ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITIES OF ESSENTIAL OILS Introduction: Reactive oxygen and nitrogen Mahmoud A. Saleh, PhD; Shavon Clark; Brooke Woodard; free radicals are produced during immune Suziat Ayomide Deolu-Sobogun activity, and are triggered by several environ- mental factors such as pollution, smoke, and sunlight. Harmful effects of these reactive species include cellular damage to RNA, INTRODUCTION the antioxidant activity of essential oils DNA, proteins and lipids. In humans several from different botanical families can be diseases including those connected with the Free radicals are produced in normal found in the scientific literature. The aim heart, lung, and the eye are associated with the and or pathological cell metabolism. of this work is to conduct comparative accumulation of reactive oxygen and nitrogen evaluation of the antioxidant properties species (ROS/RNS). Antioxidants in blood, Oxidation is essential to many living cells, and tissue fluids play an important role organisms for the production of energy of 248 essential oils. in neutralizing the normal level of oxidative to fuel biological processes. However, the damage caused by these free radicals. In an uncontrolled production of oxygen de- effort to minimize the impact of environmental rived free radicals is involved in trigger- METHODS pollution on humans, identification of natural product antioxidants has become a realistic ing many diseases such as cancer, and powerful tool in the dietary and natural rheumatoid arthritis, cirrhosis and arte- We obtained 248 essential oils of products industry. riosclerosis as well as in degenerative medicinal, herbal, wild flora and mamma- processes associated with aging. Exoge- lian oils (Essential Oil University Incorpo- Methods: 248 essential oils belonging to 18 nous chemical and endogenous metabol- rated, Charlestown, IN, USA) and tested botanical families of medicinal, herbal and them for their antioxidant capabilities. wild flora as well as 2 mammalian essential oils ic processes in the human body or in the were evaluated for their antioxidant and ROS/ digestive system might produce highly RNS radical scavenging activities using high reactive free radicals, especially oxygen DPPH (2,2-diphenyl- performance thin layer chromatography/bio- derived radicals, which are capable of picrylhydrazyl) Radical autography (HPTLC) and the DPPH (2,2- Scavenging diphenyl-picrylhydrazyl) assay. oxidizing biomolecules, resulting in cell death and tissue damage.1 Almost all The assay was carried out by mixing Results: Seven percent of the tested essential organisms are well protected against free 1.5 mL methanolic solution of each of oils were found to have very high antioxidant radical damage by oxidative enzymes the essential oils with 2.0 mL of a activity; these were further fractionated by such as superoxide dismutase (SOD) .02 mM methanolic DPPH solution at HPTLC and their chemical composition was three final concentrations (5, 25 and identified using gas chromatography/mass and catalase (CAT), or by chemicals 100 mg essential oil/mL). The mixture spectrometry. such as a-tocopherol, ascorbic acid, carotenoids, polyphenols and glutathi- was then incubated in the dark for Conclusion: The majority of the active sam- one.2 When the process of antioxidant 30 minutes at 25uC and the absorbance ples showed no more than one or two spots in protection becomes unbalanced, deteri- at 517 nm was recorded as (Asample), their TLC indicating that antioxidant activity is oration of physiological functions may using a Perkin Elmer LS 50B Lumines- only associated with certain type of chemicals. cence Spectrophotometer. A blank ex- Identified active compounds were found to be occur, resulting in diseases and acceler- oxygenated monoterpenoids, monoterpene ated aging. Antioxidant food supple- periment was also carried out applying hydrocarbons as well as monoterpene phenols. ments may be used to help the human the same procedure to a solution without (Ethn Dis. 2010;20[Suppl 1]:S1-78–S1-82) the test material and the absorbance was body to reduce oxidative damage. Nat- recorded as (A ). The free radical ural antioxidants are being extensively blank Key Words: Terpenes, Free Radicals, Oxida- scavenging activity of each solution was tive Damage, Reactive Oxygen Species studied for their capacity to protect then calculated as percent inhibition organisms and cells from damage according to the following equation: brought on by oxidative stress.3 The use of essential oils as functional ingredients % Inhibition~ ÀÁ in foods, drinks, toiletries and cosmetics 100| Ablank{Asample Ablank is becoming popular.4–7 Until recently, From the Department of Chemistry, Texas Southern University, Houston. essential oils have been studied mostly from the viewpoint of their flavor and Address correspondence and reprint fragrance chemistry for flavoring foods, DPPH/Thin Layer requests to Mahmoud A. Saleh; Depart- drinks and other goods. Few studies were Chromatography Bioautography ment of Chemistry; Texas Southern Univer- sity; 3100 Cleburne Ave; Houston, Texas published on the antioxidant activity of Assay 77004; 713-313-1912; 713-313-7824 individual essential oils, but no compre- Essential oils which showed DPPH (fax); [email protected] hensive evaluation and comparison of inhibition of $90% were examined by S1-78 Ethnicity & Disease, Volume 20, Spring 2010 ANTIOXIDANT ACTIVITIES OF ESSENTIAL OILS - Saleh et al Fig 1. Bioautography TLC of the most active antioxidant essential oils, derivatized TLC is shown on the left and DPPH stain TLC is shown on the right for each oil. Numbers represent the identified chemical structures of the active compounds listed in Figure 2 thin layer chromatography (TLC) bio- 196 mL of methanol and 7 mL of MAG Reprostar 3 camera. Antioxidant autography. Ten mL of methanolic sulfuric acid. The derivatization reagent activity was confirmed when the DPPH solution of essential oil (equivalent to was added to the tank designed for the purple color changed to yellow. 50 mg of oil) were applied to Silica gel CAMAG Immersion Device III. The 60 F254 high performance thin layer plates were then attached to the device Gas Chromatography/Mass chromatography (HPTLC) plates pur- and lowered into the solution for Spectrometry (GCMS) chased from EMD Chemicals Inc., an 3 seconds removed and dried. The Thin layer chromatography spots affiliate of Merck KGaA, (Gibbstown, plates were then heated for 5 minutes that showed radical inhibition were NJ). Each sample was applied as a in the oven at 110uC until optimal scrubbed from the plates and extracted 10 mm band onto the plate using a colorization was observed. After the with methanol. Chemical composition CAMAG Automatic TLC Sampler 4 plates cooled, pictures were taken under of each active spot was identified using system and developed in a CAMAG 366 nm and white light to record the GCMS. GCMS analysis was carried out Automatic Developing Chamber results. The documentation of the TLC on a 5890 Hewlett Packard Series II Plus (ADC2) with developing solvent of plates was carried using the CAMAG gas chromatograph equipped with a 95:5 toluene/ethyl acetate. After drying Reprostar 3 system equipped with a Hewlett Packard 5972 mass selective the plates, they were viewed under DXA252 camera with a 16 mm lens. detector. A 30 m MDN-5S (Supelco) ultraviolet light at wavelengths of Identical plates were spotted and devel- fused silica capillary column (0.25 mm 254 nm and 366 nm and documented oped as described above and then id; 0.50 mm film thickness) was used with by photography. Vanillin reagent solu- dipped in a 0.2% DPPH reagent in helium as the carrier gas. The oven tion was used for the derivatization of methanol and were left for 30 minutes temperature was started at 40uC, held the plates. The vanillin reagent was at room temperature. The plates were for 5 minutes and heated at a rate of 8uC/ prepared by adding 0.7 g of vanillin to observed under white light using CA- min to 320uC and held for 5 minutes for Ethnicity & Disease, Volume 20, Spring 2010 S1-79 ANTIOXIDANT ACTIVITIES OF ESSENTIAL OILS - Saleh et al Table 1. Antioxidant activity of the tested essential oils Essential oils causing more than 90% inhibition of DPPH at the designated concentrations Family Name 100mg/mL 25mg/mL 5mg/mL Annonaceae ylang oil (Cananga odorata), US ylang oil, Madagascar ylang oil (Cananga odorata), Madagascar Apiaceae cumin seed (Cuminum cyminum), US parsley herb, Egypt cumin seed Cuminum cyminum), Egypt coriander herb (Coriandrum sativum), US coriander seeds (Coriandrum sativum), Egypt parsley herb (Petroselinum crispum), Egypt toothpick weed (Ammi visnaga), US caraway (Carum carvi), Egypt Asteraceae helichrysum oil (Helichrysum orientale), France helichrysum oil, France blue tansy oil, Morocco davana yarrow oil (Achillea millefolium), India davana yarrow oil, India green yarrow oil (Achillea millefolium) blue tansy oil, Morocco blue yarrow oil (Achillea millefolium) blue tansy oil (Tanacetum annuum), Morocco blue tansy oil WC (Tanacetum annuum), Spain Burseraceae myrrh oil (Commiphora myrrha) sea buckthorn berry (Hippophae salicifolia) Cupressaceae blue cypress oil (Callitris intratropica), Australia cypress oil (Callitris intratropica), Nepal Fabaceae Peru balsam (Myroxylon balsamum), South America Peru balsam, South America Peru balsam, South America Geraniaceae geranium oil (Pelargonium graveolens) Lamiaceae basil oil (Ocimum basilicum), China thyme oil, Morocco thyme oil, Morocco basil oil (Ocimum