Antimicrobial Activity of Extracts Obtained from Anacardium Excelsum Againts Some Pathogenic Microorganisms
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Emir. J. Food Agric. 2011. 23 (3): 249-257 http://ejfa.info/ Antimicrobial activity of extracts obtained from Anacardium excelsum againts some pathogenic microorganisms C. Celis∗, A. García, G. Sequeda, G. Mendez and R. Torrenegra Phytochemistry Laboratory, Chemistry Department, Pontificia Universidad Javeriana, Cra 7 N 43-82 Bogotá, Colombia Abstract: In this study,we evaluated the antimicrobial activity of secondary metabolites presents in extracts and fractions of Anacardium excelsum (Anacardeaceae), using agar well diffusion assay and bioautography. As a general result was determined that Anacardium excelsum has good antimicrobial activity against gram positive microorganisms. Regarding the fractions in study, these were tested against Bacillus subtilis where the integument, seed and seed coat showed the best antimicrobial activity. Analysis by gas chromatography (GC) with mass selective detector (MSD) established the presence of some phenolic compounds like 2-(1,1-dimethylethyl)-4-(1,1,3,3-tetramethylbutyl)phenol, which is responsible of the activity found. Key words: Anacaurdium excelsum, microbial activity, phenolic compounds ﻧﺸﺎط ﻣﻀﺎدات اﻟﻤﻴﻜﺮوﺑﺎت ﻟﻤﺴﺘﺨﻠﺺ ﻧﺒﺎت Anacardium excelsum ﺿﺪ ﺑﻌﺾ اﻟﻜﺎﺋﻨﺎت اﻟﺤﻴﺔ اﻟﺪﻗﻴﻘﺔ اﻟﻤﺮﺿﺔ ﺳﻲ. ﺳﻠﻴﺲ*، ﺁ. ﻗﺎرﺳﻴﺎ، ﺟﻲ. ﺳﻴﻜﻮﻳﺪا, ﺟﻲ. ﻣﻴﻨﺪز و ﺁر. ﺗﻮرﻳﻨﻴﻨﻴﻘﺮا ﻣﺨﺘﺒﺮ اﻟﻜﻴﻤﻴﺎء اﻟﻨﺒﺎﺗﻴﺔ , ﻗﺴﻢ اﻟﻜﻴﻤﻴﺎء ، ﺟﺎﻣﻌﺔ ﺑﻮﻧﺘﻴﻔﻴﺴﻴﺎ ﺟﺎﻓﻴﺎرﻳﻨﺎﻧﺎ، آﺮا 7N 43-82ﺑﻮﻗﻮﺗﺎ, آﻮﻟﻤﺒﻴﺎ اﻟﻤﻠﺨﺺ: ﻓﻲ هﺬﻩ اﻟﺪراﺳﺔ ﺗﻢ ﺗﻘﻴﻴﻢ ﻧﺸﺎط ﻣﻀﺎدات اﻟﻤﻴﻜﺮوﺑﺎت ﻓﻰ اﻟﻤﺮآﺒﺎت اﻟﺜﺎﻧﻮﻳﺔ اﻟﻤﻮﺟﻮدة ﻓﻲ ﻣﺴﺘﺨﻠﺼﺎت وﻣﻘﺎﻃﻊ ﻧﺒﺎت (Anacardium excelsum (Anacardeaceae، ﺑﺎﺳﺘﺨﺪام ﻃﺮﻳﻘﺔ ﻓﺤﺺ ﻓﻲ ﻣﺎدة اﻻﺟﺎر bioautography . آﻨﺘﻴﺠﺔ ﻋﺎﻣﺔ ﺗﺤﺪدت ان ﻧﺒﺎت Anacardium excelsum ﻳﻤﺘﻠﻚ ﻧﺸﺎط ﺟﻴﺪ ﺿﺪ اﻟﻤﻴﻜﺮوﺑﺎت اﻟﻤﻮﺟﺒﺔ ﺿﺪ اﻟﻜﺎﺋﻨﺎت اﻟﺤﻴﺔ اﻟﺪﻗﻴﻘﺔ اﻟﻤﻮﺟﺒﺔ ﻟﺼﺒﻐﺔ ﺟﺮام. وﻓﻴﻤﺎ ﻳﺘﻌﻠﻖ ﺑﺎﻟﻤﻘﺎﻃﻊ ﻓﻘﺪ ﺗﻢ اﺧﺘﺒﺎرهﺎ ﺿﺪ ﻣﻴﻜﺮوب Bacillus subtilis ﺣﻴﺚ اﻇﻬﺮت اﻓﻀﻞ اداء وﻧﺸﺎط ﻣﻀﺎد ﻟﻠﻤﻴﻜﺮوﺑﺎت ﻓﻲ ﻣﺴﺘﺨﻠﺼﺎت اﻟﺒﺬور واﻟﻐﻄﺎء اﻟﺨﺎرﺟﻲ ﻟﻠﺒﺬور. وﺑﺎﻟﺘﺤﻠﻴﻞ ﺑﻮاﺳﻄﺔ gas chromatography وﻣﻊ ﺟﻬﺎز (mass selective detector (MSD ﺗﻢ اﻟﻌﺜﻮر ﻋﻠﻰ ﺑﻌﺾ اﻟﻤﺮآﺒﺎت اﻟﻔﻴﻨﻮﻟﻴﺔ ﻣﺜﻞ (dimethylethyl)-4-(1,1,3,3-tetramethylbutyl-1,1)-2 وهﻲ اﻟﻤﺴﺆوﻟﺔ ﻋﻦ هﺬا اﻟﻨﺸﺎط. ∗Corresponding Author, Email: [email protected] 249 C. Celis et al. Introduction The wild cashew (Anacardium excelsum; Material and Methods Anacardium rhinocarpus, Rhinocarpus excelsa), Samples of plant material were collected Ber. et Balb (Bartholomaus, 1995) is a tree in during the flowering season in the metropolitan the flowering plant Anacardiacea. The tree is area of Bucaramanga, Santander-Colombia, common in the Pacific and Atlantic watersheds, under the following coordinates 7°06'32 .51''N growing from the sea level to 1000 m, found as and 73°06'15.19''W, at elevation of 986 m. far north as Guatemala and extending south into Specimens of the plats were identified by the Ecuador. It received different names like cajuhy National Herbarium of Colombia as or cashu (Brasil), espavel (Costa Rica), nariz Anacardium excelsum under classification (Cuba), caschou (Guyana), caracoli (Panamá, number 520397 (Figure 1). The material was Ecuador, Colombia), merey (Venezuela) thoroughly washed in water; leaves, fruits, (Cáceres, 1980), (Programa Colombia forestal, flowers, bark, seed, seed coat and integument 2005). In Colombia is found in the lower valley were separated and dried to the environment for of Atrato river, in the foothills of Andes 15 days until humidity between 7 and 15% mountains and in the Colombian Amazon (Figure 1). The dried material was powdered to region (Cáceres, Meneses and Quintero, 1980). size of 5 mm and stored in sterile conditions at It is a large evergreen tree growing to 45 m 3°C until use. From 400 to 600g of dried tall, with a straight, rose-hued trunk reaching material were brought under solid-liquid 3m in diameter, brown color with medium size extraction by soxhlet technique for 15 days to lenticels equi-dimentionals, and few obtain the total extract in ethanol. Partial liquid- inconspicuous (Maderas Colombianas, 1970). liquid extraction was made in continuously The leaves are simple, alternate, oval-shaped, using solvents in different polarities. Total 15–30 cm long and 5–12cm broad (Cáceres, extracts and fractions were concentrated using Meneses and Quintero, 1980). The flowers are vacuum at 40°C (Santos et al., 2010). The produced in a pinnacle up to 35cm long, each antimicrobial activity was tested against flower small, pale green to white (Smith et al., Staphylococcus aureus and Bacillus subtilis 2004, Espinal, 1963). The true fruit is a 2–3cm (Gram positive) and Escherichia coli and long drupe shaped like a kidney supported by a Salmonella (Gram negative) (Dobner et al., green stalk (Cáceres, 1980). The nuts of wild 2003). These microorganisms were obtained cashew are a treat for monkeys but raw nuts from the strain collections of Microbiology contain oil poisonous for man. Roasted nuts are Department of Pontificia Universidad Javeriana- free from the poison and as tasty as their Bogota (Colombia). It was tested by agar well relatives, cashew nuts. In Valledupar region in diffusion on Mueller Hinton medium. Four Colombia is used cooked and ground to make a holes of 5mm diameter were made by each petri kind of edible bread called Caracoli bread dish and 50μl of extracts and fractions at (Araujo, 2008; Smithsonian, 2008). different concentrations (10, 20 and 40 mg/mL) Maturation occurs in from March to May dissolved in dimethyl sulfoxide (DMSO). The (Nichols, 1991). Anacardium is one of the most remained hole was use like positive control studied genera, determining multiple chemical (Gentemicin) or negative control (DMSO). Then and biological activities; these are summarized assay samples were incubated for two days at in Table 1. The aims of this study were to 35°C and readings were made at 24 and 48 determine the antimicrobial activity of total hours. extracts and fractions of Anacardium excelsum against some microorganisms pathogens such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella, and determinate the kind of chemical compounds related to such activity. 250 Emir. J. Food Agric. 2011. 23 (3): 249-257 http://ejfa.info/ Table 1. Anacardium genus plants with chemical and biological activity Plant Part Used Method / Assay / Determination Activity / Effect Metabolite Reference Fruit: Anacardic acids RP-HPLC. Anion superoxide. Auto Anacardic acids: acid 6- extracted and HPLC oxidation. Lipoxigenasa. DPPH. Uric Antioxidant. Kubo et al., 2006 pentadec(en) salicylic. purified. Acid generate by Xantina Oxidized Fruit: Anacardic acids HPLC. Nano-ESI-MS-MS, GC-MS and Alkyl phenols: extracted and HPLC NMR. Cinamic acid. Anion superoxide. Antioxidant. Anacardic acids, Trevisan et al., 2006 purified. Uric acid. 2-deoxi guanosine. cardanols and cardols. Induced septic shock. Plasma leakage in Extract from stem bark. Inflammatory. No reported. Olajide et al., 2004 mouse skin. Extract from bark. Agar Diffusion. Antimicrobial. Tannins, alkaloids. Akimpelu, 2001 Anacardium Fruit: Anacardic acids Acid 6- occidentale extracted and HPLC Macrodilution. Antibacterial. Kubo et al., 2003 alqu(en)ilsalicylic. purified. Anacardic acids and Fruit Extracts. RP-HPLC. Agar Dilution. Antibacterial. Kubo et al., 1995 cardanols. Anacardic acids, Fruit Extracts. RP-HPLC. UV-VIS. IR. RMN. Antimollusk. cardanols, cardol and 2- Kubo et al., 1986 methyl-cardol. Mutagenicity.Promotion-initiation in Mutagenic, carcinogenic, George and Kuttan, Fruit Extracts. No reported. mouse skin tumors. Cocarcinogenic. cocarcinogenic. 1997 Anacardium All tree (in situ). Estimation of carbon in trees. Carbon Interchange. No reported. Losi et al., 2003 excelsum Anacardium Maceration. Qualitative assay of Total Extract. Anti ulcer. Bi flavonoids. Ferreira et al., 2004 humile functional groups. Anacardium HPLC. HRGC-MSD. RMN. Citotoxicity Alkyl resorcinols and Fruit Extracts. Antitumor. Ramos et al., 2004 giganteum of human cell lines. acid 6-alkilsalic. Anti-arthritic Necrosis tumor factor. Analysis Ramprasath et al., Nuts Extracts. (rheumatoid arthritis). No reported. hispatologic and radiologic. 2006 Metabolism of bones. Anacardium Fruits Extracts in Monitoring of lipids, enzymes of lipid Anti-carcinogenic. No reported. Veena et al., 2006 semecarpus formulation ingredients. and lipoprotein metabolism. Superoxide dismutase. Catalase and Ramprasath et al., No reported. glutathione peroxidase. Lipid peroxides Antioxidant. No reported. 2005. in tissues. Glutathione reductase. 251 C. Celis et al. (a) (b) (c) (d) Figure 1. (a) Anacardium exelsum plant with flowers; (b) fruit; (c) powdered integument and (d) powdered fruit. The antibacterial effect was classified where was generated growth inhibition of based upon inhibition zone diameters, halo pathogenic microorganism. To identify the greater than 5mm present activity and halo kind of compounds with antimicrobial activity iqual or lower than 5mm without activity (Jasir was made a column chromatography according et al., 2004). Thin layer chromatography (TLC) to the results of TLC and bioautography and and bioautography was realized to fractions the fractions obtained were analyzed by GC- with microbial activity using Silica gel G60 MSD. F alumina backed plates (3cm × 8cm). Two 254 Results and Discussions TLC plate were run, in the first, the sample was The results of extractions performed with applied directly and the chromatograph was different solvents polarities are shown in Table run. The layers were developed with Vanillin 2. The efficiency for ethanol extractions in dry to determine the region with active compounds.