ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITIES OF ESSENTIAL OILS
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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 of this work is to conduct comparative
and or pathological cell metabolism.
accumulation of reactive oxygen and nitrogen
Oxidation is essential to many living evaluation of the antioxidant properties
species (ROS/RNS). Antioxidants in blood,
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
pollution on humans, identification of natural
rived free radicals is involved in trigger- METHODS
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 rated, Charlestown, IN, USA) and tested
botanical families of medicinal, herbal and
nous chemical and endogenous metabol-
ic processes in the human body or in the them for their antioxidant capabilities.
wild flora as well as 2 mammalian essential oils
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 .02 mM methanolic DPPH solution at
such as superoxide dismutase (SOD)
HPTLC and their chemical composition was three final concentrations (5, 25 and
identified using gas chromatography/mass and catalase (CAT), or by chemicals
spectrometry. such as a-tocopherol, ascorbic acid, 100 mg essential oil/mL). The mixture
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 using a Perkin Elmer LS 50B Lumines-
only associated with certain type of chemicals. oration of physiological functions may
occur, resulting in diseases and acceler- cence Spectrophotometer. A blank ex-
Identified active compounds were found to be
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 (Ablank). The free radical
Key Words: Terpenes, Free Radicals, Oxida- ural antioxidants are being extensively
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~
in foods, drinks, toiletries and cosmetics
100| Ablank {Asample A blank
From the Department of Chemistry,
is becoming popular.4–7 Until recently,
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); saleh_ma@tsu.edu hensive evaluation and comparison of inhibition of $90% were examined by
S1-78 Ethnicity & Disease, Volume 20, Spring 2010ANTIOXIDANT 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 S1-79ANTIOXIDANT 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 basilicum), US thyme oil, Spain thyme oil, Spain
thyme oil red (Thymus vulgaris), Morocco thyme oil, Germany thyme oil, Germany
red Basil oil (Ocimum basilicum) basil oil, China basil oil, China
linalool marjoram oil (Origanum marjoram) oregano oil, Turkey oregano oil, Turkey
sweet Monarda oil (Monarda didyma) catnip oil, Canada catnip oil, Canada
oregano oil (Origanum vulgare) from Turkey
thyme oil (Thymus vulgaris) from Germany
thyme oil (Thymus vulgaris) from Spain
catnip oil (Nepeta cataria) Canada
catnip oil (Nepeta cataria) USA
Lauraceae laurel leaf oil (Laureia sempervirens), Crete laurel leaf oil, Crete laurel leaf il, Crete
cinnamon bark oil (Cinnamomum verum), Ceylon cinnamon leaf il, Ceylon cinnamon leaf oil, Ceylon
cinnamon leaf oil (Cinnamomum verum), Ceylon
Myoporceae Buddha wood oil (Eremophila mitchelli), Australia Buddha wood oil, Australia Buddha wood oil, Australia
Myristicaceae Nutmeg (Myristica fragrans) Nutmeg
Myrtaceae clove bud oil (Eugenia caryophyllata), Madagascar clove bud oil, Madagascar clove bud oil, Madagascar
clove bud oil (Eugenia caryophyllata), India wild bay oil, Jamaica wild bay oil, Jamaica
allspice, Jamaica
wild by oil (Laurus nobilis), Jamaica allspice, Jamaica
allspice (Pimenta Berry), Jamaica
eucalyptus lemon oil, US
Piperaceae Cubeb Oil (Cubeb berries)
Poaceae spruce oil (Picea mariana) citronella oil, Java, Indonesia vetiver oil, Surinam
black spruce oil (Picea mariana) vetiver oil, Surinam
citronella oil (Cymbopogon winterianus), Indonesia
vetiver oil (Chrysopogon zizanioides), Surinam
Rosaceae rose oil (Rosa damascena), Bulgaria rose oil, Bulgaria rose oil, Bulgaria
Istanbul rose oil (Rosa damascena), Egypt Istanbul rose oil, Egypt
Rubiaceae coffee (Coffea arabica)
Rutaceae combava petitgrain oil (Citrus hystrix)
yuzu oil (Citrus junos)
S1-80 Ethnicity & Disease, Volume 20, Spring 2010ANTIOXIDANT ACTIVITIES OF ESSENTIAL OILS - Saleh et al
Table 1. Continued
Essential oils causing more than 90% inhibition of DPPH at the designated concentrations
Family Name 100mg/mL 25mg/mL 5mg/mL
Solanaceae chili pepper (Capsicum annuum), India chili pepper, India chili pepper, India
Moschidae musk deer (Moschus spp) musk deer
ambergris sperm whale (Physeter macrocephalus) ambergris sperm whale oil
Cedars of Lebanon flowers, Egypt, Lebanon, Spain flowering dogwood tree
gulf breeze (Oleaceae asmin)
Istanbul rose (Grandiflorum)
a run time totaling 45 minutes. The split RESULTS comparing antioxidant activity among
injector was at 220uC with a split ratio of the essential oils at each designated
1:20. Quantities of data are based on We tested the antioxidant activity of concentration. Samples having less than
peak percentage calculation using Hew- the essential oils at three different 90% inhibition were eliminated from
lett Packard Chemstation software. The concentrations of 5, 25 and 100 mg/ each group and designated as negative
compounds were identified by the mass mL using % DPPH inhibition as or insignificant antioxidant effect at
spectral library search and by comparing described in the methods section. We each concentration. Sixty of the essential
their GC retention times. used a cut off of 90% inhibition for oils were found to be active at a
Fig 2. Chemical structure of the active antioxidant compounds
Ethnicity & Disease, Volume 20, Spring 2010 S1-81ANTIOXIDANT ACTIVITIES OF ESSENTIAL OILS - Saleh et al
concentration of 100 mg/mL. Twenty they are safe to use, yet provide good in order to find possible alternatives to
seven of the essential oils were found to defense against oxidative damage and synthetic antioxidant preservatives such
be active at a concentration of 25 mg/ associated health effects. as butylated hydroxytoluene (BHT) and
mL and 17 of the oils were found to be phenolic compounds.
active at a concentration of 5 mg/mL.
(Table 1) CONCLUSIONS
Essential oils obtained from the ACKNOWLEDGMENTS
families of Asteraceae (sunflower family), Natural products are in increasing This work was supported by RCMI grant #
Fabaceae (Leguminosae), Lamiaceae R003045 and NASA/URC grants NCC
demand from the manufacturers of
165-9.
(Mint Family), Lauraceae (Cinnamon foods, cosmetics and pharmaceuticals.
family), Myoporaceae (Buddleja family), Thus the importance of conducting
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