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Antimalarial activity gardenia lutea and sida rhombifolia ijrpp


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Antimalarial activity gardenia lutea and sida rhombifolia ijrpp

  1. 1. 234 International Journal of Research in Pharmacology & Pharmacotherapeutics Available online at www.ijrpp.com Print ISSN: 2278 – 2648 Online ISSN: 2278 - 2656 IJRPP | Volume 2 | Issue 1 | 2013 Research article In vivo Antimalarial Activity of Areal Part Extracts of Gardenia lutea and Sida rhombifolia * Baye Akele University of Gondar, College of Medicine and Health, P.o.Box 196, Gondar, Ethiopia. ABSTRACT Malaria is an endemic disease which affects 40% of world’s population and it is distributed widely, mainly due to the multi-drug resistance developed by Plasmodium falciparum. It remains the leading cause of death among parasitic diseases. Resistance to all known antimalarial drugs, except for the artemisinin derivatives, has developed to various degrees in several countries. Hence, there is a huge demand to develop antimalarial drug. In line with this notion, the hydroalcoholic leaves extracts of Gardenia lutea and Sida rhombifolia were tested for their in vivo activity against Plasmodium berghei. The extracts showed significant antimalarial activity at doses of 200, 400 and 600mg/Kg. The plant extracts also exhibited safety profile at tested doses of 500, 1000 and 2000mg/Kg. To conclude, 80% methanol extracts of Gardenia lutea and Sida rhombifolia exhibits significant antimalarial activity with acceptable margin of safety. KEY WORDS: Antimalarial, Gardenia lutea, Sida rhombifolia, and safety INTRODUCTION Malaria is one of the leading killers of children under age five, accounting for almost 1 death in 10 worldwide and nearly 1 deaths in 5 in Sub-Saharan Africa [1, 2, 3]. It is estimated to account for 300 million to 500 million illnesses and nearly 1 million deaths each year. More than 80 percent of the world’s malaria deaths occur in sub-Saharan Africa with 90 percent of those deaths in children under five years of age [4]. Malaria creates significant human morbidity, suffering and economic loss, being responsible for 70 million to 80 million cases of the global malaria burden each year and it also places a tremendous burden on national health systems and individual families [4,5,6]. The proportion of government budget allocations to health varies from less than 5% in several countries in Africa, Asia and the WHO Eastern Mediterranean Region, to well over _________________________________ * Corresponding author: E-mail address: wondimakele@yahoo.com 20% in some countries in the Americas [7]. Economists estimate that malaria accounts for approximately 40 percent of public health expenditures in Africa and causes an annual loss of $12billion, or 1.3 percent of the continent’s gross domestic product [4]. The economic impact of malaria is disproportionately felt by the poor [8]. According to the World Health Organization (WHO), malaria is endemic in 91 countries, predominantly in Africa, Asia and Latin America, with about 40% of the world’s population at risk and it is distributed widely, mainly due to the multi-drug resistance developed by Plasmodium falciparum. It remains the leading cause of death due to parasitic diseases with approximately 300 million clinical cases annually resulting in huge death, primarily in children. Resistance to all known antimalarial drugs, except for the artemisinin derivatives, has developed to various degrees in several countries [9, 10].
  2. 2. 235 Baye Akele et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2012 [234-241] Drug-resistant strains of malaria have accelerated antimalarial drug research over the last two decades. There is a consensus that new drugs to treat malaria are urgently needed. Many approaches to antimalarial drug discovery are available. While synthetic pharmaceutical agents continue to dominate research, attention increasingly has been directed to natural products. Investigation of plantderived compounds is a valid strategy, and this approach can benefit from traditional knowledge of populations from malarious regions. Natural products afforded two of the most important currently available drugs to treat malaria falciparum, quinine and artemisinin. The first one, a quinoline alkaloid, was isolated from Cinchona species used for treatment of fevers and/or malaria by South America Peruvian Indians and has been a template for the synthesis of chloroquine, antimalarial drug that was used extensively. Artemisinin is responsible for the antimalarial activity of Artemisia annua, a species of millenar traditional use in China [11]. The success of artemisinin, isolated from Artemisia annua, and its derivatives for the treatment of resistant malaria has focused attention on the plants as a source of antimalarial drugs [11]. The world’s poorest are the worst affected, and many treat themselves with traditional herbal medicines. Ethnobotanical information about antimalarial plants, used in traditional herbal medicine, is essential for further evaluation of the efficacy of plant antimalarial remedies and efforts are now being directed towards discovery and development of new chemically diverse antimalarial agents. Indeed, in malaria endemic areas, plant remedies are still widely used but mostly without assurance of their efficacy. Validation of traditionally used plants to treat malaria is important and requires clinical trials which must be preceded by phytochemical and toxicological studies that are necessary to guarantee efficacy and safety of herbal preparations [12, 13]. The plant Sida rhombifolia is claimed for its promising antimalarial activity in Ethiopia, China [14] and India [15] traditional medicine. While, an in vitro study previously conducted on extracts from the fruit pulp of Gardenia lutea has shown a counter activity against 3D7-chloroquine and pyrimethamine sensitive and Dd2-chloroquine resistant and pyrimethamine sensitive Plasmodium falciparum strains [16]. These plants are claimed for their antimalarial activity based on either in vitro activity test or traditional folk knowledge though there is no remarkable in vivo studies have been reported so far to strengthen the preclinical study profile. Therefore, this study aims at investigating the in vivo antimalarial activity of extracts from a traditionally used medicinal plants, Gardenia lutea and Sida rhombifolia. METHODS Plant Collection and sample preparation The leaves Sida rhombifolia were collected in the outskirt of Woldia town, North Wollo, Ethiopia and Gardenia lutea was collected outskirt of Adirkay, Western Tigray, Ethiopia. The plants were collected from Decemer, 2011 to January 2012. Taxonomical identification was carried out at National Herbarium, Department of Biology, Faculty of Science, Addis Ababa University. The plant parts were garbled and dried in the processing room, and were then powdered and kept at room temperature in a well-closed and amber colored bottle until extracted. Each of the powdered leaves (100g) of Gardenia lutea and Sida rhombifolia were extracted by cold maceration with 500 ml of 80 %( v/v) methanol. Maceration was carried out for 48 hrs with intermittent shaking with a shaker. The extracts were then filtered with filter paper (Whatman No 3) and the marcs were remacerated. This extraction was repeated four times using 300ml hydroalcohol at a time. The successive filtrates were collected and concentrated with Rota Vapour at 400C to remove alcohol. The remaining water was dried in an oven at 400C with intermittent stringing and the dried plant extracts were weighed, packed in a glass container and kept in a refrigerator for future use. In vivo antimalarial activity In vivo antimalarial activity test of each extract was performed using a 4-day standard suppressive test [17]. This is the most widely used preliminary test, in which the efficacy of a compound is assessed by comparison of blood parasitemia and mouse survival time in treated and untreated mice [18]. Plasmodium berghei, chloroquine sensitive plasmodium strain that is most widely employed in rodent malaria parasite, was used to infect Swiss albino mice for a four day suppressive test. The P. berghei was subsequently maintained in the laboratory by serial blood passage from mouse to mouse. For the study, a donor mouse with a rising www.ijrpp.com
  3. 3. 236 Baye Akele et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2012 [234-241] parasitemia of 20% was sacrificed, and its blood was collected in a slightly heparinized syringe from the auxiliary vessels. The blood was diluted with Trisodium Citrate (TC) medium so that each 0.2 ml contained approximately 107 infected red cells [5]. Each animal received inoculum of about 10 million parasites per gram body weight, which is expected to produce a steadily rising infection in mice. The infection of the recipient mice was initiated by needle passage of the above mentioned parasite preparation, from the donor to healthy test animals via an intraperitoneal route [5]. Therefore, P. berghei infected red blood cells were intraperitoneally injected into the mice from the blood diluted with TC medium so that each 0.2 ml had approximately 10 6 - 107 infected red cells (parasite per kg of body weight). Each mouse was infected with single inoculum of 0.2 ml blood. On day 0, the test mice were injected with 0.2 ml 7 of 2X10 parasitized erythrocytes, (P. berghei ANKA strain) intraperitoneally. After 2 hr, the infected mice were weighed and randomly divided into four groups of six mice per cage. Three of the groups were made to receive extract treatments, while the fourth group received the vehicle % Paracetaemia = % Suppression = (negative control) and the fifth received chloroquine (the standard antimalarial drug). Groups 1, 2 and 3, which served as treatment groups, received the extracts orally at 200mg/Kg, 400mg/kg and 600mg/Kg doses respectively [5]. Group 4, served as a negative control, received the vehicle (7% Tween 80, 3% ethanol in water. Group 5 received the standard drug chloroquine phosphate (10 mg/kg) and served as a positive control [19]. On days 1 to 3, animals in the experimental groups were treated again (with the same dose of the extract and same route daily) as in the day 0. On day 4 (i.e. 24 hr after the last dose or 96 hr postinfection), blood smear from all test animals was prepared using Giemsa stain. Level of parasitemia was determined microscopically by counting 4 fields of approximately 100 erythrocytes per field. The difference between the mean value for the negative control group (taken as 100%) and those of the experimental groups was calculated and expressed as a percentage of suppression. Percentage parasitaemia and percentage suppression were calculated using the following formula: Number of infected RBC X100 Number of total RBC Paracetaemia in negative control- Parastemia in treatment group X100 Paracetaemia in negative control Untreated control mice typically die in about one week after infection. For treated mice the survivaltime (in days) was recorded, and the mean survival time was calculated in comparison with that of the negative group [20-23]. In vivo acute toxicity test The extracts were tested for their oral acute toxicity in mice. Three groups of mice, each group consisting of six male mice, were used for testing acute toxicity. The mice in each group were fasted over night and weighed before test. Test extracts were dissolved in 70% Tween 80 and 30% ethanol. This solution was further diluted 10-fold with sterile distilled water to give a stock solution containing 7% Tween 80 and 3% ethanol [21]. Mice in groups one and two were given orally 500,1000 and 2000 mg/kg/day of the each extracts respectively while mice in the control group (group three) were treated with the vehicle. After administration of the substance food was withheld for a further 2 hr period [22]. The mice were closely observed during the first 30 minutes after dosing. They were then observed periodically during the first 24 hr (with special attention to the first 4 hr) and once daily thereafter for a total of 14 days. Attention was given to toxicity signs including changes in skin, eyes (blinking), tremors, convulsion, lacrimation, muscle weakness, sedation, urination, salivation, diarrhea, lethargy, sleep, coma and also death. Twenty-four hours later, the % mortality and weight of mice in each group and for each test compound at each dose level was recorded [23]. The toxicity study was designed to demonstrate the approximate safe dose that could be used for subsequent experiments rather than to provide complete toxicity data on the compounds. www.ijrpp.com
  4. 4. 237 Baye Akele et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2012 [234-241] Data analysis Results of the study were expressed as mean ± standard deviation. Statistical significance for suppressive test was determined by one-way ANOVA at 95% confidence limits (p=0.05). Data on body weight and survival time were analyzed. All the data were analyzed using Microsoft office excel 2007. lutea and Sida rhombifolia extracted by maceration with 80% methanol and the percentage yields were 12.5% and 10.25% respectively. In Vivo antimalarial activity test The 4-day suppressive test, which is a commonly used and standard test for antimalarial screening [5], was used in this study. The percentage of parasitemia and percentage of inhibition of the plant extracts is depicted in table 1. RESULT Extraction Dried and pulverized leaves of 200g Gardenia Table 1: Parasitemia suppressive test of hydro alcoholic extracts of leaves extracts of Gardenia lutea and Sida rhombifolia against P. berghei in mice Test substance Dose in mg/Kg % parasitemia % inhibition 200 29.4 + 0.31 53.4 400 27.7 + 0.23 56.1 600 26.9 + 0.15 57.3 Vehicle (-) 1ml 63.1+ 0.27 00 Choroquine ( +) 10 00 100 200 27.3 ± 0.19 50.1 400 26.1 ± 0.41 52.3 600 25.2 ± 0.17 53.9 Vehicle (-) 1ml 54.7 ± 0.12 00 Chloroquine (+ ) 10 00 100 1. 2. Gardenia lutea Sida rhombifolia P ˂ 0.05 and each value is expressed as mean±SD In Vivo acute toxicity test Gross behavioral and physical observation like hair erection, urination, muscle weakness, sedation and convulsion, reduction in feeding activity in the test mice were used as indicators of acute toxicity effects. The test mice were monitored once daily for 14 days but no sign of toxicity was observed in mice treated with Gardenia lutea extract while minor effects were observed for Sida rhombifolia. Increased in body weight is observed in mice treated with hydroalcoholic extract of Sida rhombifolia. During the first 24hr of the experimental period no death occurred in any of the test groups. The result of acute toxicity study is shown in Table 2. www.ijrpp.com
  5. 5. 238 Baye Akele et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2012 [234-241] Table 2: Data for the acute toxicity studies Test substances Dose mg/Kg Wt. before test Wt. after test Gardenia lutea 500 32.5 + .12 32.2 + 0.0 0 1000 29.7 + 1.1 30.6 + 0.37 0 2000 30.4 + .28 30.4 + 0.20 0 500 29.4 + .34 31.1 + 0.26 0 1000 28.2 + .07 29.6 + 0 .11 0 2000 26.1 + .17 29.6 + 0 .39 0 1ml/100g 31.4 + .5 31.53 + 0.07 0 Sida rhombifolia Control ( -) % Mortality Key: Values are M ± SD, P<0.05 DISCUSSION The percentage yield of Gardenia lutea and Sida rhomnifolia extracts in this study were 12.5% and 10.25% respectively. Logeswari et.al [24] obtained a percentage yield of as high as 7.8% while Poojari et al [25] obtained 18.8% for Sida rhombifolia extract. Geographical and seasonal variation of the collected plant materials, method of extraction and eluting power of solvent could account for this variation. Roots and leaves extracts ( 80% methanol) of Gardenia lutea possessed moderate in vitro activity against both tested P. falciparum strains in the range 2.55 - 6.13µg/ml against K1 strain and 1.65 µg/ml against NF54 strain. However, the stem extract did not show any antimalarial activity [26]. An experiment which was done by Ahmed et al [16] demonstrated that fruit pulp extract of Gardenia lutea showed greater than 96.47 % inhibition of Plasmodium falciparum at a concentration of 50 µg/ml when activity test was performed in vitro. Roots extracts of Gardenia lutea also showed some cytotoxicity towards cultured cell lines [26]. In addition to its antimalarial activity, its stem bark 80% methanol extract of exhibited significant antimicrobial activity against common pathogens including Bacillus cereus, Neisseria gonorrhea, Shigella flexineri and Shigella dysentriea [27]. The tabulated results of this study indicated that hydro alcoholic extracts of Gardenia lutea displayed a very good in vivo activity against the P. berghei malaria parasite. The comparison analysis indicated that 200 mg/kg hydro alcoholic extract of Gardenia lutea showed statistically significant difference on day 4 parasitemia level, compared to the negative control. The antimalarial activity of Gardenia lutea dose dependent, i.e., as the dose increase from 200mg/kg to 600mg/Kg, the percentage of inhibition creeps from 53.4 to 57.3%. Hence, this experiment substantiates other in vitro studies carried out so far Ahmed et al [16], Ali et al [26] and others. This study also attests the traditional use of the plant in different parts of the world including Ethiopia. The methanol extract of the plant constituents unsaturated phenols, triterpenes and saponins [16, 27]; however, it is devoid of flavonoids, cardenolides, cyanogenic glycosides and anthraquinone [27]. Several investigations have been published in the field of antiplasmodials of plant origin related to different bioactive functional groups classified as: terpenoids, alkaloids, unsaturated fatty acids, volatile oils and phenolic compounds including flavonoids and quinines [27]. Hence, the antimalarial activity of Gardenia lutea is most probably attributed to unsaturated phenols, triterpenes, and saponins. Isolation and structural elucidation of these classes of compounds could lead to the discovery of novel antimalarial agent. Many in vitro activity tests have been done so far on the plant Sida rhombifolia which attests its wide array of activity. Sida rhombifolia exhibits very good antioxidant effect against in vitro DPPH assay method for its high phenolic content [28, 29].It has anti-inflammatory activity via xanthine oxidase inhibition [29, 30]. Furthermore, oral administration of hydroalcoholic leaves extract (400 mg/kg) of Sida rhombifolia decreased oedema induced by carrageenan injection which clearly demonstrates its anti-inflammatory activity by another mechanism [31]. The ethanol extract of dried aerial part of Sida rhombifolia produced significant (P<0.001) writhing inhibition in acetic www.ijrpp.com
  6. 6. 239 Baye Akele et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2012 [234-241] acid-induced writhing in mice at the oral dose of 250 and 500 mg/kg of body weight comparable to the standard drug diclofenac sodium at the dose of 25 mg/kg of body weight [32,33]. The crude ethanolic extract also produced the most prominent cytotoxic activity against brine shrimp Artemia salina [33]. Apart from all these experimental studies, the plant Sida rhombifolia is claimed for its promising antimalarial activity in china [14], Ethiopia [5] and India [15] traditional medicine. As clearly depicted in table 1, hydro alcoholic extracts of Sida rhombifolia showed a very good activity against the P. berghei malaria parasite. Like Gardenia lutea, 200 mg/kg hydro alcoholic extract of sida rhombifolia showed statistically significant difference on day 4 parasitemia level, compared to the negative control and its effect is dose dependent. The antimalarial activity of Sida rhombifolia is comparable to Gardenia lutea. Hence, the experimental result of this study makes clear the traditional claim and use of Sida rhombifolia for malaria treatment. The extract of dried leaf part of Sida rhombifolia showed the presence of reducing sugar, steroids, alkaloids, gums, flavonoids and glycosides [14, 24]. The leaf extract also contains ascorbic-acid, ash, beta-carotene, beta-phenethylamine, carbohydrates, fat, fiber, niacin, protein, pseudoephedrine, riboflavin, saponin, tannins, thiamin, triterpenoids and essential metals [14]. In general, many experimental works confirmed that diverse chemical classes are present in different extract of Sida rhombifolia. Hence, rigorous isolation and activity test should be done to unravel the general chemical class and the specific compound responsible for its antimalarial activity. Considering the toxicity profile of the extracts, the data indicated that the extract of Gardenia lutea has not showed significant change between weight before and after test. However, the effect in change in body weights of Sida rhombifolia extract is significant. This increase in body weight may attribute to increase in appetite of mice, the presence of several micro nutrients and immunomodulatory substances, in addition to the anti-parasitic activity [14, 24, 33]. During the first 24hr of the experimental period no death occurred in any of the plant extract. This is in line with the work of Assam et al [33] that no abnormal symptoms and death of the rats was observed up to 16 g/kg in hydro extract of Sida rhombifolia. The research finding of Assam et al [33] further illustrates the significant change of liver enzymes such as ALT, AST, ALP and CRT at higher dose. In general, Sida rhombifolia extract can be classified as non toxic since the limited dose of an acute toxicity is generally considered to be 5.0 g/kg [33].Gardenia lutea is also very safe drug at least up to the maximum dose used in this experiment, i.e., 2000mg/Kg. CONCLUSION The aqueous-methanol (1v:4v) extract of Gardenia lutea and Sida rhombifolia demonstrated effective in vivo antimalarial activity. These plants extracts also exhibited safety profile at a maximum dose of 2000mg/kg. Further research needs to be carried out to identify the active molecules and evaluate sub-acute or chronic toxicities. ACKNOWLEDGMENT The author would like to thank the Ministry of Science and Technology, Federal Democratic Republic of Ethiopian for financing this research project. REFERENCE [1] Oliveira, R.B., Souza-Fagundes, E.M., Soares, R. P., Andrade, A. A., Krettli, A.U. and Zani, C. L. (2008). Synthesis and antimalarial activity of semicarbazone and thiosemicarbazone derivatives. Eur. J. of Med. Chem., 43: 1983-1988 [2] Kalra, S., Chawla, P., Gupta, N. and Valecha, K. (2006). Screening of antimalarial drugs: an overview. Indian J. Pharmacol, 38: 5-12 [3] United Nations Children’s Fund (2007). Malaria & children: Progress in intervention coverage.Geneva, 513. http://www.unicef.org/ progressforchildren /2007n6/index_4183.html (Accessed on 03.12.2010) [4] The President’s Malaria Initiative (2009). Working with communities to save lives in Africa; Third annual report. http:// www.pmi.gov/ (Accessed on 17.11.2010) www.ijrpp.com
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