International Journal of Modern Biology and Medicine, 2012, 1(2): 108-116 International Journal of Modern Biology and Medicine

ISSN: 2165-0136 Florida, USA Journal homepage: www.ModernScientificPress.com/Journals/IJBioMed.aspx Article

Anti-Parasitic Effects of Methanolic Extracts of Artemisia annua L. against Parasites of Sarotherodon melanotheron
Obemeata Oriakpono*, Hart Aduabobo, Grace D. B Awi-Waadu, Sidney Nzeako Department of Animal and Environmental Biology, Faculty of Science, University of Port Harcourt, PMB 5323, Port Harcourt, Nigeria * Author to whom correspondence should be addressed; E-Mail: obytrees@yahoo.com. Article history: Received 28 April 2012, Received in revised form 21 May 2012, Accepted 22 May 2012, Published 23 May 2012.

Abstract: This study evaluated the antiparasitic properties of methanolic extracts of Artemisia annua. Seven hundred and fifty, ten-day-old juvenile Sarotherodon melanotheron fish samples were obtained and exposed to conditions that enhanced parasite proliferation for one week, after which fish showing signs of parasite burden were isolated and parasite load was determined by counting under a stereo-microscope. They were then separated into five groups based on samples with same number of parasites and exposed to the extract. Assessment of antiparasitic property of extract was carried out in 250 mL beakers with three replications and controls. Preliminary phytochemical screening of extracts revealed the presence of terpenoids, essential oils, flavonoids and coumarins. Results of bioactivity of extracts revealed a time dependent dislodgement of parasites from point of attachment, coagulation and death of parasite cells exposed to the extracts. Thus the secondary metabolites of A. annua and its essential oils have antiparasitic potentials against monogenetic trematodes as found in the case of monogenean parasites of S. melanotheron. This knowledge can be explored in the aquaculture industry to eliminate the use of conventional synthetic organic drugs that may be detrimental to consumers of aquaculture products. Keywords: Artemisia annua; Sarotherodon melanotheron; antiparasitic property; aquaculture; food safety.

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1. Introduction
Artemisia annua, also known as sweet Annie or wormwood in the USA belongs to the family Asteraceae. It is a vigorous growing annual weedy aromatic herb, usually single-stemmed reaching up to 2 meters in height (Simon and Quinn, 1990). A native of Asia, most probably China, it is known in China as qing hao. Recent research has shown that an infusion of the leaves can be used to treat colds, diarrhea and nose bleeds (Subhuti, 2005). Historically, the plant has been used as a worm killer and an aphrodisiac and also for the treatment of fevers and hemorrhoids in China since about 168 BC. The main active ingredient, artemisinin (or qinghaosu in Chinese) has been isolated (Zhou, 1986) and its structure correctly defined as a sesquiterpene lactone with an endoperoxide bridge (Schmid et al., 1983). Artemisinin, a crystalline compound poorly soluble in water, is currently available commercially in China and Vietnam as an anti-malarial drug effective against all multi-drug resistant strains of Plasmodium with no apparent adverse side effects (Jansen, 2006). The herb has been found to have normalizing effects on immune functions (Roberts, 2004) (perhaps a possibility that it could be used in the treatment of AIDS patients with highly compromised immune systems) and stimulates white blood cell activities. Researchers have shown that the plant is effective against a wide range of bacteria (Bone and Morgan, 1992), which include Enterobacter, Klebsiella, Streptococcus faecalis, Staphylococcus aureus, Shigella dysenteriae, and Escherichia coli. The protozoal parasites affected include Pneumocystis carini (Chen et al., 1994), and those causing Cryptosporidiasis, Amoebiasis, Schistosomiasis, Giardiasis and Leishmaniasis (Subhuti, 2005). Brisibe et al. (2008) and Allen (1997) showed that supplementing daily rations of poultry with dried pulverized leaves of A. annua was found to be effective for the treatment of coccidiosis in chickens without any adverse effects. Ridley and Hudson (1998) stated that artemisinin destroys the cells of parasitic organisms through the generation of highly reactive oxygen-based free radicals or electrophilic intermediates, by alkylating and oxidizing proteins and lipids of parasite membranes as well as inactivation of channel proteins. Collectively, these microbes and parasites cause a lot of illnesses to man and animals in the tropics, including Nigeria. Thus any inexpensive method of treatment directed against any or all of these microorganisms will be considered good for our general healthcare system. This study investigates the use of A. annua as a treatment and a preventive measure against monogenean parasites of a brackish water fish Sarotherodon melanotheron.

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2. Materials and Methods

2.1. Sample Preparation and Extraction Fresh leaves of A. annua were obtained from the garden of the University of Port Harcourt. The leaves were washed thoroughly in running tap water to remove sand and debris then sun-dried to a constant weight for three weeks, after which they were ground to a fine powder using the Ajax Milling Machine. The fine powder was subjected to Soxhlet extraction using methanol as the extracting solvent to extract the active compounds. The solvents were exhausted from the extracts using a rotary evaporator (Bichi Germany) and stored under room temperature until required for use. 2.2. Phytochemical Screening The methanolic extracts of Artemisia annua leaves were subjected to preliminary phytochemical test using standard techniques (Brian and Turner, 1975; Trease and Evans, 1996). The Buchard test was carried out for terpenoids, sudan IV test for essential oils, schinoda test for flavonoids and the ammonia UV test for coumarins. 2.3. Preparation of Stock and Working Solutions The methanolic extract of A. annua was used for the preparation of a stock solution from which the working solution used for the efficacy testing was prepared. A preliminary test was carried out to guide in the selection of the concentration of the test solutions. The stock solutions were obtained by dissolving 1 g of the extract in 5 mL of dimethyl sulfoxide (DMSO) and made up to 100 mL with water from the fish pond. Five test solutions labelled (V, W, X, Y, and Z) represented by concentrations of (5, 10, 20, 40 and 80 mg/L, respectively) were prepared from the stock solutions. Praziquantel, a commonly used antiparasitic agent in aquaculture was used for comparison at a recommended dose of 2 mg/L which was first dissolved in 5 mL of DMSO, while 5 mL of DMSO dissolved in water from the fish pond was served as control. Seven hundred and fifty, ten-day-old juvenile Sarotherodon melanotheron were obtained from an intensive culture system in a brackish water fish farm in Buguma, Rivers State, Nigeria, placed in an earthen pond and covered with a wire mesh, for a week to allow for acclimation after which they were crowded in a vessel with inadequate sanitation and water quality was allowed to deteriorate to allow for parasite proliferation. At the expiration of the second week, they were accessed for parasite load. Fish found to exhibit characters of parasite burden were isolated, for use.

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A stereo microscope was used to identify parasitized juvenile S. melanotheron from the culture system. The parasite load was counted on the gills and external surface of the fish which were then, weighed and placed into groups according to their weight and parasite load before exposure to the different concentrations of A. annua extracts in 250 mL beakers. Parasitized fish were also placed in de-ionized water containing 5 mL of DMSO in 250 mL beakers to serve as control. The exposure period of A. annua extracts, Praziquantel solution and DMSO in deionized water which served as the control treatment ranged from 1 to 6 h. After which the fish were individually re-examined for parasites. 2.5. Toxicity Test A. annua extracts were tested for their toxicity to juveniles of S. melanotheron during a 96 h assay. Fish mortality was examined at 12, 24, 48, 72 and 96 h, respectively at increasing concentrations of (80, 160, 320, 640 mg/L) of the extract to ascertain the safety margin for use as antiparasitic agent in aquaculture. For this purpose, twenty litre glass aquaria fitted with fine gravel on the basement and an aerator to enhance availability of dissolved oxygen were used and each filled with eight litres of the test solution and stocked with ten fish for each concentration of extract used, which were in triplicates. During the experimental period, physicochemical conditions were monitored and found to be within recommended ranges while observing for fish mortality.

3. Results
Preliminary phytochemical analysis of the components of the extracts revealed the presence of terpenoids, essential oils, flavonoids and coumarins (Table 1). At the expiration of the test period, microscopic examination of the exposed fish samples revealed complete dislodgements of parasites in some samples while a few samples had some parasites retained on them (Tables 2 and 3). This result was confirmed by an increase in agility of exposed fish samples. Toxicity test revealed a 40% mortality of fish samples exposed to the highest concentration (640 mg/L) of the extracts (Table 4). There was no fish mortality in lower concentrations of the extract during the 96 h assay period.

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Int. J. Modern Biol. Med. 2012, 1(2): 108-116 Table 1. Preliminary phytochemical screening of methanolic extract of Artemisia annua Compound Terpenoids Essential oils Flavonoids Coumerins
Key: + = Present, - = Absent.

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Solvent Methanol + + + +

Table 2. Parasite mortality against concentrations (milligrams per litre) of Artemisia annua and Praziquantel at different time intervals A. annua Conc. (mg/L) 1h Control (0 mg/L) 5 10 20 40 80 Praziquantel (2 mg/L) 0 0 0 0 0 0 5 Parasite mortality (%)/Time (h) 2h 0 0 0 0 12 10 12 3h 0 0 0 0 30 35 47 4h 0 9 11 20 50 57 60 5h 0 20 19 43 55 73 63 6h 0 38 49 66 71 80 90

Table 3. Total number of fish samples and total number of fish parasites counted before and after exposure to test solutions Test Solutions (mg/L) Control 5 10 20 40 80 Praziquantel Total number of fish samples 10 10 10 10 10 10 10 Total number of parasites on fish before test 50 58 55 58 60 60 60 Total number of parasites on fish after test 50 36 28 20 17 12 6

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Table 4. Toxicity test of concentrations (milligrams per litre) of Artemisia annua against fish mortality (percent) A. annua Conc. (mg/L) 12 h Control (0 mg/L) 80 160 320 640 0 0 0 0 40 Fish mortality (%)/Time (h) 24 h 0 0 0 0 40 48 h 0 0 0 0 40 72 h 0 0 0 0 40 96 h 0 0 0 0 40

4. Discussion
This study authenticates the report of Ekanem and Brisibe (2010) on the efficacy of artemisinin the active ingredient of Artemisia annua in dislodging fish parasites. With exception of the above named author, no work has been done on fish parasites using Artemisia annua. Artemisia annua has shown promising activity in vitro against some clinical bacteria isolates (Oriakpono et al., 2008), which was attributed to its composition and mechanism of action. The choice methanol as extracting solvent is because of its ability to extract essential oils and other plant metabolites as found in Table 1. Essential oils, terpenoids, flavonoids and coumarins are natural plant products found in specialized structures as oil or resin ducts, oil cells and glandular trichomes. They have been shown to be effective against Gram positive and Gram negative bacteria. Terpenoids and essential oils have a mechanism associated with membrane disruption by lipophilic compounds. Flavonoids are compounds synthesized by plants in response to microbial infection, and have been found in vitro to be effective antimicrobial agents against a wide array of microorganisms. Their effect is due to their ability to complex with extracellular and soluble proteins and to complex with bacteria cell walls. Coumarins have been found to stimulate macrophages which can have an indirect antimicrobial effect (Oriakpono et al., 2008). Artemisia annua is known to have a rich content of secondary metabolites known to dissuade herbivores from eating the leaves where they are found, including artemisinin and essential oils which have been reported to be effective against Gram positive and Gram negative bacteria, and other parasitic species in small ruminants, chickens and humans (Brisibe et al., 2008; Dixon et al., 1983; Jones-Brando et al., 2006; Mishina et al., 2007; Shingeharu et al., 2001; Turner and Ferreira, 2005). The effect of extracts of this plant on these organisms may be due to the synergistic activity of these secondary metabolites (Oriakpono et al., 2008). Previous report on the antiparasitic effect of this herb
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on fish parasite was reported on parasites of H. longifilis, this study has shown that the antiparasitic effect is not limited to H. longifilis, but also has potential against monogenetic trematodes of juvenile Sarotherodon melanotheron. The high concentrations of Artemisia annua used in this study were due to reports of its high therapeutic ratio (i.e. ability of organisms to withstand high concentrations of the herb). This study is timely considering the effects of bioconcentration, bioaccumulation and biomagnification of some of these synthetic substances in organisms and along the food chain which man consume for food and the need to avoid their continued use in the treatment of diseases in aquaculture as recommended by the Committee on mutagenicity of chemicals in food, consumer products and the environment (1999). A commonly used synthetic antiparasitic agent in aquaculture is praziquantel. Many of these synthetic agents commonly discharged into the aquatic environments have genotoxic potentials while causing toxicity and resistance to the antimicrobial or as in this case antiparasitic agent (Nnenna et al., 2010). Results of toxicity test of fish exposed to Artemisia annua extracts revealed that exposure time did not play a role in fish mortality; rather mortality was as a result of exposure to higher concentrations of the extract. Parasite mortality analysis showed that the highest numbers of parasites (80%) were killed in 80 mg/L concentration after six hours exposure to the extracts. Furthermore, results from this study have confirmed that the longer the duration of exposure, the greater the parasite mortality achievable as found in all the concentrations of the extracts used in this study. This was not the case with the toxicity test of fish mortality against exposure time. The same trend was recorded for the synthetic organic antiparasitic agent praziquantel, as exposure time increased, parasite mortality was also found to increase during the period. Thus, in formulating a natural plant based antiparasitic agent for fish, exposure time should be explored rather than concentration as seen in this study. A similar significant trend of increase in agility and activity of fish exposed to A. annua extracts as reported in the literature (Clark and Dickerson, 1997; Ekanem and Brisibe, 2008) was observed in this study. This may be attributed to the fact that a large number of blood cells (immune response) have been produced to enhance recovery by combating the stressor (parasites) which has been successfully eliminated without the destruction of the blood cells thus boosting the host protective immunity. Parasite induced fish mortality is currently one of the highest causes of mortality in culture fish, causing high losses particularly in places where appropriate chemotherapeutic agents are not readily administered (Awi-waadu et al., 2007; Faruk et al., 2004). Thus the incorporation of tested natural agents such as herbs with antiparasitic ability is hereby encouraged, as they will not only reduce the

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challenge of toxicity if used at recommended doses but will also reduce the incidence of fish mortality due to parasites.

5. Conclusions
Plants with known pharmaceutical relevance can be introduced for feeding and treatment after satisfactory test in the aquaculture industry. This will reduce the incidence of toxicity while reducing cost for the aquaculturist.

References
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