Article Type: Research Article Article Citation: Georgewill Udeme Owunari, Ezerioha Chidi Emmanuel,
and Adikwu Elias. (2020). ANTIPLASMODIAL
ACTIVITY OF KETOTIFEN-ARTEMETHER-LUMEFANTRINE ON PLASMODIUM BERGHEI INFECTED
MICE. International Journal of Research -GRANTHAALAYAH, 8(11), 251-258. https://doi.org/10.29121/granthaalayah.v8.i11.2020.2439 Received Date: 10 October 2020 Accepted Date: 30 November 2020
Keywords: Ketotifen Artemether/Lumefantrine Antiplasmodium Mice Introduction: The development of new antimalarial drugs is time-consuming and costly, thus repurposing of drugs with initial indications for possible antimalarial indication is imperative. This study assessed the antiplasmodial effect of ketotifen (KT) in combination with artemether/lumefantrine (A/L) in Plasmodium bergei infected mice. Materials and Methods: Adult mice (25-30g) were parasitized with Plasmodium berghei, grouped and treated per oral (p.o) with KT (0.1mg/kg), A/L (2.3/13.7mg/kg) and KT/A/L daily in curative, suppressive and prophylactic studies. The negative control (NC) and the positive control (PC) were treated daily p.o with normal saline (0.2mL) and chloroquine (CQ) (10mg/kg) for 4 days respectively. After treatment, blood samples were collected and assessed for percentage parasitemia level, hematological and lipid parameters. Results: The curative, suppressive and prophylactic studies showed significant decreases in percentage parasitemia levels at KT (0.1mg/kg) (p<0.01), A/L (2.3/13.7 mg/kg) (p<0.001) and KT/A/L (p<0.0001) when compared to negative control. Significant increases in mean survival times occurred at KT (0.1 mg/kg) (p<0.01), A/L (2.3/13.7mg/kg) (p<0.001) and A/L/T (p<0.0001) when compared to negative control. Significant increases in packed cell volume, red blood cells, hemoglobin, high density lipoprotein cholesterol levels with significant decreases in total cholesterol, white blood cells, low density lipoprotein cholesterol and triglyceride levels at KT (28.6 mg/kg) (p<0.05), A/L (2.3/13.7mg/kg) (p<0.01) and KT/A/L (p<0.001) when compared to negative control. Conclusion: KT may be repurposed in combination with A/L for malaria treatment.
1. INTRODUCTIONMalaria
is among the ailments ravaging the human race and its
manifestations could be severe and life-threatening. Its major impact is most
experienced in developing countries [1]. In
2018, malaria infection was 228 million which accounted for over 405,000 deaths
mostly in developing nations. The impact of malaria and its consequences is
predominant in children below the age of five and pregnant women which poses
significant health challenges [2]. Efforts
to eradicate malaria have experienced setbacks due to challenges including the
emergence of insecticide-resistant mosquitoes, drug-resistant parasites and
lack of drugs or vaccines to block parasite transmission [3]. Drug resistance has remarkably increased causing
changes in malaria treatment from chloroquine (CQ) to artemisinin-based
combination therapies (ACTs), which are currently preferred for malaria
treatment. Despite the antimalarial impact of
ACTs, setbacks due to cost and emergence of resistant parasites herald the need
for newer antimalarial drugs with different modes of action and different
structural features [4]. One of
the strategies to discover new antimalarials is to reposition, or repurpose
drugs that are already used for other indications [5]. This approach, as compared to the “de novo” drug discovery process
has advantages such as reduced cost and short time of drug development [5]. Repurposed drugs have well-documented toxicity, pharmacology and drug-drug interaction profile. Drug
repurposing is based on the principle of polypharmacology;
a paradigm in drug discovery where a drug with multiple targets and off-target
effects may have multiple mechanisms of action [6]. The repositioning
of drugs with good safety profiles will gain quick approval for newer
indications using the same route of administration [7]. It is encouraging that drug regulatory associations in
Europe and USA have launched drug repurposing programs to identify new uses for
existing medications [8]. Ketotifen (KT), a
tricyclic benzocycloheptathiophene derivate, is broadly used for allergies, asthma, and inflammatory
disorders. It blocks H1 receptors, stabilizes mast cells, and
inhibits eosinophil accumulation and degranulation that results in the further
stabilization of the cell membrane [9]. It is well absorbed after oral
administration, with peak plasma drug concentrations within 2 to 4 h [10]. In a recent study both KT and its
metabolite norketotifen were shown to be active
against schizonts and liver-stage P. berghei parasites [11]. KT
and other antihistamines have been shown to be effective in reversing CQ
resistance in P. falciparum [12], [13] and P. yoelii nigeriensis [14]. It shows higher potency in blocking oocyst
formation in mice, which suggests that KT may act more effectively on mosquito
stages of fertilization. KT affects gamete production by decreasing P. falciparum gametocytes and greatly
reduced the numbers of exflagellation centers [3]. Study shows the possible role of KT in the
treatment of P. falciparum associated malaria in combination with CQ [15].This study, assessed the antiplasmodial
activity of KT in combination with artemether/ lumefantrine (A/L) in P. berghei
infected mice 2. MATERIALS AND METHODS2.1. EXPERIMENTAL ANIMALS AND MALARIA PARASITEWistar
mice weighing 25- 30g were used. The mice were bought from the animal house of
the Department of Pharmacology, University of Port-Harcourt, Nigeria. The mice
were kept in cages and allowed to acclimatize for 2 weeks before the study
began. The mice were fed with food and water ad libitum. P. berghei was obtained from the Malaria Research
Laboratory, Centre for Malaria Research and Phytomedicine, University of
Port-Harcourt, Nigeria. The directive (2010/63/EU) of the European Union Parliament and the
Council on the handling of laboratory animals for scientific purposes was used
for this study. 2.2. DRUGSKetotifen
(KT) (Sun Pharm Industries Ltd, India), artemether/lumefantrine (A/L) (IPCA
Laboratories Ltd), and Chloroquine (CQ) (Evans Medical Nigeria Plc) were used
for this study. A/L (2.3/13.7 mg/kg) [16], Ketotifen (0.1 mg/kg) [17] CQ
(10mg/kg) [18] were used. 2.3. PARASITE INOCULATIONBlood
samples from mice were first screened to ascertain that they were parasite
free. Stock inoculum of 1 x 107P.
berghei infected erythrocytes in 0.2 mL was
prepared by diluting portions of the blood infected with P. berghei with 0.9% normal saline. This
was inoculated into each mouse via intra-peritoneal route of administration. 2.4. CURATIVE TESTThe
method proposed by Ryley and Peters (1970) [19] was used for this study. Thirty mice (Groups
I-VI) were used. Groups 11-VI were inoculated with 1 x 107P. berghei parasitized
erythrocytes intraperitoneally (i.p). After 72 hours
(3 days), the mice were treated as follows. Group I (Non-parasitized) (Normal
control), and group 11 (Negative control) were treated with normal saline
(0.2mL) respectively. Group III (Positive control) was treated with CQ
(10mg/kg) whereas groups IV – V1 were treated with KT (0.1 mg/kg), A/L
(1.1/4.6mg/kg) and KT/A/L for 4 days respectively. On
day 5, tail blood samples were collected and thin
blood films were made on microscope slides. The films were fixed with methanol
and stained with 10% Giemsa stain for 30 minutes. The stained thin blood films
were viewed under oil immersion x100 magnification and the number of
parasitized red blood cells were counted against the total number of red blood
cells in a field and percentage parasitemia calculated using the formula shown
below. 2.5. SUPPRESSIVE TESTThis test
was conducted for four days as reported by Knight and Peters (1980) [20]. The mice were inoculated i.p with blood sample (0.2mL)
containing 1 x 107 P.berghei. Afterward, the mice were randomized into 5
groups of five mice. After 3 hours, the mice were treated. Group I (Negative
control) was treated daily with normal saline (0.2mL) whereas group II
(Positive control) was treated with CQ (10mg/kg) for 4 days. Groups III - V were treated daily with KT
(0.1 mg/kg), A/L (1.1/4.6mg/kg) and KT/A/L for 4 days
respectively. On day 5, blood samples were collected, films were prepared and
percentage parasitemia determined using the formula below 2.6. PROPHYLACTIC TESTThis was
evaluated according to Peters (1965) [21]. Twenty five mice
were randomized into 5 groups of five mice each. Group I (Negative control) and group II
(Positive control) were administered daily with normal saline (0.2mL) and CQ
(10mg/kg) respectively. Groups III – V were administered daily with KT (0.1
mg/kg), A/L (1.1/4.6mg/kg) and KT/A/L respectively. On
day 4, the mice were infected i.p. with 1 x 107P. berghei parasitized
erythrocytes and treatment continued for 4 days. Blood samples were collected
from the tail on day 5 and day 7 and percentage parasitemia determined using
the formula below %
Parasitemia = × 100 %
Inhibition = ×
100 2.7. DETERMINATION OF MEAN SURVIVAL
During
the study, the mice were observed for mortality which was determined as mean
(MST) in days using the formula below MST
= 2.8. EVALUATION OF HEMATOLOGICAL AND LIPID PARAMETERS
Blood
samples from the curative test were evaluated for Red blood cell (RBC), hemoglobin (HB), pack
cell volume (PCV), high density lipoprotein cholesterol (HDL-C), white blood
cell (WBC), triglyceride (TG), total cholesterol (CHOL) and high
density lipoprotein cholesterol (LDL-C) levels using an auto analyzer 2.9. DATA ANALYSIS
Data
was analyzed using GraphPad prism 6.0 statistical software. Data was presented
as Mean±SEM. Significant difference was considered
using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Significance was considered at p<0.05;
p<0.01 and p<0.001. 3. RESULTS3.1. CURATIVE TESTCurative
test showed significant decreases in percentage parasitemia at p<0.01 and
p<0.001 in mice treated with individual doses of KT and A/L respectively
when compared to negative control. However, most significant reduction in
percentage parasitemia level at p<0.0001 occurred in mice treated with
KT/A/L when compared to negative control. The observed decrease in percentage parasitamia in KT/A/L treated mice differ (p<0.05) when
compared to CQ-treated mice (Table 1). Percentage parasitemia inhibition were
62.5% and 73.7 % in rats treated with individual doses of KT and A/L with
observed significance at p<0.01 and p<0.001 respectively when compared to
negative control. On the other hand, 90.0 % parasitemia inhibition was observed
in KT/A/L treated mice which differ at p<0.0001 when compared to negative
control (Table 1). Treatment with KT, AL, KT/A/L significantly increased MST at
p<0.05, p<0.01, and p<0.001 respectively when compared to negative
control (Table 1). 3.2. SUPPRESSIVE TESTIn the
suppression test, individual doses of KT and A/L produced significant reductions
in percentage parasitemia levels at p<0.01 and p<0.001 respectively when
compared to negative control. On the hand, KT/A/L produced most significant
reduction in percentage parasitemia level at p<0.001 when compared to
negative control. Comparatively, reduction in percentage parasitemia produced
by A/L/KF differ (p<0.05) from CQ (Table 2). Treatment with KT, A/L and
KT/A/L produced 68.0%, 75.0 % and 91. 9% percentage parasitemia inhibitions
respectively (Table 2). MST was significantly increased in mice treated with KT
(p<0.05), A/L (p<0.01) and KT/A/L (p<0.0001) when compared to negative
control (Table 2). 3.3. PROPHYLACTIC TESTTreatment
with individual doses of KT and A/L produced significant time-dependent
reductions in percentage parasitemia levels on day 5 and 7 when compared to
negative control. However, treatment with KT/A/L produced most significant
time-dependent reductions in percentage parasitemia levels on day 5 and day 7
when compared to negative control (Table 3). On day 7, individual doses of KT
and A/L produced 84.4% and 89.4 % percentage parasitemia inhibition
respectively, KT/A/L produced 99.3 percentage parasitemia inhibition whereas CQ
(Positive control) produced 91.8 % percentage parasitemia inhibition (Table 3).
MST was significantly increased in mice treated with KT (p<0.05), A/L
(p<0.01) and KT/A/L (p<0.001) when compared to negative control (Table
2). 3.4. EFFECTS ON HEMATOLOGICAL AND LIPID PARAMETERSThe
negative control shows significant decreases in RBC, HB, PCV and HDL levels
with increases in WBC, TG, CHOL, LDL-C levels when compared to non-parasitized
rats (normal control). In contrast, RBC, HB, PCV were significantly increased
whereas WBC, TG, CHOL, LDL-C were significantly decreased at p<0.01 and
p<0.001 by KT and A/L respectively when compared to negative control (Tables
4 and 5). However, KT/A/L produced most significant increases in RBC, HB, PCV and
HDL-C levels with decreases in WBC, TG, CHOL, LDL-C levels (p<0.0001) when
compared to negative control (Tables 4 and 5). The observed effects produced by
KT/A/L on RBC, HB, PCV, HDL-C, WBC, TG, CHOL, LDL-C levels differ (p<0.05)
when compared to CQ (Tables 4 and 5).
Table 1: Curative
activity of ketotifen and its combination with artemether/ lumefantrine on Plasmodium berghei-infected
mice
NC: Negative Control; CQ: Chloroquine; KT:
Ketotifen; A/L: Artemether /Lumefantrine; KT/A/L:
Ketotifen/Artemether/Lumefantrine; MST: Mean Survival Time; n=5; Data expressed
as mean ± SEM, a p<0.001 when compared to NC; b
p<0.01 when compared to NC; c p<0.0001 when compared to NC; dp<0.05 when compared to CQ. Table 2:
Suppressive activity of Ketotifen and its combination with
artemether/lumefantrine on Plasmodium berghei-infected mice
NC: Negative Control; CQ: Chloroquine; KT:
Ketotifen; A/L: Artemether /Lumefantrine; KT/A/L: Ketotifen/Artemether/Lumefantrine;
MST: Mean Survival Time; n=5; Data expressed as mean ± SEM, a p<0.001
when compared to NC; b p<0.01 when compared to NC; c p<0.0001
when compared to NC; dp<0.05
when compared to CQ. Table 3:
Prophylactic activity of ketotifen and its combination with artemether/
lumefantrine on Plasmodium berghei-infected mice
NC: Negative
Control; CQ: Chloroquine; KT: Ketotifen; A/L: Artemether /Lumefantrine; KT/A/L:
Ketotifen/Artemether/Lumefantrine; MST: Mean Survival Time; n=5; Data expressed
as mean ± SEM, a p<0.001 when compared to NC; b
p<0.01 when compared to NC; c p<0.0001 when compared to NC; dp<0.05 when compared to CQ. Table 4: Effect
of ketotifen and its combination with artemether/lumefantrine on lipid profile
of Plasmodium berghei-infected
mice
MC: Normal control; NC: Negative control; CQ:
Chloroquine; KT: Ketotifen A/L: Artemether/lumefantrine, KT/A/L:
Ketotifen/Artemether/Lumefantrine; TG: Triglyceride; CHOL: Total cholesterol;
HDL-C: High density lipoprotein cholesterol; LDL-C: Low density lipoprotein
cholesterol; n=5; Values are expressed as M±SEM, a p<0.01 when
compared to NC; b p<0.05 when compared to NC; c p<0.001 when compared to NC; dp<0.01
when compared to CQ. Table 5: Effect of ketotifen and its combination with
artemether/lumefantrine on hematological parameters of Plasmodium berghei-infected mice
MC: Normal control; NC: Negative control; CQ:
Chloroquine; KT: Ketotifen, A/L: Artemether/lumefantrine, KT/A/L:
Ketotifen/Artemether/Lumefantrine; TG: Triglyceride; CHOL: Total cholesterol;
HDL-C: High density lipoprotein cholesterol; LDL-C: Low density lipoprotein
cholesterol; n=5; Values are expressed as M±SEM, , a p<0.01 when
compared to NC; b p<0.05 when compared to NC; c p<0.001 when compared to NC; dp<0.01
when compared to CQ. 6. DISCUSSIONDrug
development is a long and complex process. It is capital intensive with no
guarantee of success. In recent years, there was a significant decline in the
number of new drugs approved for clinical use.
This necessitates the repurposing of already approved drugs, for new
indications other than their initial indications. This strategy reduces costs
and research time considerably [22]. This study evaluated the possibility of
repurposing KT as an antimalarial drug in combination with A/L on P. berghei
infected mice. The rodent parasite; P. berghei has
been used for studying the activity of antimalarial drug candidates in mice [23].
Rodent models of antimalarial study have been validated through the
identification of several conventional antimalarial drugs including quinine and
more recently artemisinin derivatives [24]. The in vivo antiplasmodial
activity of KT/A/L was evaluated using curative, suppressive and suppression
test which are validated tests for the assessment of antimalarial drug
candidates. Percentage parasitemia inhibition and mean survival time were
calculated from the curative, suppression, and prophylatic
tests with reference to other studies [25], [26]. In
this study, antiplasmodial evaluation of KT/A/L showed
reductions in percentage parasitamia levels with
increased percentage parasitemia inhibition in the suppressive and curative
tests. In the prophylactic test, KT/A/L produced time-related reductions in
percentage parasitemia levels and increased percentage parasitemia inhibitions.
Malaria associated mortality is a challenge that is prevalent in malaria
endemic regions primarily developing nations. MST is experimentally used to
assess the ability of antimalarial candidate drugs to prevent or reduce mortality
in plasmodium parasitized rodents [27]. In this study, KT/A/L increased MST in the
suppressive, curative and prophylactic tests better
than individual doses of KT and A/L. Anemia is a common malaria complication
prevalent in children and pregnant women in malaria endemic regions [28]. P. berghei
infected mice suffer from anemia because of erythrocyte destruction, either by
parasite multiplication or by spleen reticuloendotelial
cell action as the presence of many abnormal erythrocytes stimulates the spleen
to produce many phagocytes [29]. The current study observed anemia in
untreated parasitized mice (Negative control) characterized by decreased RBC,
PCV, HB with increased WBC levels. However, KT/A/L produced reduction in anemia
characterized by increased RBC, PCV, HB and decreased WBC levels. KT/A/L
produced the best effects on the aforementioned biochemical
parameters than individual doses of KT and A/L. Emerging studies suggest that
routine laboratory measurement of lipids could be a good and reliable adjunct
in the early diagnosis of malaria especially in malaria endemic areas [30]. This study observed impaired lipid profile
characterized by elevated TG, CHOL, and LDL-C and decreased HDL levels in
negative control. This observation is consistent with altered lipid profile
reported in previous findings [31]. However, lipid levels were restored in parasitized rats
treated with KT/A/L. This study shows that KT may be repurposed in combination
with A/L for the treatment of malaria. The observation in this study can be
correlated with a study that reported improved antimalarial activity when KT
was co-administered with CQ [17]. The observation in this study can also be compared with
increased antimalarial activity when KT was co-administered with CQ and sulphadoxine/pyrimethamine [15]. The precise mechanism by which KT produced antiplasmodial effect has not been elucidated. However, as
an antihistamine, it blocks H1 receptors, stabilizes mast cells, and
inhibits eosinophil accumulation and degranulation which further stabilizes
cell membrane [9]. 7.
CONCLUSION
This study shows that KT
may be repurposed in combination with A/L for the treatment of malaria. SOURCES OF FUNDINGThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. CONFLICT OF INTERESTThe authors have declared that no competing interests exist. ACKNOWLEDGMENTNone. REFERENCES
[2]
WHO,
“World Health Organization, 2019. World Malaria Report,”
https://www.who.int/malaria/en/.
[3]
Eastman
RT, Pattaradilokrat S, Raj DK, Dixit S, Deng B, Miura K et al., A Class of Tricyclic
Compounds Blocking Malaria Parasite Oocyst Development and Transmission Antimicrob Agents
and Chem, 2013; 57;425–435
[4]
Penna-Coutinho
J, Aguiar AC, Krettli AU. Commercial drugs containing flavonoids are active in mice with
malaria and in vitro against chloroquine-resistant Plasmodium falciparum Mem
Inst Oswaldo Cruz, Rio de Janeiro, 113(12): e180279, 2018
[5]
Ashburn
T.T., Thor K.B. Drug repositioning: identifying and developing new uses for
existing drugs. Nat. Rev. Drug Discov. 2004;3(8):673–683
[6]
Car
B.D. In: Polypharmacology in Drug Discovery. Peters J.-U., editor. Wiley; 2012
[7]
Kola
I., Landis J. Can the pharmaceutical industry reduce attrition rates? Nat. Rev.
Drug Discov. 2004;3(8):711–715
[8]
Nowak-Sliwinskaa P, Scapozzaa L, Altaba
AR Drug repurposing in oncology: Compounds, pathways, phenotypes
and computational approaches for colorectal cancer BBA - Rev on Can 2019; 1871
434–454
[9]
Montazeri M, Rezaei K, Ebrahimzadeh MA, Sharif1M,
Sarvi1 S, Ahmadpour E, Rahimi MT, Moreira de Oliveira
EA, Lang KL, Drug Repositioning: Concept, Classification, Methodology, and
Importance in Rare/Orphans and Neglected Diseases Journal of Appl Pharm Sci
2018; 8 (08), 157-165 [10] Grahnen A, Lonnebo A, Beck O, Eckernas
SA, Dahlstrom B, Lindstrom B. 1992. Pharmacokinetics of ketotifen after oral
administration to healthy male subjects. Biopharm.
Drug Dispos. 13:255–262 [11] Milner E, Sousa J, Pybus B, Auschwitz J, Caridha D, Gardner S et al.,
Ketotifen is an antimalarial prodrug of norketotifen
with blood schizonticidal and liver-stage efficacy.
Eur. J. Drug Metab. Pharmacokinet. 2012 Mar;37(1):17-22 [12] Basco LK, Ringwald P, Le Bras J. 1991. Chloroquine-potentiating action of antihistaminics in Plasmodium falciparum in vitro. Ann.
Trop. Med. Parasitol. 191; 85:223–228. [13] Quan H, Tang LH. 2008. In vitro
potentiation of chloroquine activity in Plasmodium falciparum by ketotifen and
cyproheptadine. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za
Zhi. 26:338 –342. (In Chinese.) [14] Singh N, Puri SK. Interaction between chloroquine and diverse pharmacological agents
in chloroquine resistant Plasmodium yoelii nigeriensis. Acta Trop. 2000; 77:185–193. [15] Ibrahim AM, Elhag ER, Mustafa SE. Ketotifen in treatment of uncomplicated falciparum
malaria. Saudi Med J. 2000;21(3):257-65 [16] Sirima SB, Ogutu B, Lusingu
JPA, et al. Comparison of artesunate-mefloquine and artemether-lumefantrine
fixed-dose combinations for treatment of uncomplicated Plasmodium falciparum
malaria in children younger than 5 years in sub-Saharan Africa: a randomised, multicentre, phase 4
trial. Lancet Infect Dis. 2016;16(10):1123-1133. [17] You L, Ni B, Cao HM. Effects of
low dose of ketotifen and chloroquine combination on the infrastructure of
chloroquine resistant strain of Plasmodium yoelii. J Shanghai Univ.
2000;4(4):338-42. [18] Somsak V, Damkaew A, and Onrak
P Antimalarial Activity of Kaempferol and Its Combination with Chloroquine in
Plasmodium berghei Infection in Mice. Jour of Path 2018; 2018;1- 7
[19] Ryley, J.F. and Peters, W. The
antimalarial activity of some quinolone esters. Ann. Trop. Med. Parasitol. 1970; 84: 209-222. [20] Knight, D.J. and Peters, W. The
antimalarial action of N-Benzyl oxydihydrotriazines and the studies on its mode of
action. Ann of Trop Med and Par. 1980; 74: 393-404. [21] Peters, W. Rational methods in the
search for antimalarial drugs. Trans. R. Soc. Trop. Med. Hyg. 1967; 61: 400-410. [23] Thomas AM, Van Der Wel AM, Thomas AW, Janse CJ, Waters AP (1998).
Transfection systems for animal models of malaria. Parasitol.
Today, 14: 248-249. [24] David AF, Philip JR, Simon IC, Reto B, Solomon N (2004). Antimalarial drug discovery: Efficacy models for
compound screening. Nassture Rev., 3: 509-520 [25] Fidock DA, Rosenthal PJ, Croft SL,
Brun R, Nwaka S. Antimalarial drug discovery: efficacy models for compound
screening. Nat Rev Drug Discov. 2004;3(6):509–520. [26] Tarkang PA, Appiah-Opong R, Ofori MF, Ayong LS, Nyarko AK. Application of multi-target phytotherapeutic concept in malaria drug discovery: a
systems biology approach in biomarker identification. Biomark
Res. 2016;4(1):25. [27] Olanlokun JO, Babarinde CO and Olorunsogo
O. O. Toxicity of Anchomanes difformis,
An Antimalarial Herb in Murine Models Eur Jour of Med Plants 2017; 20(3): 1-13 [28] Saxena R, Bhatia A, Midha K, Debnath M, Kaur P. Malaria: A Cause of Anemia and Its Effect on
Pregnancy. World J Anemia. 2017;1(2):51-62. [29] Chinchilla M, Guerrero O, Abarca G, Barrios M, Castro O. An in vivo model to study the anti-malaria
capacity of plant extracts. Rev Biol Trop. 1998;46:1–7 [30] Sirak S, Fola AA, Worku
L, Biadgo B. Malaria parasitemia and its association
with lipid and hematological parameters among malaria-infected patients
attending at Metema Hospital, Northwest Ethiopia.
Path and Lab Med Intern. 2016;8:43-50 [31] Adekunle A.S., Adekunle O.C., Egbewale B.E Serum status of selected biochemical parameters in malaria: An
animal model. Biomed Res 2007; 18 (2): 109-113
This work is licensed under a: Creative Commons Attribution 4.0 International License © Granthaalayah 2014-2020. All Rights Reserved. |