Patent Publication Number: US-2012046367-A1

Title: Essential oil composition with anti-free radical ability

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates generally to an essential oil composition and more particularly, to an essential oil composition having anti-free radical ability. 
     2. Description of the Related Art 
     Dr. Denham Harman, a professor of chemical in U.S., has shown in Drugs Aging in 1993 that excessive free radicals damage body cells and result in acceleration of aging process. Free radicals and the derivatives thereof constantly generate inside a living body because of the oxidative metabolism of a living body, the ion radiation existing in the environment and the pollutants such as air pollution and chemical substance. The skin of a human body is most easily affected by the ambient environment. For example, the mitochondria in the fibroblast in dermis of a human body will release superoxide anions (O 2   − ) when they are stimulated by the ultraviolet light existing in the environment; consequently, those excess superoxide anions will transform to free radicals that seriously damage body cells. The antioxidation defense system of human body, which is used for maintaining the equilibrium between oxidation and antioxidation, is able to retard the generation of reactive oxygen species (ROS) and free radicals; however, a large number of free radicals that generate inside the human body because of exposure of sunshine for a long period of time will deteriorate the anti-oxidative ability of skin, resulting in acute or chronic damage of skin such as photoaging, epidermal wrinkle, immune imbalance of skin and skin cancer. 
     Although many products advertise that they can scavenge free radicals and have anti-oxidative ability through applying them onto the surface of skin to increase the anti-oxidative ability of human skin, the molecules of the anti-oxidative ingredients contained in most of the aforesaid products are larger or water-soluble such that it is hard for those molecules to be absorbed into human skin. Therefore, the actual anti-oxidative ability is limited. 
     On the other hand, various kinds of blend essential oils containing anti-oxidative ingredients are most popular to consumers because they have smaller molecules that can be easily absorbed into human skin through scenting incense, application, massage and immersion. As such, the anti-oxidative ingredients contained in a blend essential oil may be absorbed by skin cells and respiratory system and then penetrate into skin tissue to all cells. Nevertheless, no objective experimental data show that those blend essential oils indeed have free radical scavenging ability and anti-oxidative ability. 
     SUMMARY OF THE INVENTION 
     The main objective of the present invention is to provide an essential oil composition having anti-free radical ability. The essential oil composition of the present invention has superior anti-free radical ability and anti-oxidative ability than a commercial blend essential oil. 
     In order to achieve the aforesaid objective, the essential oil composition of the present invention includes dl-Citronellol, Eugenol, cis/trans-Citral, Eugenol Methyl Ether and Thymol. 
     Preferably, the essential oil composition may comprise 0.5-8 wt % of dl-Citronellol, 4.5-24.6 wt % of Eugenol, 3.75-20 wt % of cis/trans-Citral, 0.15-12.5 wt % of Eugenol Methyl Ether and 4.5-25.2 wt % of Thymol. More preferably, the dl-Citronellol may be contained in Geranium essential oil, the Eugenol may be contained in Cinnamon essential oil, the cis/trans-Citral may be contained in Lemongrass essential oil, the Eugenol Methyl Ether may be contained in Pimenta racemosa essential oil and the Thymol may be contained in Origanum vulgare essential oil. 
     Because all of the chemical compounds contained in the plant essential oils and used in the essential oil composition of the present invention have good DPPH free radical scavenging ability, ABTS +  free radical scavenging ability, metal ion chelating ability and reducing property, the essential oil composition of the present invention exhibits superior anti-free radical ability and anti-oxidative ability. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention will become more fully understood from the detailed description given herein below. 
     According to the study of the present invention, an essential oil composition comprising dl-Citronellol, Eugenol, cis/trans-Citral, Eugenol Methyl Ether and Thymol provides a good anti-free radical ability. 
     An essential oil composition provided according to a preferred embodiment of the present invention comprises 0.5-8 wt % of dl-Citronellol, 4.5-24.6 wt % of Eugenol, 3.75-20 wt % of cis/trans-Citral, 0.15-12.5 wt % of Eugenol Methyl Ether and 4.5-25.2 wt % of Thymol. 
     Although phenols have good free radical scavenging ability, excessive phenols cause pungent odor. After lots of experiments, the above-mentioned ingredients and the content thereof were come out as an optimal solution in consideration of the balance between the free radical scavenging ability and the smell of the essential oil composition. 
     It will be appreciated that the contents of chemical compounds of the extracted essential oils may depend on the factors including, but not limited to, the source, cultivation, harvest time, extraction method and the extracted parts of plants for extraction. Essential oils containing an effective chemical compound in a specific content range can be used as the ingredients for the essential oil composition of the present invention. 
     More specifically speaking, for the dl-Citronellol, Geranium essential oil contains 5-40 wt % of dl-Citronellol can be used. For the Eugenol, Cinnamon essential oil contains 30-82 wt % of Eugenol can be used. For the cis/trans-Citral, Lemongrass essential oil contains 25-80% of cis/trans-Citral can be used. For the Eugenol Methyl Ether, Pimenta racemosa essential oil contains 0.1-5 wt % of Eugenol Methyl Ether can be used. For the Thymol, Origanum vulgare essential oil contains 30-84 wt % of Thymol can be used. 
     The essential oil compositions of the following examples are prepared respectively by blending the Geranium essential oil having 40 wt % of dl-Citronellol, the Cinnamon essential oil having 82 wt % of Eugenol, the Lemongrass essential oil having 80 wt % of cis/trans-Citral, the Pimenta racemosa essential oil having 5 wt % of Eugenol Methyl Ether and the Origanum vulgare essential oil having 84 wt % of Thymol in different ratios. There is no specific limitation to the method for blending these essential oils. Any method that can uniformly mix these essential oils can be used. 
     The present invention will be further described through the following examples which are used to more understand the present invention and are not used to limit the scope of the present invention. It will be apparent to one of ordinary skill in the art that many changes and modifications may be made thereto without departing from the spirit of the present invention. 
     Example 1 
     10 g of Geranium essential oil, 30 g of Cinnamon essential oil, 15 g of Lemongrass essential oil, 15 g of Pimenta racemosa essential oil and 30 g of Origanum vulgare essential oil were well blended to prepare 100 g of essential oil composition which comprises 4 wt % of dl-Citronellol, 24.6 wt % of Eugenol, 12 wt % of cis/trans-Citral, 0.75 wt % of Eugenol Methyl Ether and 25.2 wt % of Thymol. 
     Example 2 
     10 g of Geranium essential oil, 25 g of Cinnamon essential oil, 20 g of Lemongrass essential oil, 20 g of Pimenta racemosa essential oil and 25 g of Origanum vulgare essential oil were well blended to prepare 100 g of essential oil composition which comprises 4 wt % of dl-Citronellol, 20.5 wt % of Eugenol, 16 wt % of cis/trans-Citral, 1 wt % of Eugenol Methyl Ether and 21 wt % of Thymol. 
     Example 3 
     20 g of Geranium essential oil, 15 g of Cinnamon essential oil, 25 g of Lemongrass essential oil, 25 g of Pimenta racemosa essential oil and 15 g of Origanum vulgare essential oil were blended to prepare 100 g of essential oil composition which comprises 8 wt % of dl-Citronellol, 12.3 wt % of Eugenol, 20 wt % of cis/trans-Citral, 1.25 wt % of Eugenol Methyl Ether and 12.6 wt % of Thymol. 
     Comparative Example 1 
     The “Total Energy Essential Oil” of The Body Shop™, comprises Bergamot, 
     Orange Sweet, Grapefruit, Rosewood, Palmarosa, Rosemary, Mint, Ginger, Lemon, Cedarwood and Patchouli. 
     Comparative Example 2 
     The “Aroma Oil No. 6” of DHC™, comprises Tangerine, Bergamot, Orange, Neroli Essence, Rosewood, Cedarwood and Sandalwood. 
     Comparative Example 3 
     The “Energy Stimulate Mix Organic Huiles Essentielles” of Le Bon Moment™, comprises Juniper, Helichrysum, Rosewood, Rosemary and Mint. 
     Comparative Example 4 
     The “Chakra I” of Lorien Vana™, comprises Damask rose, Myrrh, Patchouli, Vetiver, Ginger and Rosewood. 
     Comparative Example 5 
     The “Chakra II” of Lorien Vana™, comprises Petitgrain, Geranium, Clary sage, Cypress, Rosewood and Grapefruit. 
     Comparative Example 6 
     The “Chakra VI” of Lorien Vana™, comprises Rose Geranium, Rosemary, Basil, Camphor, Highly Lavender and Geranium. 
     Comparative Example 7 
     The “Chakra VII” of Lorien Vana™, comprises Highly Lavender, Rosewood, Frankincense, Myrrh and Basil. 
     Comparative Example 8 
     The “Chakra VIII” of Lorien Vana™, comprises Highly Lavender, Grapefruit, Vetiver, Camphor and Bergamot. 
     The anti-free radical ability and anti-oxidative ability of the essential oil compositions of the examples and comparative examples are evaluated through the following measurements. The results are shown in Table 1 below. 
     Measurement for DPPH Free Radical Scavenging Ability 
     10 mg/mL solutions were prepared by using the compositions of examples 1-3 and comparative examples 1-8 respectively. Then, a 1.52×10 −4 M ethanol solution was prepared by dissolving 1,1-Diphenyl-2-picrylhydrazyl (DPPH) in ethanol. Thereafter, each sample mixture was prepared by vigorously shaking 500 μL of each of the aforesaid 10 mg/mL solutions and 2500 μL of the DPPH ethanol solution and placing in darkness at room temperature for 30 minutes. 
     In addition, the control mixture was prepared in the same manner as in the sample mixture, with the exception that the aforesaid 10 mg/mL solution was replaced by butyl hydroxy toluene (BHT). 
     The absorbance at 517 nm of the sample mixture and control mixture were measured respectively by UV-vis Spectrophotometer (HITACHI, UV-vis 2001). The scavenging effect of DPPH free radical was evaluated by using the following equation 1. The larger the value is, the stronger the DPPH free radical scavenging ability is. 
     
       
         
           
             
               
                 
                   
                     Scavenging 
                      
                     
                         
                     
                      
                     ratio 
                      
                     
                         
                     
                      
                     
                       ( 
                       % 
                       ) 
                     
                   
                   = 
                   
                       
                     
                       
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                           - 
                           
                             ( 
                             
                               
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                                 517 
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                         ] 
                       
                       × 
                       100 
                     
                   
                 
               
               
                 
                   [ 
                   
                     Equation 
                      
                     
                         
                     
                      
                     1 
                   
                   ] 
                 
               
             
           
         
       
     
     Measurement for ABTS +  Free Radical Scavenging Ability 
     1 mg/mL solutions were prepared by using the compositions of examples 1-3 and comparative examples 1-8 respectively. Then, 250 μL of deionized water, 250 μL of 500 μM hydrogen peroxide solution, 250 μL of 1000 μM 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) solution and 250 μL of 8.8 U/mL peroxidase were well mixed by Vortex Genie 2 Mixer (Scientific Industries). After about 10 minutes, when the stable ABTS +  cation free radicals with aquamarine color generate, 250 μL of each the aforesaid 1 mg/mL solution was added therein to prepare mixtures. Thereafter, each the aforesaid mixture was well mixed by Vortex Genie 2 Mixer and incubated for 10 minutes to prepare each sample mixture. 
     In addition, butylated hydroxyl anisole (BHA) was used as a standard for the control mixture, and the aforesaid sample mixtures was replaced by 250 μL of deionized water. 
     The absorbance at 410 nm of the sample mixture and control mixture were measured respectively by Enzyme-linked immunoassay (ELISA, Tecan Company, Sunrise-Basic-Tecan). The scavenging effect of ABTS +  free radical was evaluated by using the following equation 2. The larger the value is, the stronger the ABTS +  free radical scavenging ability is. 
     
       
         
           
             
               
                 
                   
                     Scavenging 
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                                 410 
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                       × 
                       100 
                     
                   
                 
               
               
                 
                   [ 
                   
                     Equation 
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                     2 
                   
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     Measurement for Metal Ion Chelating Ability 
     1 mg/mL solutions were prepared by using the compositions of examples 1-3 and comparative examples 1-8 respectively. Then, each mixture containing each the aforesaid 1 mg/mL solution, 750 μL of 2 mM iron (II) chloride tetrahydrate solution and 250 μl, of methanol was prepared by shaking for 30 seconds. Each the aforesaid mixture was added with 1500 μL of 5 mM ferrozine solution and placed in darkness for 30 minutes. After the reaction was completed, each mixture was subjected to centrifugation at 3000 rpm for 5 minutes and the supernatant liquid thus obtained was used as the sample mixture. 
     In addition, the control mixture was prepared in the same manner as in the sample mixture, with the exception that the aforesaid 1 mg/mL solution was replaced by ethylenediaminetetraacetic acid (EDTA) and the centrifugal process was not performed. 
     The absorbance at 562 nm of the sample mixture and control mixture were measured respectively by UV-vis Spectrophotometer (HITACHI, UV-vis 2001). The chelating effect of metal ion was evaluated by using the following equation 3. The larger the value is, the stronger the metal ion chelating ability is. 
     
       
         
           
             
               
                 
                   
                     Chelating 
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                     ratio 
                      
                     
                         
                     
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                       100 
                     
                   
                 
               
               
                 
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     Measurement for Reducing Property 
     10 mg/mL solutions were prepared by using the compositions of examples 1-3 and comparative examples 1-8 respectively. Then, 2 mM phosphate buffer solution with pH 6.6 was prepared by mixing 0.2 M sodium dihydrogenphosphate solution and 0.2 M disodium hydrogenphosphate solution. Thereafter, 500 μL of each the aforesaid 10 mg/mL solution was mixed with 250 μL of the phosphate buffer solution and 250 μL of 1% Potassium Ferricyanide. The mixture was reacted at 50° C. for 20 minutes in a water bath and then rapid cooled for 5 minutes. The obtained reactant was mixed with 250 μL of 10% trichloroacetic acid. 100 μL of the supernatant liquid thus obtained was mixed with 500 μL of deionized water and 750 μL of 0.1% ferric chloride solution, and then the obtained mixture was placed in darkness for 30 minutes to prepare the sample mixture. 
     In addition, butylated hydroxyl anisole (BHA) was used as a standard for the control mixture, and the aforesaid sample mixture was replaced by 99.8% ethanol. 
     200 μL of the sample mixture and the control mixture were added into the wells of the test plate of Enzyme-linked immunoassay with 96-well respectively, and the absorbance at 700 nm of the sample mixture and control mixture were measured respectively by Enzyme-linked immunoassay (ELISA, Tecan Company, Sunrise-Basic-Tecan). The reducing property was evaluated by using the following equation 4. The larger the value is, the stronger the reducing ability is. 
     
       
         
           
             
               
                 
                   
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                 TABLE 1 
               
               
                   
                   
               
               
                   
                 DPPH 
                 ABTS +   
                   
                   
               
               
                   
                 Scavenging 
                 Scavenging 
                 Chelating 
                 Reducing 
               
               
                   
                 ratio (%) 
                 ratio (%) 
                 ratio (%) 
                 (%) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Ex. 1 
                 94.52 ± 0.23 
                 88.47 ± 0.33 
                 62.32 ± 0.77 
                 86.88 ± 1.31 
               
               
                 Ex. 2 
                 88.02 ± 0.55 
                 83.97 ± 1.21 
                 58.08 ± 1.81 
                 80.64 ± 0.98 
               
               
                 Ex. 3 
                 82.67 ± 0.94 
                 78.31 ± 1.01 
                 56.22 ± 0.63 
                 77.62 ± 1.75 
               
               
                 Com. Ex. 1 
                 42.12 ± 1.65 
                 37.33 ± 1.82 
                 21.39 ± 2.06 
                 33.98 ± 1.87 
               
               
                 Com. Ex. 2 
                 28.24 ± 0.98 
                 27.88 ± 0.45 
                 18.65 ± 0.77 
                 25.85 ± 1.74 
               
               
                 Com. Ex. 3 
                 22.11 ± 1.82 
                 19.38 ± 2.27 
                 11.34 ± 0.69 
                 16.15 ± 0.71 
               
               
                 Com. Ex. 4 
                 38.32 ± 0.83 
                 36.18 ± 1.28 
                  28.7 ± 0.22 
                 18.28 ± 0.06 
               
               
                 Com. Ex. 5 
                 44.12 ± 0.69 
                 40.37 ± 0.24 
                 27.68 ± 0.69 
                 14.17 ± 0.13 
               
               
                 Com. Ex. 6 
                 48.35 ± 0.63 
                 44.97 ± 0.68 
                 55.31 ± 0.52 
                 20.21 ± 0.92 
               
               
                 Com. Ex. 7 
                 33.62 ± 0.12 
                 30.92 ± 0.77 
                 51.32 ± 1.37 
                 13.80 ± 1.45 
               
               
                 Com. Ex. 8 
                 28.12 ± 0.31 
                 26.41 ± 0.32 
                 19.55 ± 0.61 
                 16.61 ± 0.44 
               
               
                   
               
            
           
         
       
     
     As shown in Table 1, DPPH free radical scavenging ability, ABTS +  ee radical scavenging ability, metal ion chelating ability and reducing property of examples 1-3 are much higher than those of comparative examples 1-8. 
     In addition, a human experiment was performed by the essential oil composition of example 3 through scenting incense, and the results show that the essential oil composition of the present invention is capable of scavenging the free radicals in human blood. The bloods of 15 adults before scenting incense and after scenting incense for 10 minutes were analyzed in the human experiment. First, 0.2 mL of blood was well mixed with 0.1 mL of 0.9% saline solution, and then the mixture was heated to 37° C. so as to simulate the body temperature. Thereafter, 1 mL of the reagent, which was prepared by dissolving 2.5 mg lucigenin in 0.9% saline, was added into the mixture. The amount of free radicals in the obtained mixture was measured by Chemiluminescence Spectrometer (Tohoku Electronic Co., Ltd). The results are shown in Table 2. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Amount of free radicals 
                 Amount of free radicals 
                 Scavenging 
               
               
                 No. 
                 before scenting incense 
                 after scenting incense 
                 ratio (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 12136 
                 5554 
                 54.24% 
               
               
                 2 
                 15038 
                 3020 
                 79.92% 
               
               
                 3 
                 6283 
                 5247 
                 16.49% 
               
               
                 4 
                 20511 
                 3840 
                 81.28% 
               
               
                 5 
                 90235 
                 31152 
                 65.48% 
               
               
                 6 
                 98326 
                 2144 
                 97.82% 
               
               
                 7 
                 33897 
                 14113 
                 58.37% 
               
               
                 8 
                 59844 
                 5877 
                 90.18% 
               
               
                 9 
                 10673 
                 3725 
                 65.10% 
               
               
                 10  
                 26632 
                 8820 
                 66.88% 
               
               
                 11  
                 15351 
                 7123 
                 53.60% 
               
               
                 12  
                 47392 
                 15997 
                 66.25% 
               
               
                 13  
                 27340 
                 2687 
                 90.17% 
               
               
                 14  
                 39810 
                 2357 
                 94.08% 
               
               
                 15  
                 8991 
                 4689 
                 47.85% 
               
               
                 Mean 
                 34164 
                 7756 
                   69% 
               
               
                 Value 
               
               
                   
               
            
           
         
       
     
     As shown in Table 2, the essential oil composition of example 3 is effective in scavenging the free radicals in human blood, and the scavenging effect is excellent based on the fact that the average free radical scavenging ratio is about 70%. 
     In conclusion, the essential oil compositions of examples 1-3 have superior DPPH free radical scavenging ability, ABTS +  free radical scavenging ability, metal ion chelating ability and reducing property. Besides, the free radical scavenging effect of the present invention is excellent in view of the aforesaid human experiment. Therefore, the present invention exhibits superior anti-free radical ability and anti-oxidative ability.