International Journal of Applied Research in Natural Products Vol. 6 (4), pp. 33-42. Directory of Open Access Journals ©2008-2013. IJARNP-HS Publication
Chemical composition and biological activities of essential oils of Azadirachta indica A. Juss. El-Hawary SS1, El-Tantawy ME2, Rabeh MA1,*, Badr WK2 1
2
Department of Pharmacognosy, Faculty of pharmacy, Cairo University, Cairo, Egypt. Department of phytochemistry, National Organization of Drug Control and Research, Cairo, Egypt.
Summary. Essential oils of Neem, Azadirachta indica A. Juss. (family Meliaceae) leaves and flowers were prepared by hydrodistillation method. The chemical composition of the oil samples was investigated by GC/MS. Hydrocarbon constituted 85.36% of the leaves oil .The major compounds were β-Elemene (33.39%), γ- Elemene (9.89%), Germacrene D (9.72%), Caryophyllene (6.8%) and Bicyclogermacrene (5.23%) while the percent of the oxygenated compounds were (5.04%) mainly attributed to sesquiterpene oxide. On the other hand, flowers oil hydrocarbons constituted 63.22% composed mainly of pentacosane (18.58%), tetracosane (10.65%), β-germacrene (9.73%), βcaryophyllene (5.84%) and dodecene (4.54%) while the percent of the oxygenated compounds were 28.3% mainly attributed to octadecanol (16.7%), verdiflorol (5.32%), farnesol (1.63%) and α– terpineol (1.51%). The antioxidant properties determined by 2, 2-diphenyl-1picrylhydrazyl assays, antibacterial activity against Gram-positive and Gram-negative, antifungal and larvicidal activities were promising and in relation with the chemical composition of the essential oils. The results indicated that essential oil of flowers could be especially promising as an inexpensive source of effective antioxidant /antimicrobial /larvicidal agents tantamount to fixed oil of the neem seeds. Industrial relevance. The use of medicinal plants is a universal phenomenon. Natural products from plants are rich source to identify, select and process new drugs for medicinal use. Most of research focused on fixed oil of neem seeds but very little was concerned about volatile oils of leaves and flowers. The diverse biological activities of Neem essential oils can be applied on a large scale as antioxidant, antimicrobial and larvicidal agents comprising many important benefits including their volatility, lower level of risk to the environment than with synthetic ones. Keywords. Azadirachta indica; Neem; essential oil; GC/MS; antioxidant; antibacterial; antifungal; larvicidal.
INTRODUCTION Neem (family. Meliaceae, genus. Azaddirachta) is an evergreen tree growing in Egypt with flowers that are small, white, and fragrant. It is one of the most well known plants indigenous to India and is cultivated in tropical and subtropical regions worldwide (Bailey, 1953; Engler, 1964). Every part of the tree has been used as traditional medicine for household remedy against various human ailments, from antiquity (Chopra et al., 1956, Chopra et al., 1958, Kirtikar and Basu, 1975, Koul et al., 1990, Chatterjee and Pakrashi 1994). Neem has been considered as a potential source of many therapeutic agents; earlier research on Neem showed that it contains various active constituents with diverse medicinal properties. (Bhuiyan et al., 1997). Dholi et al., 2011 reported on its antidiabetic activity (Dholi et al., 2011). In addition, the aqueous extract of Neem leaves had shown a good therapeutic potential as anti hyperglycemic agent (Sonia Bajaj, and Srinivasan B.P 1999). Saseed et al. (2008) and El-Mahmood et al. (2010) supported the use of Neem seeds for treatment of infectious diseases especially those involving the eye and ear. Maragathavalli et al., (2012) had reported on the antimicrobial activity of the methanolic and ethanolic leaf extracts against human pathogenic bacteria. The alcoholic extract of Neem flowers has the potential of being ideal antifertility agent (Gbotolorun et al., 2008). Amer et al., 2010 have reported on the chemotherapeutic and anti viral activities of the aqueous extract, of Neem leaves and seeds. Very little was traced about the essential oil of neem so, the aim of this study was to investigate the chemical composition, antioxidant, antibacterial, antifungal and larvicidal activities of essential oils of flowers and leaves of Azadirachta indica A. Juss. commonly known as "Neem".
MATERIALS AND METHODS Plant material. leaves and flowers of Azadirachta indica were collected during October and May, respectively, from the Agriculture Research Center (Giza [29.979161N, 31.134178E], Egypt). They were identified by Prof. Dr. Monir Mohamed abd Elghany, The Herbarium, Botany department, Faculty of Science, Cairo University, Egypt. Reagents for antioxidant assay. 2, 2-diphenyl-1- picryl hydrazyl (DPPH) and Ascorbic acid were purchased from Sigma Chemicals Co. and dimethyhylsulfoxide (DMSO) was purchased from Merck. Test microorganisms. Nine bacterial strains; Staphylococcus aureus (RCMB 010027), Staphylococcus epidermidis (RCMB 010024), Streptococcus pyogenes (RCMB 010015), Neisseria gonorrhoeae (RCMB 010076), Proteous vulgaris (RCMB 010085), Klebsiella pneumoniae (RCMB 0010093), Shigella flexneri (RCMB 0100542), Pseudomonas aeruginosa (RCMB 010043) and Escherichia coli (RCMB 010056) and four fungal strains; Aspergillus fumigates ((RCMB 02564), Candida albicans (RCMB 05035), Geotricum candidum (RCMB 05096) and Trichophyton mentagrophytes (RCMB 0925) were used in this study. They were obtained from the Regional Center of Mycology and Biotechnology Antimicrobial Unite (RCMB), Cairo, Egypt. Culture media and antibiotics for antibacterial assay. Brain Heart Infusion as liquid and solid media (HiMedia), Mueller- Hinton agar (HiMedia) were used. Ampicilin (Oxoid, UK) and Gentamycin (Oxoid, UK) were used as standard antibacterial agents. Culture media and antibiotics for antifungal assay. Potato Dextrose Agar medium (HiMedia), Amphotricin B (Sigma Chemical Co., St. Louis, Mo.) was used as a standard antifungal agent. ___________________ *Corresponding Author.
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El-hawary et al.
Mosquito larvae. Culex pipiens eggs were obtained from the Insect Research Institute, Doki, Giza, Egypt and were kept in dechlorinated tap water to bread the larvae .The 3rd instars' larvae were selected for the experiments since it is the most sensitive larval stage. Preparation of the essential oils. Leaves and flowers of Azadirachta indica were subjected to hydrodistillation (Egyptian Pharmacopeia, 2005). The essential oils obtained were dried over anhydrous sodium sulphate and kept at 4◦C for analysis. Analysis of the essential oils. GC/MS analysis of the essential oil was carried out on an Agilent 6890 equipped with a mass spectrometric detector (MSD), model Agilent 5973, equipped with an HP-5MS column (30 m × 0.25 mm, 0.25 μm); programming from 80 (3 min) to 260 ◦C at 8 ̊C/min, 10 min hold; carrier gas, helium; flow rate, 1.0 ml/min; injection in split mode (60.1); injector and detector temperatures 225 and 300 ◦C, respectively. The EIMS mode at 70 ev; electron multiplier, 2500 V; ion source temperature, 250◦C; mass spectra data were acquired in the scan mode in the m/z range 50 to 700. The essential oil components were identified by comparing their mass fragmentation patterns with those of the available reference (Adams, 2009). In addition, qualitative analysis was carried out by using internal normalization method (peak area measurement) and compound identification was confirmed by electronic Wiley and NIST mass spectral data base. The retention indices (RI) of the volatile oil components were determined relative to the retention times of series of hydrocarbons. Results are given in tables 1, 2 and 3. Determination of antioxidant activity by the DPPH radical scavenging assay. The antioxidant activity of A. indica flowers' and leaves' essential oils was assayed by the DPPH free radical scavenging assay as described by Dudonné et al. (2009) with slight modifications. Briefly; 10 µL of different concentrations of essential oils (5-35 µg/ml in DMSO) was added to 190 µL of ethanolic solution of DPPH. The reaction mixtures were incubated at 37˚C for 30 min. The absorbance was measured at 517 nm using Spectramax 340 USA (molecular devices). Appropriate blank (DMSO) and standard (Ascorbic acid in distilled water) solutions were prepared and run simultaneously. The concentration of sample required for 50% scavenging of the DPPH free radical (EC50) was determined. Each determination was carried out in triplicate. The average 50% scavenging concentration was then calculated using the following equation. DPPH radical scavenging = {(A control- A sample)/ (A control)} x 100 Where, A control = Absorbance of control and A sample = Absorbance of tested sample. IC50 value (the concentration required to scavenge 50% DPPH free radicals) was calculated. DPPH scavenging activities of the extracts were expressed as the mean of EC50 ± S.D. Determination of the antibacterial activity. The antibacterial activity of essential oils of leaves and flowers of A. indica was screened by the agar disc diffusion method described by Bayer et al., 1966 and Sahoo et al., 2006 with slight modification. The bacterial cultures were grown in Brain Heart Infusion broth at 37 °C. After 6 h of growth, 100 μL of each microorganism at a concentration of 1x106 cells/mL, was inoculated on the surface of Mueller-Hinton agar plates. DMSO with a concentration up to 2% was used to dissolve the essential oils. Filter paper discs (6 mm in diameter) saturated with 20 μL of the tested essential oils or DMSO (solvent control) were placed on the surface of the inoculated plates. To evaluate the efficiency of the methodology; 50 μL of each essential oil was inserted simultaneously in a hole made in new plates. The plates were incubated at 37 °C for 24 h. The diameter of the inhibition zone was measured in millimeter, and was recorded as mean±SD of a triplicate experiment. Ampicillin (10μg), Gentamicin (10μg) discs for bacteria were used as positive standard. Cultured species producing halos equal to or greater than 7 mm were considered susceptible to the tested essential oil. Determination of the antifungal activity. The antifungal activity of essential oils of leaves and flowers of A. indica was screened was carried out as described above, with slight modification, using Potato Dextrose Agar plates. The plates were incubated at 32 °C for 72 h. The diameter of the inhibition zone was measured in millimeter, and was recorded as mean±SD of a triplicate experiment. Amphotericin B (5μg) for fungi were used as positive standard. Determination of the Minimal Inhibitory Concentration (MIC) was carried out by a serial broth dilution method described by (NCCLS, 1993). Briefly; The essential oils of leaves and flowers of A. indica were diluted in DMSO and were added to 5 ml sterile MHB tubes to give different concentrations (1.0 – 50.0 μL/ml). Later, 0.5 ml of the exponentially growing microbial broth culture of the strains that were sensitive by disc diffusion test was inoculated into respective test tubes. Another set of tubes containing only the growth medium without DMSO (control) and with DMSO (solvent control) and each of the test strains was set up separately. The tubes were incubated at 37°C for 24 h and the growth was measured by measuring optical density at 520 nm using spectrophotometer. The MIC was regarded as the lowest concentration of the extract that inhibited the growth of bacteria or fungi Determination of larvicidal activity on mosquito larvae of Culex pipiens. Third instar larvae of Culex pipiens were obtained from insect research institute, Doki, Giza. The colonies were maintained continuously at 28 ± 1 °C with 16.8 light and dark photo period in 65% ± 5% relative humidity. Larvicidal tests were performed according to the World Health Organization (WHO, 1973). The essential oils were first dissolved in DMSO. Different concentrations of the essential oils in distillated water and DMSO were prepared. For larval test, 100 larvae were collected by a strainer with fine mesh and then were transferred to a 400-ml glass beaker gently by tapping. Controls included mosquitoes from the colony exposed to water and DMSO alone. The larvae were exposed to different concentrations from 0.5- 5.0 ppm of essential oil in distilled water for 24 h. Each concentration has been replicated four times comprising 100 larvae. Glass beakers were left at room temperature and mortality was recorded after 24 h, 48h and 72h of exposure. Mortality percentage was always corrected according to Abbott's formula (1925) when the mortality among control larvae exceeded 10%. Statistical Analysis. The results were subjected to statistical analysis. Values were reported as Mean ± SD. The data were analyzed using Student’s t-test to test for differences between treatment and control where a value of P
