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Antioxidant and Anticancer Activities of Citrus Reticulate (Petitgrain Mandarin) and Pelargonium Graveolens (Geranium) Essential Oils

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Antioxidant and Anticancer Activities of Citrus Reticulate (Petitgrain Mandarin) and Pelargonium Graveolens (Geranium) Essential Oils Research Journal of Agriculture and Biological Sciences, 5(5): 740-747, 2009 © 2009, INSInet Publication Antioxidant and Anticancer Activities of Citrus reticulate (Petitgrain Mandarin) and Pelargonium graveolens (Geranium) Essential Oils Sayed A. Fayed Biochemistry Department, Faculty of Agriculture, Cairo University, Giza, Egypt Abstract: The essential oils isolated by hydro-distillation from Pelargonium graveolens (geranium) and Citrus reticulate (petitgrain mandarin) were analyzed by GC/MS and assessed for antioxidant and anticancer activities. Twenty five components of petitgrain mandarin essential oil were identified and the major components were γ-terpinene (47.89%), methyl N-methyl anthranilate (13.17%), α-terpinene (7.40%), β-phellandrene (6.26%) and trans-isolimonene (5.87%). On the other hand, Thirty two compounds constituting 99.23% of geranium essential oil have been identified. The major components were citronellol (29.90%), trans-geraniol (18.03%), 10-epi-γ-eudesmol (8.27%), isomenthone (5.44%) and linalool (5.13%). The DPPH free radical scavenging activities of petitgrain mandarin and geranium essential oils at various concentrations ranging from 25 to 200 μg/ml were determined. The essential oils of both plants reduced the concentration of DPPH free radical with an efficacy near to that of standard antioxidant (ascorbic acid). 50% effective concentration (EC50) of petitgrain mandarin essential oil (79.84 μg/ml) was higher than geranium essential oil (66.45 μg/ml). It was indicated that the antioxidant activity of the geranium essential oil was higher than that of the petitgrain mandarin essential oil. In addition to the antioxidant activity, the anticancer activity of the two essential oils on two human promyelocytic leukemia cell lines (HL-60 and NB4) using trypan blue assay were investigated in vitro. HL-60 and NB4 cell lines were treated with the essential oils samples at different concentrations ranging from 25 to 200 μg/ml. Geranium essential oil showed the highest anticancer activity with the LC50 values of 62.50 µg/ml in NB4 cell line and 86.5 µg/ml in HL-60 cell line, whereas petitgrain mandarin essential oil gave the LC50 values of 85.05 µg/ml in NB4 cell line and 105.73 µg/ml in HL-60 cell line. The results demonstrated the potential of the two essential oils for cancer treatments. Key words: Citrus reticulate, Pelargonium graveolens, Essential oils, antioxidant, anticancer. INTRODUCTION ketones, phenols and oxides. It is estimated that there are more than 1000 monoterpene and 3000 In recent years the general public has shown an sesquiterpene structures. Other compounds include increased interest in the use of herbal medicines in phenylpropenes and specific compounds containing preference to synthetic drugs. This is based on the sulphur or nitrogen. Hundreds of new natural belief that natural products are intrinsically less substances are being isolated and identified every year, dangerous and can be obtained at a lower cost [1]. but data concerning their biological activities are Many diseases, such as cancer, atherosclerosis and known for only some [5]. The volatile compounds of inflammation are caused by free radicals and lipid oils are aromatic or odoriferous that occurs in plants as peroxidation inside human bodies. This kind of risk can such or less frequently may result from the degradation be reduced by an appropriate dietary pattern including of glycerides by enzyme action [6]. They are used in a great portion of fruit and vegetables [2,3] because of perfumes, cosmetics, flavoring food and drinks and for the great amount of natural antioxidants in these plant scenting. Medicinal uses proposed by sellers of foods [4]. essential oils vary from skin treatments to remedies for Volatile oils, also known as essential oils are cancer [7]. Essential oils exhibit various and variable lipophilic compounds containing volatile aroma antimicrobial activities, including antifungal, antiviral, compounds. The constituents of the oils are mainly antibacterial, insecticidal, and antioxidant properties [8]. monoterpenes and sesquiterpines whi ch are Geranium (Pelargonium species) is an essential oil [9] hydrocarbons with the general formula (C5H 8)n. plant that belongs to the Geraniaceae family . Out of Oxygenated compounds derived from these 25 Pelargonium species, only four species are hydrocarbons include alcohols, aldehydes, esters, ethers, important in the production of essential oil. The four Corresponding Author: Sayed A. Fayed, Biochemistry Department, Faculty of Agriculture, Cairo University, Giza, Egypt E-mail: [email protected] 740 Res. J. Agric. & Biol. Sci., 5(5): 740-747, 2009 essential oil species are P. graveolense, P. GC/MS Analysis of Essential Oil: The essential oils odoratissium, P. capitatum and P. radens [10]. The were analyzed by GC-MS according to Adams[15]. geranium essential oil is largely utilized in the GC/MS analysis was performed on a Thermoquest- perfumery, cosmetic and aromatherapy industries all Finnigan Trace GC-MS equipped with a DB-5 (5% over the world. It has since become indispensable phenyl) methylpolysiloxane column (60 m \ 0.25 mm aromatherapy oil. It is one of the best skincare oils i.d., film thickness 0.25 μm). The injection temperature because it is good in opening skin pores and cleaning was 220 ºC and the oven temperature was raised from oily complexions [9-11]. Other uses of geranium essential 40 ºC (3 min hold) to 250 ºC at a rate of 5 ºC/min, oil that are becoming more and more popular include then held at 250 ºC for 2 min; transfer line temperature the treatment of dysentery, hemorrhoids, inflammation, was 250 ºC. 1 µl of sample was injected and helium heavy menstrual flows, and even cancer. The French was used as the carrier gas at a flow rate of 1.0 community is currently treating diabetes, diarrhea, ml/min. The mass spectrometer was scanned over the gallbladder problems, gastric ulcers, jaundice, liver 40 to 500 m/z with an ionizing voltage of 70 eV and problems, sterility and urinary stones with this oil. The identification was based on standard mass library that leaves are used as a form of herbal tea to de-stress, National Institute of Standards and Technology (NIST fight anxiety, ease tension, improve circulation and Version 2.0) to detect the possibilities of essential oil cure tonsillitis [11]. On the other hand, petitgrain components. mandarin essential oil is extracted from Citrus reticulata of the Rutaceae family. This essential oil has Antioxidants Activity Using DPPH Radical some great properties to help relieve stress and Scavenging Activity: The hydrogen atom-or-electron digestive problems, but is mostly used to increase donating ability of the corresponding essential oils was circulation to the skin, reducing fluid retention and to measured from the bleaching of the purple colored help prevent stretch marks. Mandarin oil is soothing to methanol solution of DPPH. This spectrophotometric the nervous system and has a tonic effect on the assay uses the stable radical, 2,2´- digestive system, while helping flatulence, diarrhea and diphenylpicrylhydrazyl (DPPH˙), as a reagent [16]. Fifty constipation [12,13]. However, few studies on antioxidant microliters of various concentrations (25, 50, 75, 100 and anticancer activities of geranium and petitgrain and 200 μg/ml) of the essential oils in dimethyl mandarin essential oils have been performed until now. sulphoxide (DMSO) were add to 5 ml of a 0.004% The objective of current study was to evaluate methanolic solution of DPPH˙. The reaction mixture antioxidant activity using DPPH radical scavenging was covered and left in the dark at room temperature. assay and anticancer activity against two human After one hour, the absorbance was read against blank promyelocytic leukemia cell lines (HL-60 and NB4) of at 517 nm. Ascorbic acid was used as standard control. Pelargonium graveolens (geranium) and Citrus The antiradical scavenging activity of the two essential reticulate leaves (petitgrain mandarin). oils were evaluated in comparison with the reference ascorbic acid as described above for extracts. The MATERIALS AND METHODS antioxidant capacity to scavenge the DPPH radical for the oils was calculated by the following equation: Plant Material: The fresh aerial parts (stems, stalks and leaves) of Pelargonium graveolens (geranium) Scavenging effect (%) = ((Ablank – Asample)/ Ablank) × 100 purchased from experimental station of medicinal plants, Faculty of Pharmacy, Cairo University, Egypt. where Ablank is the absorbance of control reaction Fresh Citrus reticulate leaves (petitgrain mandarin) (containing each reagents except the sample), and were collected from local farm in Badr Center, El- Asample is the absorbance of sample. Behira, Egypt. The percentage of Scavenging activity was plotted against the essential oil concentration to obtain the Essential Oil Isolation: Geranium and petitgrain effective concentration (EC50), defined as the essential mandarin samples (100 g) were hydro-distilled in oil concentration necessary to cause 50% scavenging. Clevenger-type apparatus [14]. The essential oils were Tests were carried out in triplicate. collected and dried over anhydrous sodium sulphate. The essential oil samples were stored in the dark at Cell Growth and Viability Assay: Human 4ºC. The amount of oil obtained from plant material promyelocytic leukemia cell lines (HL-60 and NB4), was calculated as: obtained from the American Type Culture Collection (ATCC), were cultured in RPMI 1640 medium Oil (%
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