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Original Article
Antimalarial activity of Neopetrosia exigua extract in mice Syamsudin Abdillah a, Rahmatul Wahida Ahmad b, Farid Kamal Muzaki c, Noriah Mohd Noor b,* a
Department of Pharmacology, Faculty of Pharmacy, Pancasila University, Jakarta, Indonesia Department of Basic Medical Sciences, Kulliyyah of Pharmacy, International Islamic University Malaysia, Kuantan Campus, Malaysia c Laboratorium of Ecology of Marine, Department of Biology, Institute of Technology Surabaya, Indonesia b
article info
abstract
Article history:
Objective: To evaluate the antimalarial activity of ethanolic extract of Neopetrosia exigua in
Received 3 July 2013
ICR mice. The safety of the extracts was also ICR mice by the acute oral toxicity test.
Accepted 1 August 2013
Methods: The crude ethanol extract of Neopetrosia exigua (50, 100, 200 and 400 mg/kg) was
Available online 24 August 2013
investigated for its antimalarial activity against Plasmodium berghei during early infection. The acute toxicity of the extract was also investigated. At the end of 14 days, mice were
Keywords:
sacrificed for histopathology study.
Neopetrosia exigua
Results: The extract of Neopetrosia exigua demonstrated significant ( p < 0.05) schizonticidal
Ethanolic extract
activity. The acute toxicity test showed that the extract of Neopetrosia exigua is toxicological
Antimalarial activity
safe by oral administration. Histopathological study revealed normal architecture of kidney
Acute toxicity
and liver of mice. Conclusion: The extract of sponge Neopetrosia exigua has anti-malarial activity in vivo. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Malaria during pregnancy is a major public health problem in tropical and subtropical regions throughout the world.1 Malaria causes serious illness and death amongst children and pregnant women. There are between 300 and 500 million malaria infections and 1 million malaria-attributed deaths worldwide each year.2 As malaria vaccines remain problematic, chemotherapy still is the most important weapon in the fight against the disease.3 The antimalarial drugs including chloroquine, quinine, mefloquine,
pyrimethamine, and artemisinin are currently used in malaria treatment. Part of the reason for the failure to control malaria is the spread of resistance to first-line antimalarial drugs, cross-resistance between the limited number of drug families available, and some multidrug resistance.4 Marine sponges have a potential to provide future drugs against important diseases, such as malaria, cancer and a range of viral diseases.5 Of 10,000 marine sponges, 11 genera are known to produce bioactive compounds, and only three genera (Haliclona, Petrosia and Discodermia) are known to produce anti-malarial, anticancer and anti-inflammatory
* Corresponding author. Tel.: þ60 9 5704873. E-mail address:
[email protected] (N. Mohd Noor). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.08.001
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compounds.6 Sponge from the genus of Petrosia commonly found in Situbondo waters, East Java, Indonesia is Neopetrosia sp. Marine sponge, Neopetrosia sp. is a newly revived genus name, but in the past, it might have been described as Xestospongiasp.7 They produced many potential bioactive metabolites including cytotoxicity: Renieramycin J, Araguspongine B, D, M, and three 5a,8aepidioxy sterol,7,8 antileishmanial: Renieramycin A from the Satsunan island, Japan9 and antimicrobial substance: Nethylene methyl ketone derivative of renierone, 1,6-dimethyl7-methoxy-5,8-dihydroisoquinoline-5,8-dione, renierone and mimosamycin.10 The study aims at finding out antimalarial effect in vivo the Plasmodium berghei infected mice and its safety profile in acute toxicity assay in mice when given orally.
death in animal models. The value was determined from the number of dead mice within the first 24 h and for 14 days of observation after a single dosage administration.
2.5.
Parasite inoculum
The blood of donor mice with 30e40% increase in parasitemia rate was taken through the heart, and then diluted with 0.9% of Nacl solution (1:1) up to the parasite density of 1 107. Inoculation was conducted in IP method by injecting 0.2 mL of inoculum. Inoculated mice were randomly taken into a stable that consisted of 5 mice and kept in Animal Room, Department of Basic Medical Sciences, Kulliyyah of Pharmacy, International Islamic University, in accordance with the internationally accepted principles for laboratory animal use and care.
2.
Materials and methods
2.6.
2.1.
Animal materials
A sponge of the Neopetrosia exigua (order Hadromerida, family Suberitidae) was collected by scuba diving at 8 m depth at Tanjung Pecaron Bay, near Situbondo (Indonesia). A voucher specimen, Voucher No.A24354, is deposited at Department of Biology, Faculty of Sciences, Institute Technology of Surabaya. The strain of P. berghei was kindly provided by Dr. Hashida Mohd Sidek, Centre of Bioscience and Biotechnology, Faculty of Sciences and Technology, National University of Malaysia.
In vivo assay was conducted upon ICR strain of P. berghei infected mice given with the extract of Neopetrosia exigua with the dosages of 50, 100, 200, and 400 mg/kg and compared with control group that was treated only with distilled water (containing DMSO 10% and solvent used to dilute the extract) as well as reference group that was treated with standard chloroquine with a dosage of 10 mg/kg. Percent of parasitemia was determined by using a microscope (Olympus, cover-015) from the infected red blood cells compared to 4000 RBC in random fields of the microscope.
2.2.
2.6.1.
Preparation of extract
Freezed dried or wet samples were soaked twice in ethanol. Each soaking lasted 24 h. After filtration, solvents were evaporated under reduced pressure in a rotary evaporator and the extracts were combined.
2.3.
Animals
ICR mice, male (29 2 g) and female (25 2 g), 7e8 weeks old were used in the experiment. The mice were kept in the stable and fed with standard pellet and water in libitum at Animal House. Department of Basic Medical Sciences, Kulliyyah of Pharmacy, International Islamic University Malaysia. The animals were housed under standard conditions of temperature (25 10 C) and relative humidity (60 10%), 12/12 h light/ dark cycle, and fed with standard pellet diet and tap water. Animals were fasted prior to dosing and the test substance was administered in a single dose by oral route.
2.4.
Acute toxicity assays
Acute toxicity assay was conducted by using ICR strain of mice of both sexes with body weight range of 25e30 g. The extract of Neopetrosia exigua was given with varied dosages (5000, 2500, 1250, and 625 mg/kg). Every animal model was precisely observed and recorded for any toxicity effect that occurred within the first 24 h. The observation took 14 days. Every dead mouse was observed macroscopically and microscopically for crucial organs such as liver, kidney, lung, abdomen, intestine, and heart. LD50 value referred to the dosage that caused 50% of
In vivo antimalarial assays
Early malaria infection
Early malaria infection model was used based on the method applied by Peters.11 Thirty mice of ICR strain were inoculated in IP using 0.2 mL and suspense that contained 1 106 of P. berghei in the first day (D0). Twenty four (24) hours after initiation of the infection, the mice were given the extract of Neopetrosia exigua with the dosages of 50, 100, 200, and 400 mg/ kg/bwt in an oral way. Reference group was treated with 10 mg/kg of chloroquine and control group with 0.2 ml of distilled water. The treatment was repeated after 3 days (D1eD3). On the fourth day (D-4), thin blood smear was prepared using Giemsa stain for every mouse.
2.6.2.
Established malaria infection
Established malaria infection model was used for 30 mice of ICR strain inoculated in IP of 0.2 ml and suspense that contained 1 106 of P. berghei. Seventy two hours after initiation of infection, the treatment group was orally given the extract of Neopetrosia exigua with the dosages of 50, 100, 200, and 400 mg/kg, the reference group with 10 mg/kg of chloroquine, and control group with 0.2 ml of distilled water every day for 6 days. On the seventh day, the blood was taken through the tail to prepare thin blood smear by using Giemsa stain. Observation was conducted up to 30 days after the initiation of infection to determine the survival of infected mice and the effect of the extract.
2.6.3.
Residual malaria infection
Residual malaria infection model was used for 30 mice of ICR strain that had been randomly taken into every stable, which consisted of 5 mice. The treatment group was given the
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extract of Neopetrosia exigua in an oral way with the dosages of 50, 100, 200, and 400 mg/kg, reference group with 10 mg/kg of chloroquine, and control group with 0.2 ml of distilled water for 3 days (D0eD2). On the third day, the mice were infected with suspense that contained 1 106 of P. berghei. On the seventh day, blood was taken through the tail to prepared blood smear by using Giemsa stain.
2.7.
Result
The study showed that antimalarial activity of Neopetrosia exigua had a good activity against the growth of P. berghei. Assay with chemosuppression test method showed that extracts with doses of 400 mg/kg and 200 mg/kg could suppress the growth of P. berghei by 80.69% and 60.62% compared to 98.32% inhibition of P. berghei growth using chloroquine with a dose of 10 mg/kg (Table 1). Ethanolic extract of N. exigua dose of 400, 200 and 100 mg/kg group was significantly different than dose of 50 mg/kg and vehicle (*). Oral administration of Neopetrosia exigua extract with a dose of 400 mg/kg could not increase body weight of the mice, compared the mice given with 10 mg/kg of chloroquine. On the other hand, chloroquine with doses of 200, 100, and 50 mg/ kg could decrease body weight as shown in Table 2.
3.1. Prophylactive effect of extract of Neopetrosia exigua Antimalarial test using prophylactive method showed that Neopetrosia exigua extract with doses of 400 and 200 mg/kg could inhibit the growth of P. berghei by 71.76% and 52.43%, respectively, while chloroquine group could provide P. berghei growth inhibition of 97.63%.
Table 1 e Effect of ethanolic extract of Neopetrosia exigua on chemosuppressive activity. Extract/drug Neopetrosia exigua 400 mg/kg 200 mg/kg 100 mg/kg 50 mg/kg Vehicle Chloroquine 10 mg/kg
% parasitemia % chemosuppression 1.49 0.98* 3.04 0.83* 4.81 0.92* 5.72 0.73 7.72 0.84 0.13 0.97*
Extract/drug
Dose (mg/kg)
Neopetrosia sp
400 200 100 50 0.2 mL 10
Vehicle Chloroquine
Statistical analysis
Data are expressed as mean S.E.M. and analyzed using one way analysis of variance (ANOVA) followed by Dunnett test for comparing pairs of data. The significant level was set at p < 0.05.
3.
Table 2 e Body weight of P. berghei infected mice after the administration of extract of Neopetrosia exigua.
80.69 60.62 37.43 26.04 0 98.32
Each result is mean of mice controls. * Showed extract N exigua 400 mg/kg dose, 200 mg/kg and 100 mg/kg are significantly different than dose of 50 mg/kg and the vehicle, but was not significantly different compared with chloroquine dose 10 mg/kg.
D4
D0 26.4 26.8 26.8 25.2 30.0 25.2
1.42 1.53 1.37 1.10 0.75 0.64
26.4 26.4 25.6 24.0 27.2 26.4
0.98 1.50 1.35 0.98 0.63 0.78
D0: day infection was initiated. D4: 5th day of infection.
3.2.
Curative effect of extract of Neopetrosia exigua
Antimalarial test for curative effect showed that Neopetrosia exigua extract with oral doses of 400 and 200 mg/kg in mice could survive up to 14.64 1.72 and 12.72 0.98 respectively, compared to a survival of 30.00 0.00 with chloroquine.
3.3.
Acute toxicity test
Up to the first hour of infection, all mice were still in normal condition. Three hours after the infection, the mice began to show a declining motor activity, such as the sign of silence and confusion, and deteriorating physical conditions, such as hair loss and damage. Observation in the sixth and the twelfth hours of dosing revealed declining motor activity, appetite loss, and hair damage/loss even though no death had been found after the mice were dosed with 5000 mg/kg of the extract, but all mice showed constantly declining motor activity and hair damage followed by loss of appetite for food and drink. Histopathological test on the mice treated with 5000 mg/kg of the extract and the mice in normal control group are shown in Fig. 1.
4.
Discussion
4.1.
Antimalarial assay
In vivo antimalarial assay in the mice of ICR strain was conducted using the methods of chemosuppression, prophylactive test, and rane test. Antimalarial activity was determined from the growth inhibition of P. berghei after oral administration of Neopetrosia exigua extract. Even though the rodent malaria model, P. berghei, is not exactly similar to that of the human Plasmodium parasites, it is the first step to screen most of the in vivo antimalarial activities of new molecules and new therapeutics.11 The extracts prolonged the mean survival time of the study mice indicating that the extracts suppressed P. berghei and reduced the overall pathologic effect of the parasite on the study mice (Table 4). However, neither the extracts nor the standard drug cured the infection. The extract at 400 mg/kg/day exhibited promising antimalarial activity in both chemosuppressive and prophylactive tests. The result for the prophylactive test also gave a result similar to that noticed during the chemosuppressive test (Tables 1 and 3 respectively). The ethanolic extract of N. exigua dose 400 mg/kg and 200 mg/kg group was significantly
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Fig. 1 e The histological morphology mice livers from the control (A), extract of Neopetrosia exigua (5000 mg/kg) (B), kidneys from the control (C), extract of Neopetrosia exigua (5000 mg/kg) (D) groups shown by hematoxylin and eosin staining at a magnification of 403.
different than dose 100 mg/kg, 50 mg/kg and vehicle (*) body weight. All of the three test methods showed that the extract of Neopetrosia exigua with doses of 400 and 200 mg/kg could inhibit the growth of P. berghei up to >50%, compared to the resulting growth inhibition with 100 and 50 mg/kg of the extract. The three test methods showed a difference in % of parasitemia. This is probably attributable to hospes factor, such as endurance of the mice against the growth of P. berghei. Plasmodium factor might also contribute to the mice’s endurance since P. berghei was not synchronized in the body of the mice and since only 10% of inoculated P. berghei could grow. There was a
Table 3 e Inhibition of development of P. berghei blood stages by the 4-days prophylactive test. Extract/drug Neopetrosia exigua
Vehicle Chloroquine
schizogonyeerythrocytic cycle in P. berghei, that the ring stadium and trophozoite were mostly taken as inoculums. Such character of P. berghei could contribute to its growth in the hospes body.
4.1.1.
Acute toxicity assay
Acute toxicity assay showed that the doses up to 5000 mg/kg could not induce 50% of death in mice within 24 h of dosing, with a LD50 > 5000 mg/kg. Histopathological test on the liver showed that a dose of 5000 mg/kg could lead to congestion or blood clogging and polymorphonuclear cell infiltration, namely, cell infiltration with segmented nucleus (neutrophil). No specific anomaly was observed in the control group. Mice in the group treated with a dose of 5000 mg/kgBwt died on day-14. Consequently, the damaged organ could not be
Dose (mg/kg) % parasitemia % inhibition 400 200 100 50 0.2 mL 10
2.03 3.04 5.32 6.42 7.19 0.17
1.81* 1.52* 0.98 0.89 0.82 0.42*
71.76 52.43 26.01 10.71 0 97.63
* Showed extract N exigua 400 mg/kg dose, 200 mg/kg and chloroquine 10 mg/kg are significantly different than dose of 100 mg/kg, 50 mg/kg and the vehicle.
Table 4 e Mean survival time of mice receiving various doses of extract of Neopetrosia exigua. Extract/drug Neopetrosia exigua
Chloroquine
Dose (mg/kg) 400 200 100 50 10
% parasitemia 1.58 3.42 4.71 5.83 0.14
0.97 0.58 0.92 0.83 0.98
Mean survival time (day) 14.64 12.72 7.53 5.37 30.0
1.72 0.98 0.83 0.47 0.00
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examined histopathologically. Histopathological examination on the mice showed serious damage on the hepatic cell, namely karyopyknosis e condensation of the nucleus of liver cell. In addition, sinusoidal dilatation and extensive hepatic necrosis were also found. The liver necrosis probably led to the death of mice. The great doses lead to the declining liver function, since the liver had to work hard. Urine excretion of toxic compounds through from the liver is one of the essential route of elimination. Therefore, many mechanisms underlie the renal toxicity. Mild irritation or effect of a lesion (scratch) because of foreign components in high concentration might also lead to high risk of tubular necrosis. The figure indicates a mild degeneration, namely, congestion in the kidney of the mice in control group after the dosing of extract. The congestion could probably be attributed to the daily dosing of extract and the effect of solvent chemical substance given to the mice that led to mild toxicity in the kidney of mice in control group. Mice in the group treated with 5000 mg/kg had tubular necrosis. Necrosis is a sign of serious damage in the liver, which eventually led to the death of mice. In addition, an accumulation of protein was found in the tubules. This confirms the serious renal damage. As a result, protein could not be filtered well and left in the tubules, leading to proteinuria in the mice. Serious damage in the kidney might be attributable to daily exposure of high-dose extract that lead to overwork in the kidney. Finally, the kidney could not function well. This describes the toxicity in the kidney of mice.
5.
Conclusion
In conclusion, crude extracts of Neopetrosia exigua caused strong activities against P. berghei indicating that extract of Neopetrosia exigua contain some lead antiplasmodial compounds. It would be worthwhile to isolate its active constituents and characterize their exact mode of action which can be exploited for the treatment of malaria.
Conflicts of interest All authors have none to declare.
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Acknowledgments The research was funded by Endowment B, Project Id : 12-3960874. IIUM is gratefully acknowledged.
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