Received Date: September 16, 2015; Accepted Date: November 04, 2015; Published Date: November 11, 2015
Citation:Ratananinsruang P, Taowbud BN, Ountuang P, Somsak V (2015) Effect of Nelumbo nucifera Stamen Extract on Phagocytosis and Malaria Parasite Growth Against Plasmodium Berghei Infected Mice. Malar Cont Elimination S1-004. doi:10.4172/ 2470-6965.1000S1-004
Copyright: © 2015 Ratananinsruang P. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Antimalarial drug resistant malaria parasites are causing not only the spread of malaria to new areas but also its re-emergence in areas where it had previously been eradicated. In addition, malaria-associated impairment of phagocytosis has been reported during malaria parasite infection. The present study has been carried out to investigate the effect of Nelumbo nucifera stamen extract on phagocytosis and malaria parasite growth against Plasmodium berghei infected mice. Groups of ICR mice were treated orally by gavage with N. nucifera stamen extract (500, 1,000 and 2,000 mg/kg) after infection with P. berghei ANKA. Parasitemia, percent phagocytosis and phagocytic index were determined. At these doses, N. nucifera stamen extract inhibited parasitemia in dose-dependent manner, with similar level of antimalarial activity to chloroquine (5 mg/kg). In addition, increasing of phagocytosis and phagocytic index has also been observed in dose-dependent in infected mice treated with the extracts. In particularly, the highest activities of N. nucifera stamen extract were found at dose 2,000 mg/kg. These results indicated that aqueous crude extract of N. nucifera stamens have antimalarial and improve phagocytic activity against P. berghei ANKA infected mice.
Nelumbo nucifera; Phagocytosis; Malaria; Plasmodium berghei
Malaria remains one of the world’s largest burdens of disease. With an estimated 2.5 billion people at risk, it causes 300-500 million infections and 1-3 million deaths annually, the greatest part of the latter in children under five years of age . Malaria is caused by protozoa parasite in genus Plasmodium that transmitted by female Anopheles mosquito. Five species of malaria parasites including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi are responsible for human infection, although the majority of fatal cases are caused by P. falciparum [2,3]. Although an effective vaccine is the best long term control for malaria, current research on vaccine development is still in the laboratory. Therefore, the strategy for malaria mainly focuses on drug treatment. However, antimalarial drug resistant malaria parasites are causing not only the spread of malaria to new areas but also its re-emergence in areas where it had previously been eradicated [4,5]. In addition, malaria-associated impairment of phagocytosis has been reported during malaria parasite infection [6-9]. This has prompted research towards the discovery of new antimalarial drugs with phagocytosis recruitment properties. In this respect, plant extracts are potential targets for research and development of the alternative drugs.
Nelumbo nucifera Gaertn, commonly known as lotus, is a large aquatic plant and has long been used as a medicinal herb in China, Japan, India, Korea and Thailand. Nearly every part of N. nucifera including buds, flowers, anthers, stamens, fruits, leaves, stalks, rhizomes and roots have been used as medicinal plant for treatment of cancer, heart problems, hypertension and insomnia . Catechin, quercetin, quercetin-3-O-glycoside, kaempferol-3-O-glycoside and myricetin- 3-O-glucoside have been reported as part of its major components [11,12]. Pharmacological and physiological activities including antidiabetic, cytoprotective, hepatoprotective, anti-bacterial, antioxidant, anti-hypertensive, anti-hyperlipidemia, hematopoietic system, and anti-obesity effects have been described from the extracts of N. nucifera leaves, seed and rhizome [13-16]. In particular, N. nucifera stamens are flavonoid-rich and have a variety properties including antioxidant, anti-inflammation, anti-microbial and anti-cancer [11,12,17,18]. Flavonoids are its important constituents that belong to a group of natural substances with variable phenolic structures found in fruits, vegetables, grains, flowers, tea and wine. Moreover, flavonoids have been described to increase phagocytic activity and exerted antimalarial effect in vitro against P. falciparum [19-21]. Although biological and therapeutic efficacies of N. nucifera have been reported to a certain extent, the studies on the antimalarial activity and phagocytosis during malaria infection have not yet been performed. Hence, in the present study, we investigated the effect of N. nucifera stamen extract on phagocytosis and malaria parasite growth in vivo using P. berghei infected mouse model.
The stamens of lotus (N. nucifera) were obtained from Rangsit Science Centre for Education, and authenticated by Dr. Saowanee Buatone.
Preparation of crude extract
Aqueous crude extract of N. nucifera stamens was prepared as previously described . In brief, the dried powdered sample was mixed with distilled water in a ratio of 1:5 (w:v), and heated using microwave at 360 W for 5 min. Incubation at room temperature for 30 min was subsequently performed with continuously stirring to complete extraction. Then, it was filtered through gauze and centrifuged at 10,000 g, for 10 min. The aqueous crude extract of N. nucifera stamens was stored at 4˚C.
Healthy ICR mice (female, 6-8 weeks old weighting between 30-35 g) obtained from National Laboratory Animal Center, Mahidol University, Thailand were used. The mice were conveniently housed under standard environmental condition at 22-25˚C with a 50% relative humidity and a 12hr light/dark cycle. All mice had ad libitum access to commercial feed pellets and clean water throughout the study. All animal experiments were approved and ratified by the Animal Ethic Committee, Western University.
Rodent malaria parasite
Plasmodium berghei ANKA strain (PbANKA) was used in this study. The parasite was maintained in our laboratory by weekly serial passage of 1×107 infected erythrocytes in naïve mice. Parasitemia was daily monitored by microscopic examination of Giemsa stained thin blood smear with 100× oil immersion lens.
Acute toxicity test
Acute toxicity of N. nucifera stamen extract was carried out as previously described . Groups of mice (5 mice of each) were given 500, 1,000, 2,000 and 3,000 mg/kg body weight of the extract orally. The mice were then observed for signs of toxicity which include but not limited to paw licking, salivation, stretching of the entire body, weakness, sleep, respiratory distress, coma and death in first 4hr and subsequently daily for 14 days.
Evaluation of phagocytosis
The phagocytosis was performed a method previously described with some modification . Briefly, Blood (10 μl) was collected from tail vein and gently smeared into a square of 1 cm2 on a clean microscopic slide. The slide was subsequently incubated in a moist chamber at 37˚C for 2 hours and rinsed with 0.9% normal saline solution (NSS) to remove non-adherent cells. Adherent cells were incubated with 100μl of heat-killed Saccharomyces cerevisiae (3 mg/ml) for 1 hour in a moist chamber at 37˚C and rinsed 3 times with 0.9% NSS. Subsequently, the slide was fixed with absolute methanol and stained with Giemsa solution. The slide was air-dried and examined under light microscope. The sample was analyzed under an oil-immersion objective, counting at least 100 cells in different field. The percentage phagocytosis and the phagocytic index (PI) were calculated following the formula below.
% phagocytosis = Number of phagocytic cells × 100
Total number of white blood cells
Phagocytic index (PI) = Number of engulfed yeast cells × 100
Total number of phagocytic cells
Chloroquine (CQ) was used in this study as standard antimalarial drug. The drug was freshly prepared in distilled water and administered orally by gavage. Drug dose, expressed in mg/kg of body weight, was adjusted at the time of administration according to the weight of each mouse. The dose was based on the ED90 (5.0 mg/kg) on PbANKA infected mice .
Efficacy test in vivo
The standard 4 day test was employed in this study . Randomly groups of naïve ICR mice (5 mice of each) were inoculated by intraperitoneal injection with 1×107 infected erythrocytes of PbANKA, and treated for 4 consecutive days with 500, 1,000 and 2,000 mg/kg of N. nucifera stamen extracts orally by gavage twice a day (day 0-3). Three control groups were used; the healthy control was given either with distilled water or the extract (2,000 mg/kg); the untreated control was given distilled water; the drug treatment control was given 5.0 mg/kg of CQ. On day 4 of the experiment, tail blood was collected to determine parasitemia, percent phagocytosis and phagocytic index.
Statistical analysis of the data was carried out using GraphPad Prism Software (GraphPad software, Inc., CA, USA). The one way ANOVA was used to analyze and compare the results at a 95% confidence level. Values of p<0.05 were considered significant. Results were expressed as mean + standard error of mean (SEM).
Acute toxicity test
Behavioral signs of toxicity observed in mice given 3000 mg/kg of N. nucifera stamen extract include; paw licking, salivation, stretching and reduce activity. There was however no mortality at all doses used. Therefore, 500, 1000, and 2000 mg/kg were suitable doses for using in this study.
Malaria-associated phagocytic suppression during PbANKA infection
There was a progressive increase in level of parasitemia as the days progressed from day 2 to 12 in the PbANKA infected mice (Figure 1A), and survival time was 12 days (Figure 1B). Interestingly, determination of phagocytosis showed a progressive decrease in the response to the presence of the parasites, which reached significant values on 4 after infection (Figure 1C). Moreover, decreasing of phagocytic index was also observed in the correlation with decreasing of phagocytosis (Figure 1D).
Figure 1: Malaria-associated phagocytic suppression during Plasmodium berghei ANKA infection. ICR mice (5 mice of each) were intra-peritoneally infected with 1×107 infected erythrocytes of PbANKA. (A) Parasitemia (B) percent phagocytosis and (C) phagocytic index were daily monitored. (D) Percentage survival of infected mice was also observed. Results were expressed as mean± SEM.
Antimalarial activity of N. nucifera stamen extract against PbANKA infected mice
During early malaria infection, N. nucifera stamen extract produced a dose-dependent antimalarial effect against PbANKA. The extract caused a significant (p<0.05) antimalarial when compared to the untreated control, especially at dose of 2,000 mg/kg showed the highest activity (Figure 2). The standard drug, CQ caused chemosuppression, which was similar to those of the extract treated groups.
Figure 2: Antimalarial activity of Nelumbo nucifera stamen extract against Plasmodium berghei ANKA. ICR mice (5 mice of each) were intraperitoneally infected with 1×107 infected erythrocytes of PbANKA, and given orally 500, 1,000 and 2,000 mg/kg of the extracts twice a day for 4-consecutive days. Parasitemia was subsequently measured. The results were expressed as mean± SEM. UN; untreated, CQ; 5 mg/kg of chloroquine. * p<0.05 and ** p<0.01 compared to untreated control.
Anti-phagocytic suppression of N. nucifera stamen extract during PbANKA infection
As showed in Figure 3, significant (p<0.01) suppression of phagocytosis was observed in untreated group. Interestingly, N. nucifera stamen extract exerted dose-dependent anti-phagocytic suppression in the extract treated groups, especially at a dose of 2,000 mg/kg showed the highest activity. In addition, no effect on phagocytosis was observed in normal mice treated with this extract and CQ treated group.
Figure 3: Anti-phagocytic suppression of Nelumbo nucifera stamen extract during Plasmodium berghei ANKA infection in mice. ICR mice (5 mice of each) were intraperitoneally infected with 1×107 infected erythrocytes of PbANKA, and given orally 500, 1,000 and 2000 mg/kg of the extracts twice a day for 4-consecutive days. Percent phagocytosis was then investigated. The results were expressed as mean± SEM. N; normal, N+E; normal mice treated with 2,000 mg/kg of extract, UN; untreated, CQ; 5 mg/kg of chloroquine. *p<0.05 compared to normal control.
There was a progressive increase in level of parasitemia at the days progressed from day 2 to 12 in the PbANKA infected mice (Figure 1A), and survival time was 12 days (Figure 1B). This is in line with the view that parasitemia increases progressively after inoculation or infection until the point of death in the absence of suitable treatment. Interestingly, determination of phagocytosis showed a progressive decrease in the response to the presence of the parasites, which reached significant values on 4 after infection (Figure 1C). Moreover, decreasing of phagocytic index was also observed in the correlation with decreasing of phagocytosis (Figure 1D). This could be due in part to the fact that during malaria infection, infected erythrocytes and hemozoin (malarial pigment) non-enzymatically generated large amounts of hydroxyl fatty acids that inhibit monocyte function followed by decreasing phagocytosis . Phagocytosis of hemozoin or hemozoin-containing trophozoites alters functionality of monocytes and macrophages. Monocyte ability to perform oxidative burst is compromised, bacterial killing abolished, antigen presentation altered and ability to differentiate to functional dendritic cells disturbed. Moreover, hemozoin-laden monocytes produce increased amounts of peroxidation products of polyunsaturated fatty acids and stimulate generation of several cytokines, such as TNF, IL-1beta, MIP-1alpha and MIP-1beta [28,29]. These appears to be causally related to decreasing phagocytosis and phagocytic index in our finding during PbANKA infection in mice.
During early malaria infection, the extract of N. nucifera stamens produced a dose-dependent antimalarial effect against PbANKA. The extract caused a significant (p<0.05) antimalarial when compared to the untreated control, especially at dose of 2,000 mg/kg showed the highest activity (Figure 2). The standard drug, CQ caused chemosuppression, which was similar to those of the extract treated groups. It has been reported the antioxidant potential was related to antimalarial activity in several plant extracts. Moreover, flavonoids, quercetin and kaempferol have been reported to have potent antimalarial activity against P. berghei infected mice [19,30,31]. Hence, these compounds in N. nucifera stamen extract, and its potent antioxidant activity might play a central role to inhibit PbANKA growth in vivo. Moreover, oxidative damage in order to inhibit malaria parasite of artemisinin has also been described , and might related to antimalarial activity of N. nucifera stamen extract. However, the modes of action and other mechanisms should be searched for.
As showed in Figure 3, significant (p<0.01) suppression of phagocytosis was observed in untreated group. Interestingly, N. nucifera stamen extract exerted dose-dependent anti-phagocytic suppression in the extract treated groups, especially at a dose of 2,000 mg/kg showed the highest activity. Several studies have been reported the activity of N. nucifera extract to activate innate immune response and phagocytic activity. Knowledge of properties of flavonols and polyphenolic compounds in order to protect white blood cells and macrophage from oxidative stress and activate phagocytic activity have been described . Moreover, protective effect of flavonoid-rich extract on macrophage from oxidative stress-induced apoptosis has also been reported . Hence, significant antioxidant potential and flavonoidrich stamen extract might be properties on increasing of phagocytosis during malaria infection.
It is interesting to note that N. nucifera stamen extract was found the antimalarial and anti-phagocytic suppression against P. berghei infected mice. Although the bioactive components and mechanism are yet to be identified, the results of this study provide the basis for further studies.
We thank some students of Faculty of Medical Technology, Western University for excellent assistance with animal experimentation. The authors also acknowledge Assoc. Prof. Dr. Somdet Srichairatanakool and Dr. Chairat Uthaipibull for their kindly support excellent discussion. We also thank Dr. Saowanee Buatone, Rangsit Science Centre for Education.
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