sign in sign up
<<
Home , Chili pepper

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, . 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. 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 seed (Cuminum cyminum), US , Egypt cumin seed Cuminum cyminum), Egypt herb (Coriandrum sativum), US coriander seeds (Coriandrum sativum), Egypt parsley herb (Petroselinum crispum), Egypt toothpick weed (Ammi visnaga), US (Carum carvi), Egypt Asteraceae helichrysum oil (Helichrysum orientale), France helichrysum oil, France blue tansy oil, Morocco davana yarrow oil (Achillea millefolium), 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), Burseraceae myrrh oil (Commiphora myrrha) sea buckthorn (Hippophae salicifolia) Cupressaceae blue cypress oil (Callitris intratropica), Australia cypress oil (Callitris intratropica), Fabaceae balsam (Myroxylon balsamum), Peru balsam, South America Peru balsam, South America Geraniaceae geranium oil (Pelargonium graveolens) Lamiaceae oil (Ocimum basilicum), 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 oil (Origanum marjoram) oil, 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 bark oil (Cinnamomum verum), 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 (Myristica fragrans) Nutmeg Myrtaceae 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 , Jamaica wild by oil (), Jamaica allspice, Jamaica allspice (Pimenta Berry), Jamaica eucalyptus lemon oil, US Piperaceae Cubeb Oil (Cubeb berries) Poaceae spruce oil (Picea mariana) citronella oil, Java, vetiver oil, Surinam black spruce oil (Picea mariana) vetiver oil, Surinam citronella oil ( 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) oil (Citrus junos)

S1-80 Ethnicity & Disease, Volume 20, Spring 2010 ANTIOXIDANT 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 ( 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 S1-81 ANTIOXIDANT 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 # R003045 and NASA/URC grants NCC Fabaceae (Leguminosae), Lamiaceae demand from the manufacturers of 165-9. (Mint Family), Lauraceae (Cinnamon foods, cosmetics and pharmaceuticals. family), Myoporaceae (Buddleja family), Thus the importance of conducting REFERENCES Myrtaceae (Eucalyptus family), Poaceae studies on essential oils lies not only in 1. Mau JL, Chao GR, Wu KT. Antioxidant (Grass family), Rosaceae (Rose family) the chemical characterization but also in properties of methanolic extracts from several and (Potato family) were the the possibility of linking the chemical mushrooms. J Agric Food Chem. 2001;49(11): most effective essential oils. TLC analysis contents with particular bioactive func- 5461–5467. of the most effective 17 essential oils is tional properties. The capacity of 2. Gulcin I, Buyukokuroglu ME, Oktay M, shown in Figure 1. Thin layer chroma- Kufrevioglu OI. On the in vitro antioxidative and flavors to minimize disease risk, properties of melatonin. J Pineal Res. tography regions responsible for the within the context of culinary use, has 2002;33(3):167–171. antioxidant activity were examined by not been completely evaluated. There is 3. Cazzi R, Ricardy R, Aglitti T, Gatta V, GCMS and the active chemicals respon- a strong need to understand the pre- Petricone P, De Salvia R. Ascorbic acid and b- sible for the activity were identified based ventive effect of spices and natural carotene as modulators of oxidative damage. Carcinogenesis. 1997;18(1):223–228. on their mass spectra and GC retention flavors for counter acting oxidative 4. Reische DW, Lillard DA, Eitenmiller RR. times. The chemical structure of the damages. Our work suggests that essen- Antioxidants in Food Lipids. In: Ahoh CC, identified antioxidant compounds are tial oils from spices can be considered as Min DB, eds. Chemistry Nutrition and Biotech- shown in Figure 2. Eighteen chemicals a promising future candidate food nology. New York: Marcel Dekker; 1998: were identified, seven of them were supplement. Our data showed that 423–448. aromatic hydrocarbons or phenolic com- antioxidant activity was related to the 5. McClements J, Decker EA. Lipid oxidation in oil-water emulsions: impact of molecular envi- pounds and fourteen of the identified chemical composition of the 17 most ronment on chemical reactions in heterogeneous compounds(80%)wereoxygenated active essential oils from food system. J Food Sci. 2000;65:1270–1282. monoterpenoids. commonly consumed. The results ob- 6. Ormancey X, Sisalli S, Coutiere P. Formulation All of the essential oils that were tained by the use of the DPPH method of essential oils in functional parfumery. shown to have significant antioxidant showed that some of these spices can be Parfums, Cosmetiques, Actualites. 2001;157: 30–40. activities are produced from herbal considered good sources of natural 7. Sawamura M. Aroma and functional properties plants that are commonly used as spices antioxidants. These properties are also of Japanese yuzu (Citrus junos Tanaka) essential or as food flavoring additive, therefore, very much needed by the food industry oil. Aroma Research. 2000;1:14–19.

S1-82 Ethnicity & Disease, Volume 20, Spring 2010