- Research note
- Open Access
Antimalarial combination therapies increase gastric ulcers through an imbalance of basic antioxidative-oxidative enzymes in male Wistar rats
BMC Research Notes volume 13, Article number: 230 (2020)
Antimalarials are globally used against plasmodium infections, however, information on the safety of new antimalarial combination therapies on the gastric mucosa is scarce. The aim of this study was to investigate the effects of Artesunate-Amodiaquine and Artemether-Lumefantrine on ulcer induction. Malondialdehyde (MDA), reduced glutathione (GSH) and major histological changes in male Wistar rats following ulcer induction using Indomethacin were investigated. Gastric ulcers were in four groups; Group I was administered Artesunate, group II received Artesunate-Amodiaquine, group III received Artemether-Lumefantrine, and group IV was a positive control (normal saline). Group V was the negative control consisting of healthy rats.
Antimalarial combination therapies were associated with a high gastric ulcer index than a single antimalarial agent, Artesunate. In addition, levels of MDA were significantly higher in the combination of therapies while levels of GSH were lower in comparison to Artesunate and the negative control. Microscopically, antimalarial combination therapies were associated with severe inflammation and tissue damage than Artesunate in the gastric mucosa showing that antimalarial combination therapies exert their toxic effects through oxidative stress mechanisms, and this leads to cellular damage. Findings in this study demonstrate a need to revisit information on the pharmacodynamics of major circulating antimalarial agents in developing countries.
Antimalarial single therapies (AMTs) are the aminoquinoline and artemisinin derivatives and artemisinin-based combination therapies and the development of resistance against them is a major public health threat especially in endemically infected countries with malaria parasites [1, 2]. The aminoquinoline derivatives (including quinine, chloroquine, amodiaquine, naphthoquinone, piperaquine, and mefloquine), are the prototype AMTs that have been used admist reports of varying adverse effects including toxicity concerns and increasing frequency for the development of drug resistance [3, 4]. These aminoquinolines are also aggressive to the gastric mucosa precipitating gastric ulceration [3, 5]. The derivatives of artemisinin (including artesunate, dihydroartemisinin, and artemether), are generally safer with limited side effects . Their efficacy against malaria parasites is however lower due to their lower half-life compared to that of aminoquinolines . The artemisinins have been shown to be safe on the gastric mucosal integrity [8, 9]. The artemisinin-based antimalarial combination therapies are recommended as the first-line treatment for uncomplicated malaria and this has been widely adopted . This therapy involves a combination of artemisinin and aminoquinoline derivatives into a single oral treatment. The combination of tehse two drugs ideally presents different safety challenges compared to the individual drugs comprised therein . In Africa, the use of antimalarial combinations such as Artemisinins is common for the management of malaria [12, 13], demonstrating their importance in developing countries.
The gastric mucosa is the inner protective lining of the gastric wall, made of an adherent mucus-bicarbonate-prostaglandin layer on a glandular epithelium . Its integrity and efficiency depend on the thickness of the mucus layer, continuity of glandular epithelium, adequate circulation and anti-oxidative activity of gastric tissue . The gastric mucosa is continuously exposed to endogenous and exogenous factors with protective or damaging effects [15, 16]. Drugs including antimalarials are among the exogenous substances known to affect the gastric mucosa through oxidative stress mechanisms [12, 13, 17]. Reactive oxygen species lead to lipid peroxidation through increased levels of malondialdehyde (MDA) and this disrupts the integrity of cell membranes leading to mucosal ulceration [18,19,20,21,22]. The glutathione system is an antioxidative system in the cell which prevents the accumulation of reactive oxygen species [23,24,25,26], thus hindrances to the functioning of the antioxidant system lead to increased tissue pathology. Lumefantrine, known to increase tissue oxidative stress [27, 28], has been incorporated with artemether into a combination therapy for the treatment of malaria [29,30,31,32]. However, information on the effects of this combination therapy on gastric ulcers remains to be established. The use of artesunate-amodiaquine has been associated with gastrointestinal complications like vomiting, diarrhea and abdominal pain [33, 34]. The objective of the study was to establish the gastric mucosal effects in Wistar rats of the common antimalarials used in developing countries.
This was an experimental study in which 25 adult male Wistar rats kept at Kampala International University Western Campus were assigned random numbers for experimental grouping as described previously . Animals were exposed to good husbandry practices through access to sufficient quality food and water adlibitum, exposure to daylight 12 h and sufficient spacing to minimise stress as previously described . Gastric ulcers were induced in only four experimental groups using indomethacin . Rats were fasted for about 24 h, and then orally treated with indomethacin at 40 mg/kg body weight p.o. These were then treated as follows; Artesunate 2 mg/kg i.m (n = 5) in the form Artesun® . This dosage was chosen since 2 mg/kg was very safe in subchronic studies of Artesunate ranging from 2 to 10 mg/kg [38, 39]. Artesunate-Amodiaquine per os at 4/10 mg/kg p.o in the form Winthrop® was used since this had previously been reported to have effects on gastric mucosa  while Artemether- Lumefantrine (2.3/27.4 mg/kg) from Combiart® was administered as a follow up on a previously used dosage of artemether-lumefantrine (2/12 mg/kg) on gastric ulcers . The positive control (with ulcers) was treated with normal saline at 1 ml/kg p.o. Furthermore, group five was the negative control (no ulcers) and this also received normal saline at 1 ml/kg p.o. All antimalarial agents and chemicals were procured from a licensed pharmacy in Ishaka town of Ishaka-Bushenyi municipality, Bushenyi, Uganda.
Determination of gastric ulcer index
The gastric ulcer index was determined 24 h after treatment, using standard methods . The rats were euthanized using thiopental sodium since this is ethically acceptable in experimental animals  and stomachs were harvested through a linea alba incision. The stomach was immediately opened along the greater curvature, mucosa cleaned of any debris with normal saline and pinned wide onto a wax board for ulcer counting and length taking. The ulcer counting was done using a magnifying glass (×10). Any black-red spot or line along the longitudinal axis, on the mucosa, was counted as an ulcer. The length of each counted ulcer was taken with a divider and ruler and recorded. The sum of the ulcer-lengths was recorded as the ulcer index for the particular stomach (one black/red mucosal spot was considered to be 0.5 mm). For accuracy, the average of two counting and length takings was considered for each stomach.
Determination of gastric mucosa reduced glutathione
Gastric mucosa reduced glutathione as a marker of anti-oxidative activity was determined by the method as described previously . 1 g of gastric mucosa scrapings was obtained, homogenized and then the supernatant was obtained after centrifugation at 3000 rpm, 40 °C for 10 min. The supernatant was reacted with 5, 5′-dithiol-bis-2 nitrobenzoic acid. Colorimetry was then carried out to obtain absorbance at 520 nm. The absorbance was compared with the standard curve to obtain the quantity of reduced glutathione, expressed as µm/g of mucosal tissue.
Determination of gastric mucosal malondialdehyde
Gastric mucosal malondialdehyde (MDA) was determined as a marker of lipid peroxidation, by the method as described previously . 1 g of gastric mucosal scrapings was obtained and suspended in 20 ml of butylated hydroxytoluene (0.5 M) to avoid oxidation. The sample was homogenized in Tris–HCl (20 mM) for 15 s, then centrifuged at 3000 rpm, 4 °C for 10 min to obtain a supernatant. The supernatant was then reacted with N-methyl-2-phenylindole at 45 °C. The absorbance of the solution was taken with a colorimeter (Colorimeter 254 Sherwood®) at 540 nm. The quantity of MDA in the weighed mucosa scrapings was obtained by comparison of the spectrophotometer reading with the standard curve. The MDA was expressed as µmoles/g of tissue.
The data was recorded and then entered in MS Excel version 10 for statistical analysis. Descriptive statistics were conducted to determine the homogeneity of the data on ulcer index, reduced glutathione and malondialdehyde concentrations. Data was subjected to One way ANOVA with Tukey post hoc test and information was expressed as mean ± SD and presented on graphs and a Table, while significance (P < 0.05) was reported with different superscripts (a, b, c).
Effects of Artesunate-amodiaquine treatment on gastric ulcer index, oxidative and antioxidant status
The study showed that the ulcer index was relatively the same in all experimental animals except in the positive control (P < 0.05). Ulcer index was higher in the Artesunate-amodiaquine than Artemether-lumefantrine groups although no significant differences were observed (Fig. 1a). Malondialdehyde (MDA) levels were highest in the combination groups (P > 0.05) with significantly high concentrations observed in the antimalarial combinations and Artesunate (Fig. 1b). In addition, MDA levels were lower in the negative control and no significant differences were observed with Artesunate (P > 0.05). Furthermore, levels of reduced glutathione were significantly the same (P > 0.05) in the Artesunate and the negative control (Fig. 1c). Significantly (P < 0.05) low concentrations were associated with the combined therapies of antimalarial agents with both Artesunate and the Negative control as shown in Table 1.
Gastric mucosa histopathological lesions
The macroscopic analysis showed erosion of the gastric mucosa (ulcer index) while microscopic analysis demonstrated mild inflammation in Artesunate with infiltration by inflammatory cells. Combination therapies of antimalarials i.e. Artesunate-amodiaquine and Artemether-lumefantrine were associated with diffuse vacuolations in the non-glandular stomach and acute inflammation in the glandular stomach showing that pathological lesions are widespread in the gastric mucosa. Furthermore, the positive control was associated with severe basophilic bodies and debris in the mucosa and no lesions were found in the negative control as shown in Fig. 2.
Antimalarials were able to induce gastric ulcers following indomethacin administration and this was in agreement with previous studies [14, 20, 44]. The effects of antimalarials such as amodiaquine, quinine and chloroquine on gastric ulcer has been previously reported [3, 45]. Antimalarial combination therapies (ACTs) of Artesunate-amodiaquine and Artemether-lumefantrine (Fig. 1a) showed higher gastric ulcer index than Artesunate alone. These findings raise major therapeutical challenges on the safety of ACTs due to their ability to damage the gastric mucosa and mucous layer [14, 15]. The study showed that ACTs were associated with high levels of malondialdehyde (MDA) and low concentrations of reduced glutathione (Fig. 1b, c). This showed that ACTs exert their toxic effects through an increase of oxidative stress in body tissues thus upsetting the delicate oxidative-antioxidant status responsibly for the maintenance of the integrity of cell membranes leading to mucosal ulceration [18,19,20,21,22]. Findings in this study on AMTs are contrary to single ACTs such as Artesunate which was observed to have protective effects on the gastric mucosa (Fig. 1). Findings in this study demonstrate the safety of Artesunate and this was in agreement with previous studies [8, 9, 40]. This offers a firm basis for their safety . In this study, the use of ACTs was found not to be safe, thus raising major global implications since ACTs are commonly used in the management of uncomplicated malaria .
Microscopically, Artesunate was found to be safer than ACTs (Fig. 2) showing that drugs including antimalarials are among the exogenous substances known to affect the gastric mucosa through oxidative stress mechanisms [12, 13, 17]. In addition, ACTs severe pro-oxidative stress properties stimulated vacuolations and severe inflammation. For example, Lumefantrine is a potent tissue pro-oxidant [27, 28] and it has been incorporated with Artemether into a combination therapy for the treatment of malaria [29,30,31,32]. The basic findings of this study demonstrate that ACTs might not be safe on the gastric mucosa. Furthermore, Artesunate-amodiaquine has been associated with gastrointestinal complications like vomiting, diarrhea and abdominal pain [33, 34], demonstrating that the Amodiaquine combination in the drug makes Artesunate lose its gastric protective effects (Figs. 1, 2). These findings support previous findings in which aminoquinoline derivatives including Amodiaquine have been used with reports of varying adverse effects including toxicity concerns and increasing frequency of development of drug resistance [3, 4] and are aggressive to the gastric mucosa precipitating gastric ulceration [3, 5].
The study investigated MDA-GSH axis thus to gain more information on the oxidative-antioxidant status, studies on more markers in oxidation, prostaglandins, disruption of local mucosal defense mechanisms, mucosal perfusion gastric mucus and bicarbonate secretion as well as inflammatory cytokines and molecular markers would help offer a more conclusive picture on antimalarial combination therapies.
Availability of data and materials
Data used in the study can be accessed at https://figshare.com/s/5bc742d01e28c4e23cc9.
Antimalarial combination therapies
Antimalarial single therapies
Milgram per kilogram
World Health Organisation
Blasco B, Leroy D, Fidock DA. Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic. Nat Med. 2017;23(8):917–28.
Eastman RT, Fidock DA. Artemisinin-based combination therapies: a vital tool in efforts to eliminate malaria. Nat Rev Microbiol. 2009;7(12):864–74.
Sha J, Li XL, Zhu MH, Yang AP, Chen L. Effect of nesfatin-1 on gastric acid secretion in rats. World Chin J Dig. 2013;21(16):1473–9.
AlKadi HO. Antimalarial drug toxicity: a review. Chemotherapy. 2007;53(6):385–91.
Ajeigbe KQ, Olaleye SB, Nwobodo EO. Effects of amodiaquine hydrochloride and artemisinin on indomethacin-induced lipid peroxidation in rats. Pakistan J Biol Sci. 2008;11(17):2154–8.
Price RN. Artemisinin drugs: novel antimalarial agents. Expert Opin Investig Drugs. 2000;9(8):1815–27.
Price R, van Vugt M, Phaipun L, Luxemburger C, Simpson J, McGready R, et al. Adverse effects in patients with acute falciparum malaria treated with artemisinin derivatives. Am J Trop Med Hyg. 1999;60(4):547–55.
Olaleye SB, Ajeigbe KO. Attenuation of experimental gastric ulceration by sulfadoxine-pyrimenthamine in Albino rats. J Med Sci. 2009;9(2):87–92.
Seo PJ, Kim N, Kim JH, Lee BH, Nam RH, Lee HS, et al. Comparison of indomethacin, diclofenac and aspirin-induced gastric damage according to age in rats. Gut Liver. 2012;6(2):210–7.
World Health Organisation. Guidelines for the treatment of malaria. 3rd ed. Geneva: World Health Organisation; 2015.
Hombhanje FW, Huang Q. Artemisinin-Naphthoquine combination (ARCO®): an overview of the progress. Pharmaceuticals. 2010;3(12):3581–93.
Bukirwa H, Yeka A, Kamya MR, Talisuna A, Banek K, Bakyaita N, et al. Artemisinin combination therapies for treatment of uncomplicated malaria in Uganda. PLoS Clin Trials. 2006;1(1):e7.
Kamya MR, Yeka A, Bukirwa H, Lugemwa M, Rwakimari JB, Staedke SG, et al. Artemether-Lumefantrine versus Dihydroartemisinin-Piperaquine for treatment of malaria: a randomized trial. PLoS Clin Trials. 2007;2(5):e20.
Wallace JL. Prostaglandins, NSAIDs, and gastric mucosal protection: why doesn’t the stomach digest itself? Physiol Rev. 2008;88(4):1547–65.
Silva MIG, Sousa FCF de. Gastric Ulcer Etiology. http://cdn.intechopen.com/pdfs-wm/22939.pdf. 2011.
Atuma C. Gastrointestinal mucosal protective mechanisms mudolatory effects of Heliobacter pyroli on the gastric mucus gel barrier and mucosal blood flow in vivo. 2000;60.
Attaran A, Barnes KI, Curtis C, D’Alessandro U, Fanello CI, Galinski MR, et al. WHO, the global fund, and medical malpractice in malaria treatment. Lancet. 2004;363(9404):237–40.
Kwiecien S, Jasnos K, Magierowski M, Sliwowski Z, Pajdo R, Brzozowski B, et al. Lipid peroxidation, reactive oxygen species and antioxidative factors in the pathogenesis of gastric mucosal lesions and mechanism of protection against oxidative stress—induced gastric injury. J Physiol Pharmacol. 2014;65(5):613–22.
El-Beltagi HS, Mohamed HI. Reactive oxygen species, lipid peroxidation and antioxidative defense mechanism. Not Bot Horti Agrobot Cluj-Napoca. 2013;41(1):44–57.
Bhattacharyya A, Chattopadhyay R, Mitra S, Crowe SE. Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol Rev. 2014;94(2):329–54.
Lushchak VI. Free radicals, reactive oxygen species, oxidative stress and its classification. Chem Biol Interact. 2014;224:164–75.
Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol. 2003;552(2):335–44.
Cnubben NH, Rietjens IMC, Wortelboer H, van Zanden J, van Bladeren PJ. The interplay of glutathione-related processes in antioxidant defense. Environ Toxicol Pharmacol. 2001;10(4):141–52.
Guilford F, Hope J. Deficient glutathione in the pathophysiology of Mycotoxin-Related illness. Toxins. 2014;6(2):608–23.
Leeuwenburgh C, Fiebig R, Chandwaney R, Ji LL. Aging and exercise training in skeletal muscle: responses of glutathione and antioxidant enzyme systems. Am J Physiol. 1994;267(2 Pt 2):R439–45.
Perricone C, De Carolis C, Perricone R. Glutathione: a key player in autoimmunity. Autoimmun Rev. 2009;8(8):697–701.
Abolaji A, Adesanoye O, Awogbindin I, Farombi E. Endocrine disruption and oxidative stress implications of artemether–lumefantrine combination therapy in the ovary and uterus of rats. Hum Exp Toxicol. 2016;35(11):1173–82.
Abolaji AO, Eteng MU, Omonua O, Adenrele Y. Influence of coadministration of artemether and lumefantrine on selected plasma biochemical and erythrocyte oxidative stress indices in female Wistar rats. Hum Exp Toxicol. 2013;32(2):206–15.
Denis MB, Tsuyuoka R, Lim P, Lindegardh N, Yi P, Top SN, et al. Efficacy of artemether? lumefantrine for the treatment of uncomplicated falciparum malaria in northwest Cambodia. Trop Med Int Heal. 2006;11(12):1800–7.
Färnert A, Ursing J, Tolfvenstam T, Rono J, Karlsson L, Sparrelid E, et al. Artemether-lumefantrine treatment failure despite adequate lumefantrine day 7 concentration in a traveller with Plasmodium falciparum malaria after returning from Tanzania. Malar J. 2012;11(1):176.
Leeuwenburgh C, Fiebig R, Chandwaney RJ, Ji LL. Aging and exercise training in skeletal muscle: responses of glutathione and antioxidant enzyme systems. Am J Physiol—Regul Integr Comp Physiol. 1994;267(2 36-2):R439–45.
Stover KR, King ST, Robinson J. Artemether-Lumefantrine: an option for malaria. Ann Pharmacother. 2012;46(4):567–77.
Anvikar AR, Sharma B, Shahi BH, Tyagi PK, Bose TK, Sharma SK, et al. Artesunate-amodiaquine fixed dose combination for the treatment of Plasmodium falciparum malaria in India. Malar J. 2012;11(97):1–8.
Schramm B, Valeh P, Baudin E, Mazinda CS, Smith R, Pinoges L, et al. Tolerability and safety of artesunate-amodiaquine and artemether-lumefantrine fixed dose combinations for the treatment of uncomplicated Plasmodium falciparum malaria: two open-label, randomized trials in Nimba County. Liberia Malar J. 2013;12(250):1–13.
Eze ED, Afodun AM, Kasolo J, Kasozi KI. Lycopene improves on basic hematological and immunological parameters in diabetes mellitus. BMC Res Notes. 2019;12(1):805.
Rifat-uz-Zaman MS, Khan MS. Gastroprotective and antisecretory effects of Nigella sativa and its extracts in indomethacin-treated rats. Pakistan J Biomed Sci. 2004;7(6):995–1000.
Olaleye SB, Ajeigbe KO, Emikpe BE. Effect of sulfadoxine-pyrimethamine and artesunate on gastric acid secretion and parietal cell mass in rats. African J Biomed Res. 2012;15(1):23–8.
Bigoniya P, Sahu T, Tiwari V. Hematological and biochemical effects of sub-chronic artesunate exposure in rats. Toxicol Reports. 2015. https://doi.org/10.1016/j.toxrep.2015.01.007.
Verma S, Kumar VL. Attenuation of gastric mucosal damage by artesunate in rat: modulation of oxidative stress and NFκB mediated signaling. Chem Biol Interact. 2016. https://doi.org/10.1016/j.cbi.2016.07.027.
Ajeigbe KO, Emikpe BO, Olaleye SB. Effects of artemisinin, with or without lumefantrine and amodiaquine on gastric ulcer healing in rat. J Basic Clin Physiol Pharmacol. 2018. https://doi.org/10.1515/jbcpp-2017-0145.
Kasozi KI, Namubiru S, Safiriyu AA, Ninsiima HI, Nakimbugwe D, Namayanja M, et al. Grain amaranth is associated with improved hepatic and renal calcium metabolism in type 2 diabetes mellitus of male wistar rats. Evid-Based Complement Altern Med. 2018;2018:1–10.
Jaeschke H, Mitchell JR. Use of isolated perfused organs in hypoxia and ischemia/reperfusion oxidant stress. Methods Enzymol. 1990;186(C):752–9.
Esterbauer H, Cheeseman KH.  Determination of aldehydic lipid peroxidation products: Malonaldehyde and 4-hydroxynonenal. In: Methods in Enzymology. 1990: 407–421.
Sinha K, Sil PC. Targeting oxidative stress and inflammation in NSAIDs induced gastropathy: a plausible therapeutic approach. Inflamm Cell Signal. 2015;2(e763):1–6.
Ajeigbe KO, Emikpe BO, Olaleye SB. Augmentation of gastric acid secretion by chloroquine and amodiaquine in the rat stomach. Niger J Physiol Sci. 2012;27(June):89–94.
The authors wish to acknowledge the technical support of Professor S.B Olaleye at the University of Ibadan, Nigeria, during the conceptual development of the study.
Ethics approval and consent to participate
This was acquired from the Kampala International University Western Campus Ethics and Scientific review board. Consent to participate was not applicable to this study.
Consent to publish
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Kalange, M., Nansunga, M., Kasozi, K.I. et al. Antimalarial combination therapies increase gastric ulcers through an imbalance of basic antioxidative-oxidative enzymes in male Wistar rats. BMC Res Notes 13, 230 (2020). https://doi.org/10.1186/s13104-020-05073-7
- Pharmacodynamics of antimalarial agents
- Malaria in developing countries
- Gastric ulcers