African Journal of Biochemistry Research Vol. 4(6), pp. 167-174, June 2010 Available online at http://www.academicjournals.org/AJBR ISSN 1996-0778 ©2010 Academic Journals Full Length Research Paper Characterization of antioxidant and antimicrobial compounds of cinnamon and ginger essential oils G. S. El-Baroty 1, H. H. Abd El-Baky 1, R. S. Farag 2 and M. A. Saleh 3 1Department of Biochemistry, Faculty of Agriculture, Cairo University, Cairo, Egypt. 2Department of Plant Biochemistry, National Research Centre, Dokki, Cairo, Egypt. 3Department of Chemistry, Texas Southern University, Houston, Texas, USA. Accepted 6 May, 2010 Essential oils obtained from the bark of Cinnamomum zeylanicum (cinnamon) and the rhizomes of Zingiber officinale (ginger) were characterized by analytical TLC and GC/MS, and their antimicrobial and antioxidant compounds were detected by TLC-bio-autography assays. Essential oil of cinnamon bark (CEO) was found to be a unique aromatic monoterpene-rich natural source, with trans -cinnamaldehyde (45.62%) as the major constituents. Ginger oil (GEO) was characterized by high content of sesquiterpene hydrocarbons, including β-sesquiphellandrene (27.16%), caryophyllene (15.29%), zingiberene (13.97%), α-farnesene (10.52%) and ar -curcumin (6.62%). CEO and GEO oils showed significant inhibitory activity against selected strains of bacteria and pathogenic fungi, with MIC values ranging from 20 to 120 µg/ml depending upon the microbial species. Cinnamaldehyde (CA) and eugenol in cinnamon bark oil and β-sesquiphellandrene, caryophyllene and zingiberene in ginger rhizome oil were identified as the most active antibacterial components, with the aid of bioautography on TLC and GC-MS. Also, both oil exhibited appreciable in vitr o antioxidant activity as assessed by 2, 2'-diphenyl-1- picrylhydrazyl (DPPH) radical scavenging and β-carotene bleaching methods, compared to α- tocopherol, BHT and BHA. Guided isolation through TLC-autography using 0.05% DPPH and β- carotene/linoleic acid as a detection reagent led to identified CA and eugenol as main active antioxidant compounds in CEO. The significant antimicrobial and antioxidant activities of both oils suggest that it could serve as a source of compounds with preservative phenomenon. Key words: Antimicrobial, bioautographic assay, antioxidant, essential oils, ginger, cinnamon. INTRODUCTION The oxidative stress (OS), induces by reactive oxygen atherosclerosis, cirrhosis and cataracts (Halliwell and species (ROS), can be described as a dynamic imba- Gutteridge, 2000). On other hand, the spread of drug lance between the amounts of free radicals generated in resistant pathogens is one of the most threats to the body and levels of antioxidants to quench and successful treatment of microbial diseases. In addition, or/scavenge them and protect the body against their the consumption of food contaminated with food-borne deleterious effects (Shirwaikar et al., 2006). Excessive microorganisms can pose a serious threat to human amounts of ROS may be harmful because they can health. The existence of microorganisms causes spoilage initiated biomolecular oxidations which lead to cell injury and results in reduction of the quality and quantity of and death, and create oxidative stress which results possessed food (Anwar et al., 2009). Therefore, there numerous diseases and disorders such as aging, cancer, has been a growing considerable interest to identify new sources of safe and inexpensive antioxidant and anti- microbial potential of natural origin (Abdel-Baky and El Baroty, 2008; Anwer et al., 2009). *Corresponding author. E-mail: [email protected]. Ginger (Z ingiber officinale , Zingiberaceae) and cinna- mon ( Cinnamomum zeylanicum , Lauraceae) are widely Abbreviation : TLC , Thin layer chromatography. been consumed as spices and food preservation. They 168 Afr. J. Biochem. Res. are added to food products in the form of essential oils interpretation of their mass spectra and confirmed by mass spectral and various extracts (Yu et al., 2007). Also, both spices library search using the National Institute of Standards and have been employed as a folk remedy to treat of several Technology (NIST) database (Massada, 1976; Adams, 2007). diseases, disorders and ailments (Geiger, 2005; Senhaji et al., 2007). Since long time, cinnamon and ginger have Antioxidant activity been used to treat dyspepsia, gastritis, blood circulation disturbance and inflammatory diseases in many countries DPPH scavenging assay (Wang et al., 2009). Also, they show potential antipyretic, The ability of the essential oil to scavenge DPPH radical was antiallergenic, analgesic, antitussive (Gurdip et al., 2008) assessed as described by Tagashira and Ohtake (1998). Briefly, and chemopreventive activities (Sabulal et al., 2007). different concentrations of tested oil (ranged 5 - 200 µg) in 1 ml of This potential activity was believed to be attributed to the ethanol were added to 25 ml of methanolic DPPH (100 mmol/L -1) major compounds in oils such as cinnamaldehyde and solution. The mixtures were incubated in the dark at 27 ± 1°C, then zingiberene, and their activity could be multiple (Ali et al., the absorbance was measured at 517 nm for 90 min, at 15 min 2005; Singh et al., 2008; Anwer et al., 2009). However, intervals, against a blank (pure methanol). BHT, BHA and α- tocopherol (2 – 40 µg/ml) were used as reference standards. The cinnamon and ginger are locally named karfa and radical scavenging activity of the each oil was calculated from a zingabil in several Arabic countries such as Egypt, and calibration curve. All tests were run in triplicate and averaged. The are used as common beverages like tea (in tea-bags oil concentration providing 50% inhibition (IC 50 ) was calculated from form) among many people in particular in winter season, a graph representing the inhibition percentage against oil due to their protective effect and curative remedy for concentration. numerous disorders. Therefore, the aim of this study was to characterize the chemical constituents, and antimi- β-Carotene-linoleic acid bleaching crobial and antioxidant properties of essential oils obtained from two spices, viz. ginger (Z . officinale ) and The ability of the oils to prevent the bleaching of β-carotene-linoleic cinnamon ( Cinnamomum zeylanicum ). These beneficial acid was assessed as described by Abd El Baky and El Baroty characteristics could increase food safety and shelf life of (2008). An aliquot, β-carotene (0.2 mg) in chloroform (2 ml), linoleic acid (30 µL) and Tween-20 (200 mg) were transferred into round- fatty-rich foods and processed food products. bottom flask. Once the solvent was evaporated, 250 ml ultra-pure water was added and the resulting mixture was stirred vigorously. Then, 1 ml of aliquot essential oil (containing 5 – 200 µg oil and 10 MATERIALS AND METHODS mg Tween-20) was added to 50 ml reagent mixture and tested samples were subjected to thermal auto-oxidation at 50°C. At Essential oil distillation intervals up to 300 min, the absorbances of the reaction mixture (4 ml) were measured at 470 nm against a blank (1% Tween-20 The bark of C. zeylanicum and rhizome of Z. officinale were solution). All samples were done in triplicates. Antioxidant purchased from the local spices store, Egypt. About 100 g of each capacities of algal extracts were compared with those of BHT, BHA spice were subjected to hydro-distillation for 3 h using a Clevenger- and α-tocopherol (at 2-100 µg ml -1) and control. Inhibition of type apparatus. The obtained essential oil was dried over bleaching β-carotene (I %) was calculated as follows: anhydrous Na 2SO 4, filtered and stored at –10°C in a sealed vial until use. Inhibition percentage (%I) = (Abs initial –Abs sample / Abs initial ) x 100 TLC rapid antioxidant assay: TLC plates were used to detect the Determination of total phenols most active constituents in both essential oils based on spraying the separated compounds on TLC plates either with β-carotene/ The content of total phenols in the plant essential oils were linoleic acid reagent as described by Abdel Baky and El Baroty calculated using the Folin-Ciocalteau reagent method as described (2008) or with 0.05 % DPPH radical in methanol (Jaime et al., by Singleton and Rossi (1965). Eugenol as the reference standard 2005), to locate the antioxidant compounds. The protection against was used for preparation of calibration curve. the bleaching β-carotene gave orange spots and scavenging DPPH radical gave pale yellow colored spots were considered positive results. Gas chromatography/mass spectrometry (GC-MS) analysis The analysis of the essential oils were performed using GC/MS Antimicrobial assay system consisted of a HP 5890 series II gas chromatograph, HP 5972 mass detector and Agilent 6890 series auto-sampler (Agilent Preparation of bacterial cultures Technologies, USA). A Supelco MDN-5S capillary column (30 m × 0.25 mm i.d., 0.5 µm film thickness) was used with helium as the Four species of Gram-positive bacteria ( Bacillus subtilis ATCC carrier gas, at a flow rate of 1 ml/min. GC oven temperature was 6633; Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC programmed at an initial temperature of 40°C for 5 min, then heated 27840 and Micrococcus luteus ATCC 4698) and two Gram-negative up to 140°C at 5°C/min and held at 140°C for 5 min, then heated to bacteria ( Klebsiella pneumoniae ATCC 13883 and Serratia 280°C at 10°C/min and held for 5 additional minutes. Injector and marcescens ATCC 13880) were routinely used for the antimicrobial detector temperatures were 250°C. Diluted sample (1/100, v/v in assay in our laboratory. These specific strains were recommended heptane) of 1.0 µl were injected automatically. Mass spectrometry for antibacterial screening purpose (NCCLS, 1990). The bacteria was run in the electron impact mode (EI) at 70 eV. The components were sub-cultured on nutrient agar at 37°C prior to overnight growth were identified based on the comparison of their GC retention times, in nutrient broth.