel blood-replenishing potential of Azadirachta indica fruit juice, which would need to be explored and exploited in further investigation. Fruit juice of Azadirachta indica thus may have dual potentials of clearing malaria parasites and replenishing depleted RBC due to malaria pathogenesis. Fruit juice of Azadirachta indica may also have an additional economic advantage over conventional malaria drugs by eliminating prescriptions for malaria patients and alleviating the metabolic burden on the liver and kidneys.
To confirm whether the novel blood-replenishing potential of Azadirachta indica fruit juice is a holistic one (i.e. affecting other blood components), we evaluated the levels of the White Blood Cells (WBC). As shown in Fig. 5, infection with Plasmodium beighei (NK 65) parasites significantly decreased (p˂0.05) the levels of WBC in the infected animals which are consistent with the results of other investigators, particularly in humans [61, 63, 73, 74]. WBCs are necessary for combating infectious agents with the potential of causing diseases [75, 76]. Their decrease in blood circulation leads to a lowering of the body's immune system and further increases one's susceptibility to various diseases [77]. Perhaps, malaria infection lowers body immunity through depletion in the WBCs circulation as previously shown by other studies [63, 78, 79]. Interestingly, we further observed that treatment with Azadirachta indica fruit juice led to a significant increase (p˂0.05) in the mean values of WBC in the treated animals. Fruit juice of Azadirachta indica may be useful in boosting the immune system through elevation of WBCs. Perhaps, it could further be recommended for people in low-income regions who are not able to afford costly multivitamins to enhance their protection against various diseases, which usually result due to lowered immune system.
In conclusion, this present study has identified novel therapeutic potentials of Azadirachta indica fruit juice which would require further investigations for full deployment into pharmaceutical development of natural products and new therapies for malaria infection. The antiplasmodial activity of the juice extract can be attributed to the fatty acid compositions and phytochemical constituents. The yellow colour will appeal to children and the sweet taste of the juice will find a very useful application in pediatrics and anti-malarial syrup production with additional nutritional and energy value. This is necessary as the high cost and toxic side effects of synthetic and orthodox medicine have created the challenge for the development of strategies and novel organic candidates for nutritional intervention in health and diseases.