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Sky Journal of Biochemistry Research Vol. 2(6), pp. 37 – 41, November, 2013
Available online http://www.skyjournals.org/SJBR
2013 Sky Journals
Full Length Research Paper
occidentale) leaf extract enriched with zinc in wistar
Udedi S. C1*., David E. E2., Igwilo I. O1., Ekwealor K. E3., Enemali M. O4., Bamidele, T. O4., Ifemeje J. C.5 and Asogwa
1Department of Applied Biochemistry, Nnamdi Azikiwe University, Awka, Nigeria. 2Department of Biochemistry, Renaissance University, Enugu, Nigeria. 3Department of Botany, Nnamdi Azikiwe University, Awka, Nigeria. 4Department of Biochemistry, Nasarawa State University, Keffi, Nigeria. 5Department of Biochemistry, Anambra State University, Uli, Nigeria.
Accepted 9 October, 2013
This study investigated the antidiarrhoeal activity of zinc, aqueous extract of cashew leaf and zinc enriched leaf
extract. Diarrhoea was induced in wistar albino rats by the administration of 2 ml of castor oil. The negative
control group received normal saline while the positive control group received loperamide- a standard
antidiarrhoeal drug. Zinc (15, 30 and 45 mg/kg), aqueous extracts of cashew leaf (100, 250 and 500 mg/kg) and
Zinc plus extract (500 mg/kg extract with 15 mg/kg zinc), were administered orally to different groups of rats.
Diarrhoealfaeces and colonal nitric oxide concentration were determined. The lowest dose of 15 mg/kg zinc
showed the highest significant reduction in both the diarrhoealfaeces and colonal nitric oxide concentration
relative to the castor oil control group. Aqueous extract (500 mg/kg) had the highest reduction in
diarrhoealfaeces and colonal nitric oxide concentration relative to the castor oil control group.There was a
higher reduction in diarrhoealfaeces and colonal nitric oxide concentration in extract with zinc group than that
obtained for the cashew leaf alone. An excellent antidiarrhoeal activity was obtained on the co-administration of
loperamide and zinc. The result showed that zinc, cashew leaf and cashew leaf extract enriched with zinc are
good antidiarrhoeal agents but the later gave the best antidiarrhoeal activity.
Key words: Anarcadium occidentale, antidiarrheal activity, castor oil, nitric oxide, nitric oxide synthase.
Diarrhoea is the passing out of increased amount of
pathological conditions of diarrhoea and an effector loose stool as a result of an irritation of the mucous substance in both laxatives and antidiarrhoeal agents membrane of the intestine, resulting in imbalance (Mourad, 1999). Nitric oxide (NO) is synthesised from L- between secretion and absorption of water (Kazi and arginine by the enzyme nitric oxide synthase (NOS). This Henry, 2006). Nitric oxide, an endogenous signaling complex reaction involves the transfer of electrons from molecule has been considered a mediator of the NADPH, via the flavins FAD and FMN in the carboxy- terminal reductase domain,tothehaem in the amino- terminal oxygenase domain, where the substrate L- arginine is oxidised to L-citrulline and NO (Andrew and Mayer,1999). The enzyme is a homodimeric protein with 125- to 160-kDa per monomer and the haem is responsible for the dimerization (Andrew and Mayer, 1999). The dimer is structurally stabilized by a zinc ion MATERIALS AND METHODS
which is situated at the oxygenase domain interface of the dimer (Fischmann et al., 1999). The zinc ion is Animals and diet
tetrahedrally coordinated by four cysteine (two from each monomer - Cys109 and Cys104). In mammals, NOS Wistar albino rats obtained from the animal house of appears as 3 isozymes: neuronal NOS (nNOS), cytokine- Veterinary Department of University of Nigeria, weighing inducible NOS (iNOS) and endothelial NOS (eNOS). The between 64 – 72 g were used for the study. The animals zinc ion is found at a region which connects the N- were acclimatized at room temperature in a standard wire terminal hook and the subunit core. The coordination of cages for 7 days prior to commencement of the zinc arranges the N-terminal hooks so that they interact experiment. During the period of study, the animals were with their own subunit. However, when there is no zinc fed with standard diet and water. In this study, all the ion present, two of the thiolate ligands (cysteines) form a animals experimentation were carried out according to disulfide bond connecting the two subunits (Craneet al., 1999). Little wonder both nitric oxide and zinc is implicated in diarrhoea. It is a public health problem, generally caused by poor sanitation and hygiene and kills Collection of plant materials
some 194,000 Nigerian children under five every year (UNICEF, 2012). As diarrhoeal diseases are oral in Fresh leaves of cashew tree were collected. Botanic nature, one of the simplest and most inexpensive barriers identification was performed at the Department of Botany, to infection is hand washing with soap or ash at critical Nnamdi Azikiwe University Awka, Nigeria and voucher times, such as before handling food and after defecation or changing a diaper. Hygiene promotion and proper hand washing is the most cost effective of public health intervention against diarrhoeal diseases. However, Preparation of aqueous cashew leaf extract
poverty is inextricably linked to poor sanitation and hygiene in developing countries. It therefore appears that Fresh samples of cashew leaves were washed, chopped poor sanitation and hygiene persist as poverty persists in into smaller pieces, air dried in the laboratory and then developing countries and the use of antidiarrhoeal drugs ground to powder. 300 g of the powder was mixed with are indispensable. Antidiarrhoeal drugs are expensive 250 ml distilled water for 24 h. The mixture was filtered and are not easily accessible by majority of the rural poor using a glass funnel plugged with a white sieve cloth. The people who still rely on traditional healing practices and resulting filtrate was evaporated using gentle heating at medicinal plants for their daily health care needs 20°C using water bath. A deep brownish coloured (10 g) (Jebunnessa et al., 2009), despite immense technological concentrated extract was obtained which was kept in advancement in modern medicine. Cashew is a multipurpose tree of the Amazon that grows up to 15 m high. It has a thick and tortuous trunk with branches so winding that they frequently reach the ground. Cashew Chemicals
trees are often found growing wild on the drier sandy soils in the central plains of Brazil and are cultivated in Loperamide (reference antidiarrhoeal drug), castor oil many parts of the Amazon rainforest (Abulude et al., (laxative) were purchased from Golden pharmaceutical, 2010). Cashew leaf or bark tea is still widely used Ltd, Enugu, Nigeria. Zinc gluconate, citrullin solution, throughout the tropics as an effective diarrhoea and colic diacetylmonoxime, potassium persulphate, trichloroacetic remedy, considered gentle enough for children (Aiswariya acid, saline, acetate buffer and urease solution were of et al., 2011). Zinc deficiency often develops with purchased from Sigma Chemical Company Ltd, St. Louis, abdominal pain, vomiting and fever (Li et al., 1999) indicating that the intestine is one of the tissues most sensitive to zinc deficiency. Intestinal zinc loss has been reported in children with dehydrating diarrhoea (Bhutta et Induction of diarrhea
al., 2000). The objective of this work therefore, is to establish a scientific basis for the use of the cashew leaf The method proposed by Meite et al. (2009) was used to as an antidiarrhoeal agent and to establish its further induce diarrhoea. Adult rats were fasted for 18 h and Table 1. The effect of zinc on castor oil induced diarrhoea in wistar albino rats.
Total amount of
Total amount of
Concentration of nitric
Result = mean S.E.M. asignificantly higher compared to normal control. bSignificantly lower than the castor oil control group. cSignificantly lower than normal group. P < 0.01. Table 2. Effect of aqueous cashew leaf extract on castor oil induced diarrhea.
Total amount of
% reduction of
Concentration of nitric
oxide (mg/100 ml)
Values are expressed as Mean ± S.E.M. asignificantly higher compared to normal control. bsignificantly lower than the castor oil control group. csignificantly lower than normal group. P < 0.01. divided into groups of four animals each. Following the Data analysis
oral administration of 2 ml castor oil to induce diarrhea, the animals were treated and placed in separate wired Data were analyzed using the students‘t’ test distribution. cages for observation. The cages were covered with filter The results were expressed as a mean ± S.E.M. At 99% paper to identify both solid and diarrhoealfaeces. The confidence interval, p < 0.01 was considered statistically total faeces and wet diarrhoealfeaces were recorded over a period of three hours. The percentage diarrhoea inhibition was calculated as function of the castor oil % inhibition = (control-test) × 100/control. The effect of zinc (15, 30, 45 mg/kg) on castor oil induced diarrhoea in wistar albino rats are shown in Table 1. At 15 The colonal nitric oxide concentration was determined by mg/kg, zinc significantly decreased (p < 0.01), the total measuring colonalcitrullin concentration using a method number of wet faeces by 44% (6.67 ± 1.53) upon described by Gornall and Hunter (1941) since both administration of castor oil (12.0 ± 1.73). Colonal nitric products were produced in equimolar concentration. oxide concentration level also decreased significantly Briefly, the colon was excised after 3 h of treatment, from (1.62 ± 1.73) mg/100 ml castor oil control group to instilled isotonic saline and incubated with acetate buffer pH 5.0. Urease solution and trichloroacetic acid were The effects of aqueous cashew leaf extract on castor oil added to break down urea and for deproteinisation induced diarrhea are shown in Table 2. The aqueous respectively. After a brief heating, potassium per sulphate extract of cashew leaf showed significant antidiarrheal was added to the filtrate to develop citrullin color. The activity (p < 0.01) against castor oil induced diarrhoea in absorbance was read at 440 nm and citrullin rats. It reduced the number of diarrhoealfeaces produced concentration obtained using a citrullin standard by castor oil administration from (12.0 ± 1.73) 100% control group to (11.6 ± 1.53) 3%, (8.67 ± 1.15)28%,
40 Sky J. Biochem. Res.
Table 3. The effect of zinc enriched cashew leaf extract on castor oil induced diarrhea.
Total amount of
Total amount of
% inhibition of nitric
Results are expressed as mean S.E.M. asignificantly higher compared to normal control. bSignificantly lower than the castor oil control group. cSignificantly lower than normal group. P < 0.01. (7.00 ± 1.00) 42% when experimental animals were significantly reducing both diarrhoealfaeces and colonal respectively administered 100, 250 and 500 mg/kg of leaf nitric oxide from castor oil control group 12.00 ± 1.73 and 1.62 ± 1.73 mg/100 ml to (4.33 ± 1.53) 64% and (1.16 ± The effects of zinc enriched cashew leaf extract on castor oil induced diarrhea are shown in Table 3. Zinc-enriched leaf extractsgave 9.30 ± 3.06and 4.33 ± 1.53 as the total amount of faeces and total amount of wet faeces Conclusion
respectively while the % inhibition of nitric oxide synthase and the concentration of nitric oxide are 64% and 1.16 ± The results strongly established that zinc, cashew leaf extract and zinc-enriched cashew leaf extracts are good Discussion
As shown in Table 1, zinc-enriched loperamide at 75%
Abulude FO, Ogunkoya MO, Akinjagunla YS (2010). Phytochemical
reduction (3.00 ± 0.70) has a better antidiarrheal activity Screening of Leaves and Stem of Cashew Tree Anacardium than loperamide alone with diarrhoea reduction of 66% occidentale. Electr J. Environ. Agric. Food Chem., 9(4): 815-819. (4.00 ± 2.65). Colonal nitric oxide concentration showed Aiswariya G, Reza H, Radhika G, Rahul VS (2011). Study for the same pattern. Although the pathophysiological Antihelminthic Activity of cashew apple Anacardium occidentale extracts. Int. J. Pharma. Sci. Rev. Res., 6(1): 44-47. mechanism that either link zinc deficiency with severe (1999). Enzymatic function of nitric oxide diarrhoea or explain the efficacy of zinc in reducing diarrhoea is not yet well understood, successful clinical Bhutta ZA, Bird SM, Black RE, Brown KH, Gardner JM, Hidayat A, studies concluded that the possible mechanism for the Khatun F, Martorell R, Ninh NX, Penny ME, Rosado JL ,Roy SK, Ruel M, Sazawal S, Shankar A (2000). The therapeutic effects of beneficial effect of zinc on the duration of diarrhea oral zinc in acute and persistent diarrhea in children in developing involves improved absorption of water and electrolyte by countries: pooled analysis of randomized controlled trials. Am. J. Clin. the intestine and faster regeneration of gut epithelium (Mourad, 1999). Another publication established that zinc Crane BR, Rosenfeld RJ, Arvai AS, Ghosh DK, Ghosh S, Tainer JA, Stuehr DJ, Getzoff ED (1999). N-terminal domain swapping and inhibits cAMP-induced calcium-secretion by inhibiting metal ion binding in nitric oxide synthase dimerization. EMBO. J., basolateral potassium channel in in-vitro studies with rat ileum (Kazi and Henry, 2006). The leaf extract (Table 2) Fischmann TO, Hruza A, Niu XD, Fossetta JD, Lunn CA, Dolphin E, is believed to stimulate the re-absorption of water from Prongay AJ, Reichert P, Lundell DJ, Narula SK, Weber PC (1999). Structural characterization of nitric oxide synthase isoforms reveals the intestinal lumen resulting to the normalization of the striking active-site conservation. Nat. Struc. Biol., 6(3): 233-242. deranged water transported across the mucosal cells Gornall AG, Hunter AA (1941). Colorimetric method for the which are seen in the type of faeces produced. Cashew determination of citrullin. Biochem. J., 35(5-6): 650-658. leaf extracts also reduced the colonal concentration of Jebunnessa A, Shaikh BU, Mahabuh MZ, Rasheda A (2009). Antidiarrhoeal activity of ethanolic extracts of Mitragynadiversifolia. J. nitric oxide, the endogenous mediator of diarrhoea from castor oil control group of 1.62 ± 1.73 mg/100 ml to 1.56 Kazi M, Henry J (2006). Zinc in the treatment of acute diarrhea: current ± 0.07, 1.47 ± 0.10, 1.40 ± 0.05mg/100ml when 100, 250, status and assessment. Gastroenterol., 130: 2201-2205. 500 mg/kg of leaf extracts where administered. Though Li C, Yoji T, Masefumi W (1999). Nitric oxide synthase inhibitor attenuates intestinal damage induced by zinc deficiency in rats. Nutr. the reduction was not significant, the result suggests that the extracts might have an inhibitory effect on colonal Meite S, Ngnessan J, Guina G (2009). Antidiarrheal Activity of Ethyl nitric oxide synthase. As shown in Table 3, zinc-enriched Acetate Extract of Moringamorindoides in rats. Trop. J. Pharm. Res., leaf extracts showed a better antidiarrheal activity by Mourad FH (1999). Role of nitric oxide in intestinal water and electrolyte UNICEF (2012). Water, Sanitation and Hygiene in Nigeria. Annual
Original Article Characterization of Staphylococcus species isolated from raw milk and milk products (lben and jben) in North Morocco Abdrezzak Bendahou,1,2 Mariam Lebbadi,3 Latifa Ennanei,2 Fatima Z. Essadqui,1 Mohammed Abid.2 1Département de Sécurité Alimentaire & Environnementale, Institut Pasteur, Tanger, Maroc 2Département de Recherche scientifique, Laboratoir