INTRODUCTION
Malaria is one of the oldest and most life threatening parasitic diseases that human has faced till now. The in- fection is caused by plasmodia species that are transmit- ted by the female Anopheles mosquitoes. Five species of Plasmodium have been identified which cause disease in human with different clinical manifestations. However, out of the five species, Plasmodium falciparum is con- sidered to be potentially fatal in delayed treatments1–2. Despite continuous global attempts to fight parasitic in- fections, malaria still remains one of the major public health problems in tropic and subtropic areas3. In 2015, there were estimated 214 million new cases of malaria, and approximately 438,000 deaths; the majority of them were children4. In spite of launching malaria elimination programme (elimination phase started from 2010) in Iran, the infection is still, more or less, a health problem in the southeast of the country1. According to the recent report
of WHO, in total 1243 confirmed malaria cases were re- corded during 2015 in Iran4.
Antimalarial drugs such as chloroquine, quinine, artemisinine and primaquine are currently being used to prevent and treat human malaria. However in the past 40 years, malaria parasites have rapidly developed resistant to the most of the antimalarial drugs2. Plasmodium falciparum became resistant to chloroquine (CQ) in Iran since 19835–6. Difficulty of producing efficient antimalaria vaccines and increasing drug-resistant strains, highlight the urgent need to search for novel, effective, easily avail- able and safe alternative antimalarial drugs for both pro- phylaxis and treatment of malaria.
Plasmodium berghei is a trustable malaria parasite model for in vivo tests in the field of antimalarial drug studies and drug resistance in malaria parasites, and based on the obtained results, it has been accepted for prelimi- nary study as human malaria parasite. Moreover, attain- ment and manipulation the parasite is more simple and Research Articles
Antimalarial efficacy of low molecular weight chitosan against Plasmodium berghei infection in mice
Aref Teimouri
1, Afsane Motevalli Haghi
1, Mehdi Nateghpour
1, Leila Farivar
1, Haleh Hanifian
1, Sara Ayazian Mavi
1& Roghayeh Zare
21Department of Medical Parasitology and Mycology, School of Public Health; 2Department of Epidemiology and Biostatistics, Tehran University of Medical Sciences (TUMS), Tehran, Iran
ABSTRACT
Background & objectives: Despite continuous global attempts to fight parasitic infections, malaria still remains one of the major human life threatening diseases. Difficulty of producing efficient antimalaria vaccines and increasing drug-resistant strains, highlight the urgent need to search for a new alternative antimalaria drug. The aim of this study was to find a new agent against malaria parasite with maximum efficacy and minimum range of side-effects. For this, the antiplasmodial activity of commercial chitosan, a natural carbohydrate polymer, was evaluated on Plasmodium berghei via in vivo experiments. This is the first report that to highlight antimalarial effects of low molecular weight chitosan against P. berghei in vivo.
Methods: Low molecular weight chitosan with 95% degree of deacetylation was melted in normal saline with 1%
(w/v) acetic acid for preparing 10, 20, 40 and 80 mg/kg concentrations of chitosan, which were then examined for their antimalarial efficacy in P. berghei infected mice.
Results: The study showed that differrent concentrations of chitosan exhibited significant antimalarial effect (p=
0.002) when compared with the control group. Also, analysis of mice survival time showed significant differences between 20 and 80 mg/kg concentrations of used chitosan in comparison to negative control group.
Interpretation & conclusion: The results of this study showed that the chitosan has potent antimalarial activity and could be suggested as an alternative antimalarial drug component.
Key words In vivo; Iran; low molecular weight chitosan; malaria; Plasmodium berghei
safe than other human malaria parasites.
Chitin is a long-chain carbohydrate polymer and com- monly found in nature as structural element in exoskel- eton of crustaceans and many other organisms, like arthropods and fungi7. Commercial chitosan is produced by deacetylation of chitin which is characterized by its wide applications8. In vitro and in vivo experiments have demonstrated considerable effectiveness of chitosan, against wide range of micro-organisms including bacte- ria, fungi, yeasts and parasites whithout justifiable effects on mammalian cells9–10.
Necessity for developing a new agent with maximum antimalarial efficacy and minimum side-effects encour- aged us to carry out this study with the aim of evaluating antiplasmodial activity of commercial chitosan on P. berghei in vivo. This is the first study reporting anti- malarial effects of low molecular weight chitosan against P. berghei infection in mice.
MATERIAL & METHODS Animals
Male white small saurian mice, weighing 20–25 g (supplied by the Animal Laboratory in Pasteur Institute of Iran) were used in this study. The mice were kept in normal temperature and light-dark cycle situation, with unlimited food and tap water supplies.
Ethical considerations
Animal experiments were done according to ethical standards formulated in the Helsinki Declaration, and ap- proved by Ethical Committee of the Pasteur Institute of Iran for the use of laboratory animals.
Parasite culture
Chloroquine sensitive strain of P. berghei (NICD strain) that was stored in liquid nitrogen (originally ob- tained from Haffkine Institute, India) was maintained by blood passage in mice under laboratory conditions as de- scribed by Nateghpour et al6.
Chitosan solution preparation
Low molecular weight chitosan (Mol. wt. 50,000–
190,000 Da, Sigma-Aldrich, Cat No.:448869–50G, St.
Louis, MO, USA) with 95% degree of deacetylation was melted in normal saline with 1% (w/v) acetic acid to a final concentrations of 1, 2, 4 and 8 mg/ml. All concen- trations were kept overnight at room temperature to reach complete dispersion. The pH of all tubes was then ad- justed to 7 with 1 mol NaOH11.
Inoculation of malaria parasites
A number of mice were used as infected donors and as parasite reservoir. Mice infected via the intraperito- neal route were allowed to build parasitaemia level up to
~ 22% (till four days after the infection). The parasitaemia of the donor mice was first determined and infected blood was collected into the heparinized tubes directly from heart via the cardiac puncture using ether as anesthesia. The standard four-day suppressive method described in a study by Peters12 was used. The mice were randomly divided into six groups with six mice in each group. On the first day each mouse was inoculated by intraperitoneal injec- tion with 0.2 ml of infected blood suspension diluted in physiological saline, and containing about 106 P. berghei parasitized erythrocytes per ml. Treatment was initiated 2 h after the injection of the infected erythrocytes on Day 0 (D0, inoculation day) and continued daily until Day 3 (for four days). The negative control group was given 0.2 ml of normal saline with 1% (w/v) acetic acid. The posi- tive control group was given 0.2 ml of chloroquine (20 mg/kg) subcutaneously, while groups 3–6 were adminis- tered single dose of 0.2 ml chitosan subcutaneously, with concentrations of 1, 2, 4 and 8 mg/ml per day which equaled to 10, 20, 40 and 80 mg/kg respectively. Thin blood films were made by collecting blood from the tail of each mouse on the D4, D7and D14 days. Smears were fixed with methanol and stained with 3% Giemsa at pH 7.2 for 30 min. The percent parasitaemia was determined by counting the number of parasitized erythrocytes out of 10,000 blood cells. Then, average suppression percent- age of parasitaemia was calculated for each concentra- tion by comparing the control, using the modified method described by the Peters and Robinson13 formula:
Where, A is the mean percentage parasitaemia in nega- tive control group; and B is the mean percentage parasitaemia in treated group.
Evaluation of mean survival time
Mean survival time (MST) is another factor that is usually used to calculate the efficacy of antimalarial ac- tivity of drugs14. Mortality was checked daily and period of survival time was recorded for each mouse in all groups and the MST was assessed for each group by using the following formula:
Data analysis
Descriptive results were expressed as mean ± stan- dard deviation. The statistical differences was calculated by Mann-Whitney U-test and survival analysis using the statistical package for the social sciences (SPSS) soft- ware (SPSS IBM version 21, Chicago, IL, USA).
RESULTS Antiplasmodial studies
As shown in Fig. 1, the antimalarial activity of the low molecular weight chitosan against P. berghei in white small mice was quite obvious due to considerable sup- pression of parasitemia on Days 4, 7 and 14. The differrent concentrations of chitosan showed significant antimalarial efficacy (p = 0.002) when compared with the control group treated with chloroquine (20 mg/kg/day) (Table 1).
Survival analysis
In the four-day suppressive test, the mean survival time in all groups treated with different concentrations of chitosan was longer than negative control group. Groups with concentration 20 mg/kg and 80 mg/kg, and with p- value 0.018 and 0.029 respectivly showed significant dif- ferences in comparison with the negative control group
(Table 2). The number of dead mice before Day 15 is shown as number of death.
DISCUSSION
Malaria is one of the most perilous and important in- fectious diseases in the world. Since developing effec- tive antimalarial vaccines is not an easy task and faced with several problems, chemotherapy still is the first-line action against the disease2. The development of resistance against these chemotherapeutic agents necessitates fur- ther search for new antimalarials. In addition to the need of developing new antimalarial drugs, it is also important to prove the efficacy and safety of natural agents and tra- ditional medicinal plants which are used against malaria15. It is well-known that natural agents and traditional medi- cine used against malaria and other diseases play impor- tant role in some endemic countries lacking access to es- sential medicines16. For example, the herbal extract of Iranian Flora Artemisia khorassanica has been success- fully tested in vivo for its antiplasmodial activity through artemisin composition, which is globally used as a stan- dard malaria treatment2. Similiar studies establish the ef- ficacy of traditional remedies and natural agents16–18.
The present study was carried out to evaluate the an- timalarial activity of chitosan, a natural carbohydrate poly- mer, in vivo on P. berghei. Chitin and chitosan have been
Table 1. Antimalarial effect of chitosan against P. berghei in concentrations of 10, 20, 40 and 80 mg/kg
Dose Percent parasitaemia (Mean ± SD) Suppression (%)
(mg/kg) Day 14 Day 7 Day 4 Day 14 Day 7 Day 4
10 7.17 ± 0.72 11.12 ± 0.93 17.28 ± 0.88 55.49 40.23 24.86
20 5.62 ± 0.35 8.78 ± 0.98 11.68 ± 0.51 65.11 52.78 49.20
40 8.50 ± 0.54 12.28 ± 0.42 16.90 ± 0.75 47.20 33.96 26.52
80 7.70 ± 0.43 10.10 ± 0.58 13.92 ± 0.67 52.17 45.70 39.49
Negative control 16.1 ± 0.52 18.6 ± 0.44 23 ± 0.61 – – –
Positive control 0.0 0.0 0.0 100 100 100
Fig. 1:Percentage chemosuppression of P. berghei induced malaria in mice.
Table 2. The effects of chitosan on survival time of mice infected with P. berghei using 10, 20, 40 and 80 mg/kg concentrations
Antimalarial Dose Mean survival Number
agents (mg/kg) time (Days) of death
Chitosan 10 12.33 ± 3.07a 3
Chitosan 20 14.83 ± 0.4a* 1
Chitosan 40 12.67 ± 3.67a 2
Chitosan 80 13.67 ± 3.26a* 1
Negative control [Normal 10 9.83 ± 4.99 5 saline with 1% (w/v)
acetic acid]
Positive control (Chloroquine) 20 15 ± 0 0
aCompared to negative control; *p <0.05.
explored as an antimicrobial agent against a wide range of organisms like algae, bacteria, yeasts and fungi in in vivo and in vitro experiments9–10. Although, the precise mechanism of the antimicrobial action of chitin, chitosan, and their derivatives is not fully understood; but different mechanisms have been proposed, the most passable be- ing the interaction between negatively charged microbial cell surface and positively charged chitin/chitosan mol- ecules19–22. Another posed mechanism is the binding of chitosan with microbial DNA via the penetration of chitosan into the nuclei of the microorganisms causing inhibition of mRNA and proteins synthesis23. The third mechanism is the chelation of metals, suppression of spore elements and binding to necessary nutrients required for microbial growth24. All of these properties, with the non- toxicity feature, make chitosan suitable for the pharma- ceutical production for present and future uses. In many studies, the antimicrobial activity (antibacterial, antiviral and antifungal) of low molecular weight chitosan has been reported at pH values lower than 6. However, only a few studies have been performed to evaluate antiparasitic effect of chitosan23–24.
Recent studies have shown considerable antileishmanial activity of chitin/chitosan against Leish- mania infantum LIPA 137, but not against L. infantum LIPA 155/1025. Results of other studies have shown that chitosan with 0.025% concentration inhibited the growth of fungi and bacteria including Helminth osporium, Fusarium oxysporum, Alternaria alternata and Escheri- chia coli22. Another study revealed that combination of chitosan and an antiparasitic drug prolonged the releas- ing time in gastrointestinal tract in Cryptosporidium in- fections26. Results of a comparative study among differ- ent types of fungal chitosan against protoscolices of hydatid cyst using in vitro experiments showed their scolicidal activity equivalent to commercial chitosan22.
Yarahmadi et al27, evaluated the effect of chitosan on the viability of Giardia lamblia cysts that resulted in 100%
mortality rate in 180 min of exposure in concentration of 400 µg/ml of chitosan. Similiar studies showed that 1.250 µg/ml of chitosan after 360 min exposure time could com- pletely inhibit the viability of Trichomonas gallinae28.
On the other hand, nanomedicine is expecting to present an exact delivery system and offers the ability of enhancing the quality of chloroquine by decreasing its toxicity, increasing bioavailability and potent of distri- bution. Tripathy et al29 conjugated chloroquine to chitosan–tripolyphosphate (CS–TPP) nanoparticles to prepare nanochloroquine (Nch) particles by inotropic gelation ranging from 150–300 nm. They showed that efficacy of Nch is more than chloroquine against P.
berghei NK65 infection in Swiss mice. In addition they proved that Nch is able to keep tissues safe from oxida- tive damage, caused by P. berghei infection andis capable to decrease free radical generation and also increases an- tioxidant enzymes activity. The results of their study showed that chitosan conjugated CQ delivery is more potent than only CQ in decreasing parasitemia and host pathology. This study established that CQ in 68.5 mg/kg body weight is not as effective as same amount of CQ conjugated with chitosan in attenuating the parasitaemia29–31. The present study revealed that chitosan alone can reduce parasitemia and its efficacy may be increased when used as a component in combination thrapy.
Another parameter to evaluate antimalarial activity of plant extracts is mean survival time. The prolonged survival time of cured infected mice with chitosan in comparison to the non treated group (negative control) revealed consid- erable anti-plasmodial activities of the agent, but not as effective as chloroquine; in four-day suppressive test.
As mentioned previously, very few studies about anti- parasitic effect of chitosan are available, and this work is the first report of using chitosan alone in treatment of murine malaria. The low molecular weight chitosan was successfully tested in vivo for its anti-malarial activity.
The results indicated that the various concentrations of chitosan exhibited significant antimalarial effect (p=0.002) when compared with the negative control groups. Some natural substances, possess significant ef- ficacy against the parasitic agents with low toxicity and can be used, more or less, as a safe treatment against murine malaria infection. Among the natural agents, chitosan has a reasonable efficacy to be supposed as a new therapeutic product. However, more studies are needed to analyse further aspects of chitosan and its exact effectiveness.
CONCLUSION
The results of this study showed that the low mo- lecular weight chitosan has potent antimalarial activity and could be selected as an alternative natural antima- larial drug candidate. More investigations on different plasmodia species, animal hosts and combination between chloroquine and chitosan on chloroquine-sensitive and chloroquine-resistant strains of P. berghei are necessary to elucidate the antimalarial activity of chitosan and its natural components.
Conflict of interest
All the authors declare that they have no conflict of interests.
ACKNOWLEDGEMENTS
This research received full financial support from Tehran University of Medical Sciences and Health Ser- vices grant (Project no.: 94–01–6126962), Tehran, Iran.
The authors would like to thank Ms. M. Amirzadeh and Mr. R. Eskandary for their useful technical support.
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Correspondence to: Dr Mehdi Nateghpour, Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences (TUMS), Tehran, Iran.
E-mail: [email protected]
Received: 20 June 2016 Accepted in revised form: 29 September 2016
