SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF CASHEW LEAVE

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SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF CASHEW LEAVE

CHAPTER TWO

Literature review

African flora in general and Abuja in particular, have an important reserve of aromatic, food and medicinal plants. It was demonstrated that medicinal plants play an important role in the African pharmacopoeia. About 80% of Africans have recourse to traditional medicine that involves the use of plants’ active principles, to treat most of diseases (Alagesaboopathi, 2011). Nigeria is covered with a large number of plant species, some of which have been used for centuries in folkloric medicines to diagnose, prevent and treat various ailments (ElMahmood et al., 2010) some of these plants such as Guava, Calotropis procera, Trema orientalis, and Cnidoscolus aconitifolius, have been investigated for heir antibacterial properties and have recorded positive results (Akin-Osanaiye et al., 2018; Akin-Osanaiye and Okholoma, 2018; Akin-Osanaiye et al., 2016; Akin-Osanaiye et al., 2015).

Anacardium occidentale has a height of 5-10 m, but in clay land can reach up to 20 m. It has a crooked trunk of 25-40 cm in diameter. The leaves are oval, obovais, leathery, glabrous; rosy when young; it has vinaceas flowers, arranged in terminal panicles (Lorenzi, 2008). The family is rich in important secondary metabolites with varieties of interesting biological activities (Abu-Reidah et al., 2015).

Several ethnobotanical studies have focused on identifying medicinal plants species. Among these plant species, Anacardium occidentale L. has an important place (Chabi-Sika et al., 2014). Its leaves, bark, roots, stem are traditionally used for the treatment of numerous diseases such as, allergy, cough, stomach ache, diarrhoea, skin infections (Chabi-Sika et al., 2013; Chabi-Sika et al., 2014). Besides these medicinal uses, cashew plays several other important roles. Its wood is used mainly in carpentry, as firewood, or turned into charcoal, whereas the resins are used in the manufacture of plastics and natural insecticides (Akinwale, 2000).

Phytochemicals are plant metabolites (Sofowora, 1993) which act as natural defence systems for host plants, and also provide characteristic colour, aroma and flavour in specific plant parts. They are a group of non-nutrient compounds that are biologically active when consumed by human. Many phytochemicals are health-promoting and are prevent many diseases (Birt, 2006).

CHAPTER THREE

MATERIALS AND METHODS

Materials Plant Material

The leaves of A. occidentale L., were collected in Giri, Abuja and was taken for botanical identification in the herbarium of University of Abuja Biological Garden. The leaves were dried under room temperature till constant weight was obtained; the dried leave sample was grinded to powder form using a blending machine for effective extraction with solvents.

Test Organisms

Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were obtained from the Department of Microbiology, University of Abuja Teaching Hospital.

Methods Extraction of Plant Material

The powdered A. occidentale was extracted using sterile distilled water and ethanol following the procedure described by Bankole et al., (2012). One hundred gram of A. occidentale leaf powder was soaked in 500 ml of 96% ethanol at room temperature. For the aqueous extraction, 100 g of A. occidentale leaf powder was soaked in 500 ml of sterile distilled hot water. The mixtures were allowed to stand for 24 h after which they were filtered using a fine mesh cloth. The solvent was evaporated to dryness using water bath at 40oC.

Phytochemical Constituents

All the extracts were subjected to standard phytochemical qualitative screening for secondary metabolites as described by Trease and Evans (2002), Sofowora (1993) and Harborne (1998).

Confirmation of purity and Viability of test Organisms

The pure isolates of Staphylococcus aureus was sub-cultured from nutrient agar slants on Mannitol Salt Agar and observe for growth after 24 h at 37°C, yellow colonies was seen on the mannitol salt agar. Pure isolates of Escherichia coli was subcultured from nutrient agar slants onto Eosine Methyl Blue Agar and observe for growth after 24h at 37°C, colonies with metallic green sheen was seen. Pure isolate of Pseudomonas aeruginosa was subcultured from nutrient agar slants onto Cetrimide Agar and observe for 24 h at 37 °C, blue-green colonies was seen.

Preparation of Test Inoculum

The test bacteria from the agar slant was subcultured on nutrient agar by stocking and incubated at 37 oC for 18-24 h. A bacteria suspension was made using the 18-24h culture in 4 ml of physiological saline; its density was adjusted to match the 0.5 McFarland density.

Antimicrobial Activity of plant Extract

The ethanol and aqueous extracts were reconstituted in sterile distilled water into different concentrations (500 mg/ml, 250 mg/ml, 125 mg/ml and 62.5 mg/ml) by doubling dilution. The antibacterial activity of the different concentrations of the extracts was carried out using agar well diffusion assay technique of Bankole et al. (2012).

Aliquot (20 µl) of the bacterium in physiological saline was spread on already solidified Mueller Hinton agar surface using a sterile cotton swab. The surface of the medium was allowed to dry for 3 min and sterile 6mm cork borer was used to bore holes on agar plate (two each), the base of each hole was sealed with a drop of molten agar to avoid diffusion of extract under agar. Some (20 µl) of each extract concentrations was dispensed into each hole. The plates were allowed to undisturbed for about 15 min before they were incubated at 37°C for 24 h. The diameter of zones of inhibition was measured using a transparent ruler and the zone of inhibition was measured in mm.

Minimum Inhibitory Concentration (MIC)

The MIC of A. occidentale extract was determined using broth tube dilution method described by Kacaniova et al., (2011). Eight (8) sterile test tubes were labelled 1 through 8 and arranged in a rack. A. occidentale extract control tube and growth control tube was used as a quality control. Nine (9) ml of sterile nutrient broth was added to each tube, and then 1 ml of A. occidentale extract was added to test tube number 1 and growth control tube with sterile pasture pipettes. Then serial dilutions were performed by transferring 1 ml of A. occidentale extract into the second tube with separate sterile pasture pipette and vortexed for homogenization. After thorough mixing, 1 ml was transferred with another sterile pasture pipette from tube 2 and tube 3. The procedure was repeated until the 7th tube. The growth control tube received no A. occidentale extract. One (1) ml of the culture of 0.5 McFarland standard of test bacteria was added to each tube. Tubes were then incubated at 37 °C for 24 h and observed by visual inspections for the presence and absence of growth.

Minimum Bactericidal Concentration (MBC)

To determine the MBC, incubated tubes showing no visible sign of growth/turbidity in MIC, were sub-cultured onto sterile Mueller Hinton agar plates by streak plate method and incubated at 37 °C for 24 h. The least concentration of extract that did not show growth of test organisms was considered as the minimum bactericidal concentration (Kacaniova et al., 2011). The inoculated plates were scored as bactericidal if no growth occurred after 24 h; bacteriostatic if there is light to moderate growth.

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