(Mus musculus L.) Infected by Plasmodium berghei ANKA
Prawesty D. Utami1*, M. Taufan Wiryakusuma2, Nugroho Y. Abriyanto3, Anindya K. Winahyu4,Azarine Neira Avisha5
1Departement of Parasitology, Faculty of Medicine, Hang Tuah University, Surabaya, Indonesia
2,3,4,5Faculty of Medicine, Hang Tuah University, Surabaya, Indonesia
*Corresponding Author: [email protected]
Abstract
Malaria causes rupture of erythrocytes, thereby decreasing the amount of hemoglobin. The effect of malaria on the picture of leukocyte count remains unclear. Malaria disease management is also limited due to resistance. Curcuma (Curcuma xanthorrhiza Roxb.) which is known to contain curcumin and flavonoids that have antiplasmodium, antiinflammation and antioxidant effects. This research want to preceive the effect of curcuma extract on leukocyte and haemoglobin level on male BALB/C mice (Mus musculus L.) infected by Plasmodium berghei ANKA. This research was conducted experimentally post-test only control group on five groups of mice. One group was normal while four other groups were inoculated with Plasmodium berghei ANKA. One out ouf 4 inoculated group was given aquadest and three groups were treated with curcuma extract at 150 mg/KgBW, 100 mg/KgBW, and 50 mg/KgBW dose for four days. On the fifth day, a blood test was performed to determine the number of leukocytes and haemoglobin. The statistical test showed p = 0,001 (significant) which proved hypothesis that giving curcuma extract influenced the number of leukocytes and hemoglobin on male BALB / c inoculated with Plasmodium berghei ANKA. Group with extract dose 150 mg/KgBW showed the most effective decrease in parasitemia levels and significantly lower leukocyte and haemoglobin levels. Curcuma extract (Curcuma xanthorrhiza Roxb.) may affect the number of leukocytes and haemoglobin of male BALB/C mice (Mus musculus L.) infected with Plasmodium berghei ANKA.
Keywords: Malaria, curcuma (Curcuma xanthorrhiza Roxb.), Leukocyte, haemoglobin, Plasmodium berghei ANKA.
INTRODUCTION
Malaria is a global health problem especially in tropical and subtropical countries, which is caused by the Plasmodium parasite and transmitted by Anopheles mosquito(Paniker & Ghosh 2013;
UNICEF & World Health Organization 2015).
Laboratory findings of malaria infection that are often found are changes in blood parameters especially leukocytes and erythrocytes. The pathophysiologic mechanism for malaria induced anaemia involve destruction of infected erythrocyte, liberation of parasite and erythrocyte materials into circulation and the host reactions to these events (Ekaidem & Akpan 2016). Severe anemia and tissues damage may occur in the phase of hyperparasitemia or excessive parasite growth (Wahlgren & Perlmann 2005; The Ministry of Health Republic of Indonesia 2015; UNICEF &
World Health Organization 2015).White blood cell
(WBC) counts during malaria are generally characterized as being low to normal, a phenomenon that is widely thought to reflect localization of leukocytes away from the peripheral circulation and to the spleen and other marginal pools, rather than actual depletion or stasis.
Leukocytosis is typically may be associated with concurrent infections and/or poor prognosis (McKenzie et al. 2005; Gansane et al. 2013). Some studies illustrate that malaria toxin glycosyl phosphatidylinositol play directly on immunological response by activation monocytes and macrophages and trigger the release of pro- inflammatory cytokines (Tangpukdee et al. 2008).
Although vector control and use of chemoprophylates agents, are common preventive /curative measures against malaria infection;
attention is also being focused on theuse of effective and safe natural remedies as alternative strategies (Fairhurst & Dondorp 2016; WHO 2016). Indonesia has various medicinal plants that
have showed some therapeutic effects, such as curcuma (Curcuma xanthorriza Roxb.). It contains many chemical substances such as flavonoids and curcumin. In vitro experiments of Curcuma xanthorriza Roxb. showed some potentials of curcumin and flavonoid as antioxidant, antimalarial, and anti-inflammatory. Moreover, they can also be used as antibiotic and anti-fungal (Rosidi et al. 2014; Jain, Sood, & Gowthamarajan 2013; Rosidi et al. 2016).
As an antioxidant and anti-inflammation, flavonoids show the ability to bind free radicals and to prevent the formation of free radicals. As an antimicrobial, flavonoid compounds can also inhibit the formation of proteins, so as to inhibit microbial growth (Rosidi et al. 2014; Jain, Sood, &
Gowthamarajan 2013; Eva 2015; Rosidi et al.
2016). At the molecular level, curcumin increases intracellular ROS production and inhibits the plasmodial activity of histone aceyltransferase, the enzyme needed for chromatin remodeling and transcriptional activation of malaria parasites (Haddad, Sauvain, & Deharo 2011). Although it can increase intracellular ROS, curcumin it self also has a low antioxidant effect (Jain, Sood, &
Gowthamarajan 2013). This study therefore is aimed at investigating the impact of Curcuma xanthorriza Roxb extract on some haematological indices (leucocytes and haemoglobin) of Plasmodium berghei ANKAinfected mice model.
MATERIALS AND METHODS
This true laboratory experimental research used apost test only randomized control group design. The population of this study were male mice (Mus musculus L.) BALB / c, which were obtained and treated at the Institute of Tropical Disease Laboratory, Airlangga University.
Plasmodium berghei ANKA (PbA) strain was obtained from the Institute of Tropical Disease Airlangga University, andwas inoculated in experimental mice. The ethical research approval was obtained from the Research Ethics Commission of Hang Tuah University, Surabaya (No. 6 / HC / EC / KEPUHT / 2017).
Animal Samples
The samples on this research were male mice (Mus musculus L.) BALB / c aged 7-9 weeks.Adult mice (aged ± 50 days) have better body condition and immunological response compared to mice with aged ˂ 7 weeks (49 days) or > 9 weeks (63 days). The average weight of mice aged 7-9 weeks
ranged from 18 - 25 grams. Male mice have more stable biological condition compared to female mice whose biological condition is affected by the estrous cycle. Adaptation to the environment was carried out for 7 days in all groups of mice and maintained its condition, food and drink. Every six mice were placed in a plastic tub sized 30 x 20 cm that were given chaff mats. All mice were allocated to metabolic cages and kept in an air-conditioned environment (22– 24 °C) with 12-h alternating periods of light/dark and free access to food and fresh water (Nurul 2010; Darmawan 2014).
This study used 5 groups of mice. The sample size was calculated based on the Federer WT formula (k-1) (n-1) ≥ 15(Federer 1966). yieldedfive mice for each group, with a total of 25 mice. (1) The first group (G1) was not infected with PbA and was given Aquadest for four days (Day 1 – Day 4);
(2) The second group (G2)was infected with PbA and was given Aquadest for four days (Day 1 – Day 4) (3) The third group (G3) was infected with PbA and obtained 150 mg / kg / day extract of curcuma rhizome (Curcuma xanthorriza Roxb.) orally for 4 consecutive days (Day 1 – Day 4); (4) The fourth group (G4) was infected with PbA and obtained 100 mg / kgBW / day extract of curcuma rhizome (Curcuma xanthorriza Roxb.) orally for 4 consecutive days (Day 1 – Day 4); (5) The fifth group (G5) wasinfected with PbA and obtained 50 mg / kgBW / day extract of curcuma rhizome (Curcuma xanthorriza Roxb.)orally for four consecutive days (Day 1 – Day 4).
Inoculation of P.berghei ANKA
Plasmodium berghei ANKA was one of malaria parasite in rodents, and it had similar biological nature and sensitivity to drugs with malaria species that infected humans, especially P.
falciparum. The blood stage forms of both parasites were stored in liquid nitrogen after in vivo passages in BALB/C mice. When the percentage of parasitemia from blood donor mice reached ≥20%, the bloodwas diluted with pbs, then 200 μl of diluted blood was injected intraperitoneally to all groups of mice. The infected mice were marked and put in a cage according to the treatment group.
Naive mice were infected intraperitoneally (i.p.) with 106 infected red blood cells (iRBC) and their parasitemia level were monitored daily (Blanco et al. 2008; Souza, Hafalla, & Riley 2009;
Widyawaruyanti et al. 2014).
Curcuma xanthorriza Roxb. Extract
Curcuma xanthorriza Roxb. was obtained from the traditional market in Surabaya. Extraction process and identification of the plant taxonomy was done by the Faculty of Pharmacy, Widya Mandala University Surabaya (No. 060/ LJ- FF/I/2017). Extraction process was started by washing all fresh rhizome of Curcuma xanthorriza Roxb, then cut into small pieces, and dried. After drying, the pieces were inserted into the milling with a large hole to filter 0.75 mm as preparation for maceration. A total of 5 kg of fresh rhizome, macerated with 1 L of 96% ethanol to get 500 mg extract. Immersion was done for 24 hours while stirring occasionally. The extract produced was filtered with a Buchner funnel to obtain the filtrate.
The residue is re-marred in the same way four times. The extract obtained was then concentrated with rotary evaporators until the viscous extract was obtained. The extract was left at room temperature or heated in a low temperature heater to allow the remainder of ethanol to vapor out and it was extracted into a dry paste (Hutomo &
Winarno, 2005). Preparation of Curcuma xanthorriza Roxb.solution was done by mixing rhizome extract Curcuma xanthorriza Roxb. with aquadest and CMC-Na solution as a solvent.
The solution was then given to the treatment group every day orally using intragastric sonde.In this study,the dosagesof Curcuma xanthorriza Roxb. were150 mg / kgBW of mice / day, 100 mg / kgBWof mice / day, and 50 mg / kgBWof mice / day. The dose was determined based on the similar study that used 100 mg / kgBW dose of curcuma extract (Widyawaruyanti et al. 2014).
Anesthesia
Animals were anesthetized with ketamine (83mg/g) and xylazine(13mg/g) administered intraperitoneally for all of the blood drawingprocedures including tail blood samples.
Parasitemia Examination
Parasitemia was determined daily in all infected groups throughout the study period.
Blood smears were prepared from a drop of blood of the animal’s tail on slides and stained with Giemsa. The slides were allowed to dry and then fixed with methanol. After fixing, the slides were allowed to dry and then stained with 10% Giemsa in methanol for 30 minutes. The slides were then rinsed with water and then allowed to dry. The percentage of parasitemia was described as the
number of parasitized red blood cells (pRBC) in 1,000 erythrocytes on at least ten random microscopic fields. The percentage of parasitemia was calculated using the following formula(Adetutu et al. 2016) :
Blood Examination
Blood tests to determine the number of leukocytes and hemoglobin performed on day 5 after the mice terminate. Blood sampling was done on the heart of mice, following these protocol:
animal positioned on its back, the abdomen wasopened and the heart visualized through the membranous part of thediaphragm. A 21 gauge needle and 3cc syringe were used to obtain 0.5 ml of blood from the left ventricle. The blood was then immediately transferred to 20ml capillary pipettes containing the appropriate anticoagulants for cell counting. EDTA anti-coagulated blood samples were used to obtaina complete blood count with a Mindray Auto Hematology Analyzer.
Statistical Analysis
The one way ANOVA test was used to analyze and compare the results at a 95% confidence level if normality and homogenity were obtained. If the data were not met the parametric criteria, the data were then analyzedusing Kruskal Wallis test, followed by the Mann-Whitney U test at = 0,05.
RESULTS
Acute toxicity test
The experimental mice ingested with extract of rhizome of Curcuma xanthorriza Roxb.
in all doses did not show any indication of gross physical or behavioral changes, such as hair erection, reduction in feeding and motor activities, weight loss, lacrimation, diarrhea, depression or abnormal secretions within 24 hour of monitoring period. No fatalities occurred within the four days observation period.
Level Parasitemia
Normality test results indicate that the normality requirement is not fulfilled because p <
0,05. This study was using non parametric analysis number of infected erythrocytes X 100
1000 erythrocytes
Kruskal Wallis to analyze the difference of % parasitemia between the groups.
Figure1. Changes Level Parasitemiaof Plasmodium berghei ANKAInfected & Non Infected Mice With Rhizome Extract Of Curcuma xanthorriza Roxb
G1 : not infected with PbA and was given Aquadest for four days
G2 : infected with PbA and was given Aquadest for four days (Day 1 – Day 4)
G3 :infected with PbA and obtained 150 mg / kg / day extract of curcuma rhizome (Curcuma xanthorriza) Roxb.)orally for 4 consecutive days (Day 1 – Day 4)
G4 : infected with PbA and obtained 100 mg / kgBW / day extract of curcuma rhizome (Curcuma xanthorriza Roxb.) orally for 4 consecutive days (Day 1 – Day 4)
G5 : infected with PbA and obtained 50 mg / kgBW / day extract of curcuma rhizome (Curcuma xanthorriza Roxb.)orally for four consecutive days (Day 1 – Day 4).
Level of parasitemia in D0 showed that there was no significant difference (p > 0,05), G1 did not show parasite growth because the mice in this group were not infected with PbA and got aquadest. Mean value of % parasitemia on D1 – D4 between groups of this research showed that G2 (infected with PbA and was given Aquadest for four days) has the highest % parasitemia and G3 (infected with PbA with 150 mg/KgBW extract curcuma) has th e lowest % parasitemia.
Leucocyte Count
Normality test is not fulfilled because p >
0,05, we used a nonparametric analysis Kruskal Wallis to analyze the difference of leucocyte count between the groups. Haematological changes associated withadministration of rhizome extract of Curcuma xanthorriza Roxb to Plasmodium berghei ANKAinfected mice were investigated.
The effects rhizome of Curcuma xanthorriza Roxbextractto infected and uninfected mice on leucocyte count are shown in Table 1.
Table 1. Changes In Leucocyte Count of Plasmodium berghei ANKA Infected & Non Infected Mice With Rhizome Extract Of Curcuma
xanthorriza Roxb
Group Mean (/µL) Std.
Deviation
G 1 4620 931
G 2 16980 1881
G 3 21280 2705
G 4 28600 2026
G 5 30700 2328
p = 0,001, p <α (α = 0,05)
Descriptive and statistical analysis above shows that there are significant differences in the groups. The lowest leukocyte count was found in G1 (4620) and the highest leukocyte count was in G5 (30700). Based on Mann whitney post hoc analysis showed that the number of leukocytes
between groups ( G1 with G2 – G5; G2 with G4 – G5; G3 with G4 – G5) was significantly different (p <0.05), but there were no significant differences between groups G2 with G3 and G4 with G5 (p>
0,05).Several treatment groups were found to have significant differences in leukocyte counts among other treatment groups. This may be due to differences in the administration of extracts with different doses and containing different levels of bioactive components as well.
Hemoglobin Level
The data of hemoglobin level shows normal distribution, so the analysis used is non parametric analysis, Kruskal Wallis. Haematological changes associated with administration of rhizome extract of Curcuma xanthorriza Roxb to Plasmodium berghei ANKA infected mice were investigated.
The effects rhizome of Curcuma xanthorriza Roxb extract to infected and uninfected mice on hemoglobin level are shown in Table 2 :
Table 2. Changes In Hemoglobin Level of Plasmodium berghei ANKAInfected & Non Infected Mice With Rhizome Extract Of Curcuma
xanthorriza Roxb
Group Mean (g/dL) Std. Deviation
G 1 11,51 0,159
G 2 11,36 0,204
G 3 11,28 1,268
G 4 8,80 0,803
G 5 10,08 0,120
p = 0,003 , p <α (α = 0,05)
Descriptive analysis and statistics in the table above shows the difference in the hemoglobin level. The lowest hemoglobin level was found in G4group (8,80) and the highest hemoglobin levelwas in G1 group (11,51). Based on Mann whitney post hoc analysis showed that the number of hemoglobin level between groups ( G1 with G2 – G5; G2 with G4 –G5) was significantly different (p <0.05), but there were no significant differences between groups G2 with G3; G3 with G4- G5; and G4 with G5 (p> 0,05). Several treatment groups were found to have significant differences in hemoglobin level among other treatment groups.
This may be due to differences in extract doses and containing different levels of bioactive components as well.the other cause of the difference is also due
to the development of malarial parasites in erythrocytes.
DISCUSSION
High level parasitemia infections are often associated with malaria severity which triggers a defense mechanism that inhibits parasite multiplication and an important factor in the survival of the host. The chemical content of curcuma (Curcuma xanthorrhiza Roxb) includes flavonoids and curcumin has an anti-tumor, antioxidant, and antiplasmodium roles. Curcumin has two roles both anti oxidant and pro oxidant activities. Curcuma has anti-malarial activity through inhibition of the parasitic hystone acetyltransferase enzyme which plays a role in the transcription process and also through increased production of ROS in parasites. ROS can increase the expression of CD 36 on the surface of monocytes / macrophages, which facilitates the process of nonopsonic phagocytosis of infected erythrocytes. In addition to the antimalarial effects of curcuma, this plant also has the effect of regulating the immune response (immunomodulators) in various immune cells (Mimche, Taramelli, & Vivas 2011; Jain, Sood, &
Gowthamarajan 2013)
Based on the results of statistical analysis on the number of leukocytes it can be seen that G3 (mice group given a dose of curcuma extract 150 mg / KgBW) showed a lower leukocyte count than G4 (mice group given 100 mg / KgBW) and G5 (group of mice given a dose of curcuma extract of 50 mg / KgBB). Leukocytes count in the G3 (mice group given a dose of curcuma extract 150 mg / KgBW) did not show a significant difference in the G2 (the group of mice inoculated with P. berghei and received aquadest). The negative control group had significantly lowest leukocyte counts compared to other groups infected with Plasmodium. This result caused by existence of erythrocytes containing Plasmodium triggers the immune system's initial response to eliminate Plasmodium, which is played by leucocyte cell such as macrophages, neutrophils and lymphocyte.
Descriptive analysis showed that the positive control group (G2) had a lower leukocyte count than all treatment groups that received curcuma extract (G3, G4 and G5), although statistically the number of leukocytes between G2 and G3 was not significantly different. Some treatment groups on this study were found to have significant differences in leukocyte counts among other treatment groups. This might occur because of
differences in the administration of extracts with different doses and containing different levels of bioactive components.
The groups that received curcuma extracts were found to experience a drastic increase in leukocyte counts probably due to ongoing parasite growth which triggered the work of phagocytic cells and curcumin activity was effective against increased leukocyte counts (Reddy et al. 2005;
Jain, Sood, & Gowthamarajan 2013). Bioactive components contained in herbal medicines such as curcuma can activate the RNA and protein synthesis pathways that stimulate proliferation of leukocyte cells (neutrophils, eosinophils, monocytes, and lymphocytes) (Reddy et al. 2005;
Jain, Sood, & Gowthamarajan 2013). The increase in the number of leukocytes in the group that received the extract was accompanied by a decrease in the level of parasitemia. The lowest reduction in parasitemia was found in the group who received a dose of 150 mg / KgBW (G3) compared to the group who received other doses.
In this study it can be concluded that administration of curcuma extract will increase the number of leukocytes associated with activity to phagocyte infected erythrocytes. In the positive control group there was an increase in leukocytes caused by the malaria infection process, but the increase was lower than the group that received the extract. This is probably due to the absence of Curcuma extract, so that leukocyte cell proliferation did not occur as much as the group that received Curcuma extract.
Based on the results of hemoglobin examination showed that the group infected with PbA showed a decrease in hemoglobin compared to the negative control group that was not infected.
The decrease in hemoglobin count can be caused by two main factors such as increased erythrocyte destruction and decreased erythrocyte production.
Increased destruction of erythrocytes is caused by:
(1) rupture of pRBC (parasitized RBC); (2) phagocytosis of erythrocytes, either erythrocytes containing parasites or erythrocytes that have not been infected; (3) hypersplenism, contributes to the initial phase of acute malaria by eliminating parasites from erythrocytes without lysis or ingestion of erythrocytes with a shorter life span of the erythrocytes; and (4) autoimmune extravascular hemolysis (Perkins et al. 2011)
The decrease in hemoglobin level in the group that received the extract was greater than the positive control, although the difference was not significant between positive control (G2) and G3
and G5. This is because the active ingredients in Curcuma extract have antimalarial activity that can increase immune cell activity, especially phagocytic cells and the production of ROS, which can reduce the number of erythrocytes and hemoglobin (Reddy et al. 2005; Haddad, Sauvain,
& Deharo 2011). This allegation is supported by data on decreasing levels of parasitemia in the group that received curcuma extract. The group that received a dose of 150 mg / KgBW (G3) showed the lowest level of parasitemia compared to other groups and a hemoglobin level that was almost the same as positive control.
CONCLUSION
Based on the results of the statistical data analysis test and the interpretation of the results of the study, it can be concluded that:
1. Plasmodium berghei ANKA infection can reduce hemoglobin and increase leucocyte count in BALB / c male mice (Mus musculus L.).
2. The administration of ginger rhizome extract (Curcuma xanthorrhiza Roxb.) Has an effect on increasing leukocytes and decreasing hemoglobin in male BALB / c mice (Mus musculus L.) inoculated with Plasmodium berghei ANKA.
3. The administration of temulawak extract (Curcuma xanthorrhiza Roxb.) At a dose of 150 mg / KgBB is the most effective dose due to the increased inhibitory effect of parasitemia of BALB / c male mice (Mus musculus L.) infected with Plasmodium berghei ANKA.
Acknowledgement
This research can be held because of funding support from LPPM and the Faculty of Medicine, University of Hang Tuah
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