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Essential oil composition and antioxidant activities of Curcuma aromatica Salisb.
Food and Chemical Toxicology 48 (2010) 1757–1760
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Essential oil composition and antioxidant activities of Curcuma aromatica Salisb. Sharif M. Al-Reza a,b, Atiqur Rahman b,*, M.A. Sattar b, M. Oliur Rahman c, Hasan M. Fida d a
Department of Biotechnology, Daegu University, Kyoungsan, Kyoungbook 712-714, Republic of Korea Department of Applied Chemistry and Chemical Technology, Islamic University, Kushtia 7003, Bangladesh c Department of Botany, University of Dhaka, Dhaka 1000, Bangladesh d Department of Process Engineering and Applied Science, Dalhousie University, Halifax, Nova Scotia, Canada B3J 2X4 b
a r t i c l e
i n f o
Article history: Received 20 January 2010 Accepted 6 April 2010
Keywords: Curcuma aromatica Salisb. Essential oil Antioxidant activity Total phenolics
a b s t r a c t The chemical composition of the hydro-distilled essential oil from leaves of Curcuma aromatica Salisb. was analysed by GC–MS. Twenty-three compounds representing 94.29% of the total oil were identified. The antioxidant activities of the oil and various extracts of C. aromatica were evaluated by using 2,2diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical-scavenging assays. The oil and methanol extract showed potent DPPH radical-scavenging activities (IC50 = 14.45 and 16.58 lg/ml, respectively), which were higher than butylated hydroxyanisole (IC50 = 18.27 lg/ml). The extracts also exhibited remarkable superoxide radical-scavenging activities (IC50 = 22.6–45.27 lg/ml) and the activity in the methanol extract was superior to all other extracts (IC50 = 22.6 lg/ml). Furthermore, the amount of total phenolic compounds was determined and its content in ethyl acetate extract was the highest as compared to other extracts. The results indicate that the oil and extracts of C. aromatica could serve as an important bio-resource of antioxidants for using in the food industries. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Many edible plant products such as fresh fruits, vegetables and some seafoods contain large amounts of antioxidants such as polyphenols, which have been widely used as additives to avoid the degradation of foods. Also, antioxidants have an important role in preventing a variety of stress-related diseases and ageing because these are closely related to the active oxygen and lipid peroxidation (Noguchi and Niki, 1999). Antioxidants have been used for the prevention and treatment of free radical-related disorders (Middleton et al., 2000). There is considerable evidence that the antioxidants contained in fruits; vegetables and beverages play an important role in the maintenance of health, and in prevention of disease. The safety of synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate, is now under scrutiny (Sasaki et al., 2002). Thus, the food industry is undertaking the rapid development and use of natural antioxidants, especially those of plant origin, to replace synthetic food additives. Among these various kinds of natural substances, essential oils from aromatic and medicinal plants receive particular attention as potential natural agents for food preservation. Moreover, essential oils are proven to have various pharmacological effects, such as spasmolytic, carminative, hepatoprotective, antiviral and anticarcinogenic effects (Bowles, 2004). * Corresponding author. Tel.: +880 71 62201 5x2266; fax: +880 71 62399. E-mail address: [email protected] (A. Rahman). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.04.008
Although it remains unclear which of the compounds, of medicinal plants are the active ones, phenolics recently have received increasing attention because of some interesting new findings regarding their biological activities. From pharmacological and therapeutic points of view, the antioxidant properties of phenolics, such as free radical-scavenging and inhibition of lipid peroxidation, are the most crucial. Even though a variety of herbs and plants are known to be the sources of phenolic compounds, studies isolating phenolics and evaluating their antioxidative effects have rarely been carried out (Löliger, 1991; Chu et al., 2000). Curcuma aromatica Salisb. belongs to genus Curcuma consists of about 70 species of rhizomatous herbs. Foliage dies down in late in autumn and the rhizomes remain dormant in winter. The inflorescence appears in early spring from the base of the rhizome. The peduncle grows to about 8–10 in. tall. Leaves appear after the flowers. When in full growth the plants can reach a height of about 1 m tall. Leaves are broad and very decorative. Good for cut-flower use with a vase life of about 10 days for a fresh stem. Most of the Curcuma species are distributed throughout tropical and subtropical regions of the world and are widely cultivated in Asian countries. It is widely used as a flavouring agent, condiment and a source of yellow dye. Medicinally, it possesses strong antimicrobial effect. It is a well-listed drug in Ayurveda and other indigenous systems of medicine. The rhizomes of C. aromatica possess a reputed property to promote health conditions by arresting ageing and have immunomodulatory effects. From ancient times, it is being used as an antibiotic against various microbial infections (Wealth of
1758
S.M. Al-Reza et al. / Food and Chemical Toxicology 48 (2010) 1757–1760
India, 2001). Historically, rhizomes are used as tonic, carminative, and externally in combinations with astringents, bitters and aromatics to bruises, in sprains and in snake-bite. They are also used for skin eruptions and infections and to improve complexion (Chopra et al., 1956). C. aromatica has been reported to exert various medicinal activities such as promoting blood circulation to remove blood stasis and for the treatment of cancer (Shi et al., 1981). The aim of this study was to determine the chemical composition of the hydro-distilled essential oil from leaves of C. aromatica by GC–MS, to evaluate the antioxidative properties of the essential oil and various organic extracts.
2.5.2. Superoxide radical (O2) scavenging activity Superoxide radicals were generated in vitro by the xanthine oxidase (XOD). The scavenging activity of the sample was determined using the nitro-blue tetrazolium (NBT) reduction method. In this method, O2 reduces the yellow dye (NBT2+) to produce the blue formazan, which was measured spectrophotometrically at 560 nm. Antioxidants are able to inhibit the blue NBT formation (Cos et al., 1998). The results were calculated as the percentage of inhibition according to the following formula:
Ið%Þ ¼ 100½1 ðS SBÞ=ðC CBÞ where S, SB, C, and CB are the absorbance of the sample, the blank sample, the control, and the blank control, respectively. 2.6. Assay for total phenolics
2. Materials and methods 2.1. Plant material The leaves of C. aromatica were collected from Islamic University Campus, Kushtia, Bangladesh in December 2007. The taxonomic identification of plant materials was confirmed by a senior plant taxonomist Dr. M. Oliur Rahman, Department of Botany, University of Dhaka, Bangladesh and a voucher specimen (DACB 32562) has been deposited in Bangladesh National Herbarium.
2.2. Isolation of the essential oil The air-dried leaves (200 g) of C. aromatica were subjected to hydro-distillation for 3 h using a Clevenger type apparatus. The oil was dried over anhydrous Na2SO4 and preserved in a sealed vial at 4 °C until further analysis.
2.3. Preparation of organic extracts The air-dried leaves (50 g) of C. aromatica were extracted with hexane, chloroform, ethyl acetate and methanol separately at room temperature for 7 days and the solvents were evaporated by vacuum rotary evaporator. The extraction process yielded in hexane (6.5 g), chloroform (6.2 g), ethyl acetate (6.4 g) and methanol (5.7 g) extracts. Solvents (analytical grade) for extraction were obtained from commercial sources (Sigma–Aldrich, St. Louis, MO, USA).
2.4. Gas chromatography–mass spectrometry (GC–MS) analysis The GC–MS analysis of the essential oil was performed using a GC–MS (Model QP 2010, Shimadzu, Japan) equipped with a ZB-1 MS fused silica capillary column (30 m 0.25 i.d., film thickness 0.25 lm). For GC–MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as a carrier gas at a constant flow rate of 1 ml/min. Injector and mass transfer line temperature were set at 220 and 290 °C, respectively. The oven temperature was programmed from 50 to 150 °C at 3 °C/min, then held isothermal for 10 min and finally raised to 250 °C at 10 °C/min. Diluted samples (1/100, v/v, in methanol) of 1 ll was manually injected in the split less mode. The relative percentage of the oil constituents was expressed as percentage by peak area normalization. Identification of components of the essential oil was based on their retention indices, relative to a homologous series of n-alkane (C8–C20) on the ZB-1 capillary column under the same operating conditions and computer matching with the Wiley 6.0 libraries, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature data (Adams, 2007).
Total phenolic contents of the plant extract and the oil were determined as described (Tsai et al., 2008), with Folin–Ciocalteu reagent and garlic acid used as a standard. An aliquot (0.2 ml) of the oil or the extract solution was added to a volumetric flask. Then, 46 ml distilled water and 1 ml Folin–Ciocalteu reagent was added, and the flask was shaken thoroughly. After 3 min, a 3 ml solution of Na2CO3 (7.5%) was added, and the mixture was allowed to stand for 2 h with intermittent shaking. Absorbance was measured at 760 nm on a UV–VIS 755B spectrophotometer (Shanghai, China). The results were expressed as milligrams of gallic acid equivalents (GAEs) per gram of extract. 2.7. Statistical analysis The essential oil and different organic extracts were assayed for their antioxidant activities. Each experiment was run in triplicate, and mean values were calculated. Analysis of variance for individual parameters was performed by Duncan’s test on the basis of mean values to find out the significance at p < 0.05.
3. Results and discussion 3.1. Chemical composition of the essential oil The hydro-distillation of dried C. aromatica leaves gave yellowish essential oil (yields 0.7%, w/w). The identified compounds, qualitative and quantitative analytical results by GC–MS, are shown in Table 1, according to their elution order on a ZB-1 capillary column. Twenty-three constituents accounting for 94.29% of total oil compositions were identified. The major components were camphor
Table 1 Chemical composition of the essential oil of C. aromatica.
2.5. Antioxidant activity 2.5.1. DPPH assay DPPH radical-scavenging activity of the oil and extracts was measured on the basis of the scavenging activities of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma–Aldrich, St. Louis, MO) free radical (Cuendet et al., 1997). Various concentrations of test extracts were added to 2.9 ml of a 0.004% (w/v) methanol solution of DPPH. After 30 min of incubation period at room temperature, the absorbance was measured against a blank at 517 nm. Inhibition free radical DPPH in percent (I%) was calculated in following way:
I% ¼ ðAblank Asample =Ablank Þ 100 where Ablank is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound. IC50 values (concentration of sample required to scavenge 50% of free radicals) were calculated from the regression equation. Synthetic antioxidant reagents, butylated hydroxyanisole (BHA) and L-ascorbic acid were used as reference positive controls and all tests were carried out in triplicate.
a
No.
Compounds
RIa
% RAb
Identificationc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Cyclohexane Dimethylacetamide Camphor Vinyldimethylcarbinol Bomeol 3-Hexen-l-ol Anisaldehyde 2-Methoxy-4-vinylphenol Vinyl cyclopentylacetate Germacrene D Veridiflorol Ledol Cubenol Epiglobulol Nerolidol b-Famesene b-Pinene oxide Cyclopentylmethanol Cucumber alcohol Caryophyllene oxide Nerolidyl acetate trans-Z-a-Bisabolene epoxide Hexahydrofamesyl acetone Total
719 620 1121 600 1088 868 1171 1293 1116 1490 1569 1530 1580 1574 1564 1440 1020 903 1175 1561 1754 1531 1816
1.03 0.63 26.32 12.21 16.45 0.36 0.21 0.23 0.43 3.45 2.38 3.84 5.59 1.26 2.15 1.83 0.18 0.44 5.19 6.33 1.45 1.14 0.19 94.29
RL, MS RI, MS RL, MS RL, MS RL, MS RL, MS RI, MS RI, MS RL, MS RI, MS RI, MS RI, MS RI, MS RL, MS RI, MS RI, MS RI, MS RI, MS RL, MS RI, MS RI, MS RI, MS RI, MS
Retention indices relative to n-alkanes C8–C20 on ZB-l capillary column. Relative area (peak area relative to the total peak area). c Identification: MS, comparison of mass spectra with MS libraries; RI, comparison of retention index with bibliography. b
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(26.32%), borneol (16.45%), vinyldimethylcarbinol (12.21%), caryophyllene oxide (6.33%), cubenol (5.59%), cucumber alcohol (5.19%), ledol (3.84%), germacrene D (3.45%), veridiflorol (2.38%) and nerolidol (2.15%), b-farnesene (1.83%), nerolidyl acetate (1.45%), epiglobulol (1.26%), trans-Z-a-bisabolene epoxide (1.14%) and cyclohexane (1.03%). Additional notable constituents included limonene (8.6%) in the leaf oil, caryophyllene oxide (8.7%), patchouli alcohol (8.4%) and elsholtzia ketone (6.0%) in the petiole oils and curzerenone (10.9%) in the rhizome oil (Choudhary et al., 1996). The major constituents of the leaf oil from Northeast India were found to be camphor (28.5%), curzerenone (6.2%), 1,8-cineole (6.05%), and a-turmerone (2.55%) while the rhizome oil consisted mainly of camphor (32.3%), curzerenone (11.0%), a-turmerone (6.7%), arturmerone (6.3%) and 1,8-cineole (5.5%) (Bordolol et al., 1999). The results reported by these researchers are particularly in agreement with the results presented here except for the amounts of the compounds. However, it is noteworthy that the composition of the essential oils from a particular species of plant can differ between harvesting seasons, extraction methods, and geographical sources, and that those from the different parts of the same plant can also differ widely (Burt, 2004).
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Gutteridge, 1999). The scavenging activities of the extracts on superoxide radical are shown in Fig. 1. All the extracts exhibited strong superoxide radical-scavenging activities. The highest superoxide radical-scavenging activities was found in methanol extract (IC50 = 22.60 lg/ml). Ethyl acetate and chloroform extract exhibited IC50 values 24.56 and 33.96 lg/ml, respectively. Hexane extract showed negligible superoxide radical-scavenging activity (IC50 = 45.27 lg/ml). These results imply that organic extracts of C. aromatica leaves are superoxide scavengers and their capacity to scavenge superoxide may contribute to their antioxidant activity. 3.4. Total phenolics content
Superoxide radical is known to be very harmful to cellular components as a precursor of the more reactive oxygen species, contributing to tissue damage and various diseases (Halliwell and
The amounts of total phenolics in the extracts were determined spectrometrically according to the Folin–Ciocalteu method and calculated as garlic acid equivalents. The standard curve equation is, y = 0.01428x + 0.1372. The amounts of total phenols found in the plant extracts were shown in Fig. 2. The amount of total phenolics was highest in the methanol extract, followed by the relatively non-polar ethyl acetate, chloroform and hexane extracts. The key role of phenolic compounds as scavengers of free radicals is emphasised in several reports (Madsen et al., 1996; Moller et al., 1999). Phenolic antioxidants are products of secondary metabolism in plants, and the antioxidant activity is mainly due to their redox properties and chemical structure, which can play an important role in chelating transitional metals, inhibiting lipoxygenase and scavenging free radicals (Decker, 1997). Indeed, the results given in Fig. 2 showed that the phenolic content was high in polar extracts. It seems clear that presence of polar phenolics is fundamental in the evaluation of free radical-scavenging. Moreover, radical-scavenging activity is one of various mechanisms contributing to overall activity, thereby creating synergistic effects. On the other hand, total antioxidant activities of the non-polar extracts (HE, CHE) could also be attributed to the volatile components, since they are still present in these extracts. Some mechanisms are available for the mode of action of phenolic compounds in antioxidant activity test systems. One of them has been put forward by Ramirez-Anguiano et al. (2007). According to this group, the oxidation of diphenols to quinines is a very fast reaction, which might occur in seconds. Even when only a few quinines are formed before the preparation of the extract, they become to the low molecular weight compounds and might react spontaneously with other phenols, generating molecules like dopachrome, indolic compounds, catechol dimers and other higher polymers yielding radical-scavenging degradation products. Therefore, it could be concluded that the phenolic compounds were highly involved in the antioxidant activity found in organic ex-
Fig. 1. Antioxidant activities of essential oil and extracts of C. aromatica. ( ), DPPH radical-scavenging activity; ( ), superoxide radical-scavenging activity. Data are given as mean ± S.D. (n = 3). EO, essential oil; MEE, methanol extract; EAE, ethyl acetate extract; CHE, chloroform extract; HAE, hexane extract; AA, ascorbic acid (control); BHA, butylated hydroxyanisole (control).
Fig. 2. The amount of total phenolics in C. aromatica. Data are given as mean ± S.D. (n = 3). MEE, methanol extract; EAE, ethyl acetate extract; CHE, chloroform extract: HAE, hexane extract.
3.2. Scavenging activity of DPPH radical The DPPH free radical is a stable free radical, which has been widely used as tool to estimate free radical-scavenging activity of antioxidants. Antioxidants, on interaction with DPPH, either transfer electrons or hydrogen atoms to DPPH, thus neutralizing the free radical character (Archana et al., 2005). The color of the reaction mixture changes from purple to yellow, and a decrease in absorbance. The DPPH radical-scavenging activity of the essential oil and the organic extracts are shown in Fig. 1. Lower IC50 value indicates higher antioxidant activity. Polar extracts exhibited stronger activity than non-polar extracts. Of all samples studied, the essential oil and methanol extract had the strongest free radical-scavenging activity with an IC50 value of 14.45 and 16.58 lg/ml, respectively. The ethyl acetate (IC50 = 30.70 lg/ml) and the chloroform extract (IC50 = 38.86 lg/ml) showed moderate DPPH radicalscavenging activity, while hexane extract showed little activity (IC50 = 45.97 lg/ml). 3.3. Superoxide radical-scavenging activity
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tracts of C. aromatica leaves and also able to enhance or complement their activity. 4. Conclusions This study concludes that the antioxidant and radical-scavenging activity of the essential oil and organic extracts of C. aromatica leaves indicate towards its strong protective role against oxidative diseases and possible use of C. aromatica leaves as a natural antioxidant, food supplement and potential pharmaceutical application. Further work is needed to fully understand the variables that can affect the evaluation of the antioxidant capacity by different methodologies. Conflict of Interest The authors declare that there are no conflicts of interest. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy, fourth ed. Allured Publishing Corporation, Carol stream, Illinois, USA. Archana, B., Dasgupta, N., De, B., 2005. In vitro study of antioxidant activity of Syzygium cumini fruit. Food Chem. 90, 727–733. Bordolol, A.K., Sperkova, J., Leclercq, P.A., 1999. Essential oils of C. Aromatica Salisb. from Northeast India. J. Essen. Oil Res. 11, 537–540. Bowles, E.J., 2004. The Chemistry of Aromatherapeutic Oils, third ed. Allen and Unwin Academic, Crows Nest, NSW. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in foods – a review. Int. J. Food Microbiol. 94, 223–253. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants, first ed. CSIR, New Delhi. p. 84. Choudhary, S.N., Ghosh, A.C., Saikia, M., 1996. Volatile constituents of the aerial and underground parts of C. Aromatica Salisb. from India. J. Essen. Oil Res. 8, 633–638.
Chu, Y., Chang, C., Hsu, H., 2000. Flavonoid content of several vegetables and their antioxidant activity. J. Sci. Food Agric. 80, 561–566. Cos, P., Ying, L.Y., Calomme, M., Hu, J.H., Cimanga, K., Van Poel, B., Pieters, L., Vlietinck, A.J., Vanden Berghe, D., 1998. Structure–activity relationships and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers. J. Nat. Prod. 61, 71–76. Cuendet, M., Hostettmann, K., Potterat, O., 1997. Iridoid glucosides with free radical scavenging properties from Fagraea blumei. Helv. Chim. Acta 80, 1144–1152. Decker, E.A., 1997. Phenolics: prooxidants or antioxidants? Nutr. Rev. 55, 396–398. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. Oxford University Press, Oxford. Löliger, J., 1991. The use of antioxidants in foods. In: Aruoma, O.I., Halliwell, B. (Eds.), Free Radicals and Food Additives. Taylor and Francis, London, pp. 121– 150. Madsen, H.L., Nielsen, B.R., Bertelsen, G., Skibsted, L.H., 1996. Screening of antioxidative activity of spices. A comparison between assays based on ESR spin trapping and electrochemical measurement of oxygen consumption. Food Chem. 57, 331–337. Middleton, E.J., Kandaswami, C., Theoharides, T.C., 2000. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52, 673–751. Moller, J.K.S., Madsen, H.L., Altonen, T., Skibsted, L.H., 1999. Dittany (Origanum dictamnus) as a source of water-extractable antioxidants. Food Chem. 64, 215– 219. Noguchi, N., Niki, E., 1999. In: Papas, M.P. (Ed.), Diet Nutrition and Health, 20th ed. CRC Press, Florida. Ramirez-Anguiano, A.C., Santoyo, S., Reglero, G., Soler-Rivas, C., 2007. Radical scavenging activities, endogenous oxidative enzymes and total phenols in edible mushrooms commonly consumed in Europe. J. Sci. Food Agric. 87, 2272– 2278. Sasaki, Y.F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., Taniguchi, K., Tsuda, S., 2002. The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat. Res. 519, 103–119. Shi, J.H., Li, C.Z., Liu, D.L., 1981. Experimental research on the pharmacology of Curcuma aromatica volatile oil. Zhongyao. Tongbao. 6, 36–38. Tsai, T.H., Tsai, T.H., Chien, Y.C., Lee, C.W., Tsai, P.J., 2008. In vitro antimicrobial activities against cariogenic streptococci and their antioxidant capacities: a comparative study of green tea versus different herbs. Food Chem. 110, 859– 864. Wealth of India, 2001. A Dictionary of Indian Raw Materials and Industrial Products. NISCOM (CSIR), New Delhi. pp. 262–264.
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Essential oil composition and antioxidant activities of Curcuma aromatica Salisb. Sharif M. Al-Reza a,b, Atiqur Rahman b,*, M.A. Sattar b, M. Oliur Rahman c, Hasan M. Fida d a
Department of Biotechnology, Daegu University, Kyoungsan, Kyoungbook 712-714, Republic of Korea Department of Applied Chemistry and Chemical Technology, Islamic University, Kushtia 7003, Bangladesh c Department of Botany, University of Dhaka, Dhaka 1000, Bangladesh d Department of Process Engineering and Applied Science, Dalhousie University, Halifax, Nova Scotia, Canada B3J 2X4 b
a r t i c l e
i n f o
Article history: Received 20 January 2010 Accepted 6 April 2010
Keywords: Curcuma aromatica Salisb. Essential oil Antioxidant activity Total phenolics
a b s t r a c t The chemical composition of the hydro-distilled essential oil from leaves of Curcuma aromatica Salisb. was analysed by GC–MS. Twenty-three compounds representing 94.29% of the total oil were identified. The antioxidant activities of the oil and various extracts of C. aromatica were evaluated by using 2,2diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical-scavenging assays. The oil and methanol extract showed potent DPPH radical-scavenging activities (IC50 = 14.45 and 16.58 lg/ml, respectively), which were higher than butylated hydroxyanisole (IC50 = 18.27 lg/ml). The extracts also exhibited remarkable superoxide radical-scavenging activities (IC50 = 22.6–45.27 lg/ml) and the activity in the methanol extract was superior to all other extracts (IC50 = 22.6 lg/ml). Furthermore, the amount of total phenolic compounds was determined and its content in ethyl acetate extract was the highest as compared to other extracts. The results indicate that the oil and extracts of C. aromatica could serve as an important bio-resource of antioxidants for using in the food industries. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Many edible plant products such as fresh fruits, vegetables and some seafoods contain large amounts of antioxidants such as polyphenols, which have been widely used as additives to avoid the degradation of foods. Also, antioxidants have an important role in preventing a variety of stress-related diseases and ageing because these are closely related to the active oxygen and lipid peroxidation (Noguchi and Niki, 1999). Antioxidants have been used for the prevention and treatment of free radical-related disorders (Middleton et al., 2000). There is considerable evidence that the antioxidants contained in fruits; vegetables and beverages play an important role in the maintenance of health, and in prevention of disease. The safety of synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate, is now under scrutiny (Sasaki et al., 2002). Thus, the food industry is undertaking the rapid development and use of natural antioxidants, especially those of plant origin, to replace synthetic food additives. Among these various kinds of natural substances, essential oils from aromatic and medicinal plants receive particular attention as potential natural agents for food preservation. Moreover, essential oils are proven to have various pharmacological effects, such as spasmolytic, carminative, hepatoprotective, antiviral and anticarcinogenic effects (Bowles, 2004). * Corresponding author. Tel.: +880 71 62201 5x2266; fax: +880 71 62399. E-mail address: [email protected] (A. Rahman). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.04.008
Although it remains unclear which of the compounds, of medicinal plants are the active ones, phenolics recently have received increasing attention because of some interesting new findings regarding their biological activities. From pharmacological and therapeutic points of view, the antioxidant properties of phenolics, such as free radical-scavenging and inhibition of lipid peroxidation, are the most crucial. Even though a variety of herbs and plants are known to be the sources of phenolic compounds, studies isolating phenolics and evaluating their antioxidative effects have rarely been carried out (Löliger, 1991; Chu et al., 2000). Curcuma aromatica Salisb. belongs to genus Curcuma consists of about 70 species of rhizomatous herbs. Foliage dies down in late in autumn and the rhizomes remain dormant in winter. The inflorescence appears in early spring from the base of the rhizome. The peduncle grows to about 8–10 in. tall. Leaves appear after the flowers. When in full growth the plants can reach a height of about 1 m tall. Leaves are broad and very decorative. Good for cut-flower use with a vase life of about 10 days for a fresh stem. Most of the Curcuma species are distributed throughout tropical and subtropical regions of the world and are widely cultivated in Asian countries. It is widely used as a flavouring agent, condiment and a source of yellow dye. Medicinally, it possesses strong antimicrobial effect. It is a well-listed drug in Ayurveda and other indigenous systems of medicine. The rhizomes of C. aromatica possess a reputed property to promote health conditions by arresting ageing and have immunomodulatory effects. From ancient times, it is being used as an antibiotic against various microbial infections (Wealth of
1758
S.M. Al-Reza et al. / Food and Chemical Toxicology 48 (2010) 1757–1760
India, 2001). Historically, rhizomes are used as tonic, carminative, and externally in combinations with astringents, bitters and aromatics to bruises, in sprains and in snake-bite. They are also used for skin eruptions and infections and to improve complexion (Chopra et al., 1956). C. aromatica has been reported to exert various medicinal activities such as promoting blood circulation to remove blood stasis and for the treatment of cancer (Shi et al., 1981). The aim of this study was to determine the chemical composition of the hydro-distilled essential oil from leaves of C. aromatica by GC–MS, to evaluate the antioxidative properties of the essential oil and various organic extracts.
2.5.2. Superoxide radical (O2) scavenging activity Superoxide radicals were generated in vitro by the xanthine oxidase (XOD). The scavenging activity of the sample was determined using the nitro-blue tetrazolium (NBT) reduction method. In this method, O2 reduces the yellow dye (NBT2+) to produce the blue formazan, which was measured spectrophotometrically at 560 nm. Antioxidants are able to inhibit the blue NBT formation (Cos et al., 1998). The results were calculated as the percentage of inhibition according to the following formula:
Ið%Þ ¼ 100½1 ðS SBÞ=ðC CBÞ where S, SB, C, and CB are the absorbance of the sample, the blank sample, the control, and the blank control, respectively. 2.6. Assay for total phenolics
2. Materials and methods 2.1. Plant material The leaves of C. aromatica were collected from Islamic University Campus, Kushtia, Bangladesh in December 2007. The taxonomic identification of plant materials was confirmed by a senior plant taxonomist Dr. M. Oliur Rahman, Department of Botany, University of Dhaka, Bangladesh and a voucher specimen (DACB 32562) has been deposited in Bangladesh National Herbarium.
2.2. Isolation of the essential oil The air-dried leaves (200 g) of C. aromatica were subjected to hydro-distillation for 3 h using a Clevenger type apparatus. The oil was dried over anhydrous Na2SO4 and preserved in a sealed vial at 4 °C until further analysis.
2.3. Preparation of organic extracts The air-dried leaves (50 g) of C. aromatica were extracted with hexane, chloroform, ethyl acetate and methanol separately at room temperature for 7 days and the solvents were evaporated by vacuum rotary evaporator. The extraction process yielded in hexane (6.5 g), chloroform (6.2 g), ethyl acetate (6.4 g) and methanol (5.7 g) extracts. Solvents (analytical grade) for extraction were obtained from commercial sources (Sigma–Aldrich, St. Louis, MO, USA).
2.4. Gas chromatography–mass spectrometry (GC–MS) analysis The GC–MS analysis of the essential oil was performed using a GC–MS (Model QP 2010, Shimadzu, Japan) equipped with a ZB-1 MS fused silica capillary column (30 m 0.25 i.d., film thickness 0.25 lm). For GC–MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as a carrier gas at a constant flow rate of 1 ml/min. Injector and mass transfer line temperature were set at 220 and 290 °C, respectively. The oven temperature was programmed from 50 to 150 °C at 3 °C/min, then held isothermal for 10 min and finally raised to 250 °C at 10 °C/min. Diluted samples (1/100, v/v, in methanol) of 1 ll was manually injected in the split less mode. The relative percentage of the oil constituents was expressed as percentage by peak area normalization. Identification of components of the essential oil was based on their retention indices, relative to a homologous series of n-alkane (C8–C20) on the ZB-1 capillary column under the same operating conditions and computer matching with the Wiley 6.0 libraries, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature data (Adams, 2007).
Total phenolic contents of the plant extract and the oil were determined as described (Tsai et al., 2008), with Folin–Ciocalteu reagent and garlic acid used as a standard. An aliquot (0.2 ml) of the oil or the extract solution was added to a volumetric flask. Then, 46 ml distilled water and 1 ml Folin–Ciocalteu reagent was added, and the flask was shaken thoroughly. After 3 min, a 3 ml solution of Na2CO3 (7.5%) was added, and the mixture was allowed to stand for 2 h with intermittent shaking. Absorbance was measured at 760 nm on a UV–VIS 755B spectrophotometer (Shanghai, China). The results were expressed as milligrams of gallic acid equivalents (GAEs) per gram of extract. 2.7. Statistical analysis The essential oil and different organic extracts were assayed for their antioxidant activities. Each experiment was run in triplicate, and mean values were calculated. Analysis of variance for individual parameters was performed by Duncan’s test on the basis of mean values to find out the significance at p < 0.05.
3. Results and discussion 3.1. Chemical composition of the essential oil The hydro-distillation of dried C. aromatica leaves gave yellowish essential oil (yields 0.7%, w/w). The identified compounds, qualitative and quantitative analytical results by GC–MS, are shown in Table 1, according to their elution order on a ZB-1 capillary column. Twenty-three constituents accounting for 94.29% of total oil compositions were identified. The major components were camphor
Table 1 Chemical composition of the essential oil of C. aromatica.
2.5. Antioxidant activity 2.5.1. DPPH assay DPPH radical-scavenging activity of the oil and extracts was measured on the basis of the scavenging activities of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma–Aldrich, St. Louis, MO) free radical (Cuendet et al., 1997). Various concentrations of test extracts were added to 2.9 ml of a 0.004% (w/v) methanol solution of DPPH. After 30 min of incubation period at room temperature, the absorbance was measured against a blank at 517 nm. Inhibition free radical DPPH in percent (I%) was calculated in following way:
I% ¼ ðAblank Asample =Ablank Þ 100 where Ablank is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound. IC50 values (concentration of sample required to scavenge 50% of free radicals) were calculated from the regression equation. Synthetic antioxidant reagents, butylated hydroxyanisole (BHA) and L-ascorbic acid were used as reference positive controls and all tests were carried out in triplicate.
a
No.
Compounds
RIa
% RAb
Identificationc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Cyclohexane Dimethylacetamide Camphor Vinyldimethylcarbinol Bomeol 3-Hexen-l-ol Anisaldehyde 2-Methoxy-4-vinylphenol Vinyl cyclopentylacetate Germacrene D Veridiflorol Ledol Cubenol Epiglobulol Nerolidol b-Famesene b-Pinene oxide Cyclopentylmethanol Cucumber alcohol Caryophyllene oxide Nerolidyl acetate trans-Z-a-Bisabolene epoxide Hexahydrofamesyl acetone Total
719 620 1121 600 1088 868 1171 1293 1116 1490 1569 1530 1580 1574 1564 1440 1020 903 1175 1561 1754 1531 1816
1.03 0.63 26.32 12.21 16.45 0.36 0.21 0.23 0.43 3.45 2.38 3.84 5.59 1.26 2.15 1.83 0.18 0.44 5.19 6.33 1.45 1.14 0.19 94.29
RL, MS RI, MS RL, MS RL, MS RL, MS RL, MS RI, MS RI, MS RL, MS RI, MS RI, MS RI, MS RI, MS RL, MS RI, MS RI, MS RI, MS RI, MS RL, MS RI, MS RI, MS RI, MS RI, MS
Retention indices relative to n-alkanes C8–C20 on ZB-l capillary column. Relative area (peak area relative to the total peak area). c Identification: MS, comparison of mass spectra with MS libraries; RI, comparison of retention index with bibliography. b
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(26.32%), borneol (16.45%), vinyldimethylcarbinol (12.21%), caryophyllene oxide (6.33%), cubenol (5.59%), cucumber alcohol (5.19%), ledol (3.84%), germacrene D (3.45%), veridiflorol (2.38%) and nerolidol (2.15%), b-farnesene (1.83%), nerolidyl acetate (1.45%), epiglobulol (1.26%), trans-Z-a-bisabolene epoxide (1.14%) and cyclohexane (1.03%). Additional notable constituents included limonene (8.6%) in the leaf oil, caryophyllene oxide (8.7%), patchouli alcohol (8.4%) and elsholtzia ketone (6.0%) in the petiole oils and curzerenone (10.9%) in the rhizome oil (Choudhary et al., 1996). The major constituents of the leaf oil from Northeast India were found to be camphor (28.5%), curzerenone (6.2%), 1,8-cineole (6.05%), and a-turmerone (2.55%) while the rhizome oil consisted mainly of camphor (32.3%), curzerenone (11.0%), a-turmerone (6.7%), arturmerone (6.3%) and 1,8-cineole (5.5%) (Bordolol et al., 1999). The results reported by these researchers are particularly in agreement with the results presented here except for the amounts of the compounds. However, it is noteworthy that the composition of the essential oils from a particular species of plant can differ between harvesting seasons, extraction methods, and geographical sources, and that those from the different parts of the same plant can also differ widely (Burt, 2004).
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Gutteridge, 1999). The scavenging activities of the extracts on superoxide radical are shown in Fig. 1. All the extracts exhibited strong superoxide radical-scavenging activities. The highest superoxide radical-scavenging activities was found in methanol extract (IC50 = 22.60 lg/ml). Ethyl acetate and chloroform extract exhibited IC50 values 24.56 and 33.96 lg/ml, respectively. Hexane extract showed negligible superoxide radical-scavenging activity (IC50 = 45.27 lg/ml). These results imply that organic extracts of C. aromatica leaves are superoxide scavengers and their capacity to scavenge superoxide may contribute to their antioxidant activity. 3.4. Total phenolics content
Superoxide radical is known to be very harmful to cellular components as a precursor of the more reactive oxygen species, contributing to tissue damage and various diseases (Halliwell and
The amounts of total phenolics in the extracts were determined spectrometrically according to the Folin–Ciocalteu method and calculated as garlic acid equivalents. The standard curve equation is, y = 0.01428x + 0.1372. The amounts of total phenols found in the plant extracts were shown in Fig. 2. The amount of total phenolics was highest in the methanol extract, followed by the relatively non-polar ethyl acetate, chloroform and hexane extracts. The key role of phenolic compounds as scavengers of free radicals is emphasised in several reports (Madsen et al., 1996; Moller et al., 1999). Phenolic antioxidants are products of secondary metabolism in plants, and the antioxidant activity is mainly due to their redox properties and chemical structure, which can play an important role in chelating transitional metals, inhibiting lipoxygenase and scavenging free radicals (Decker, 1997). Indeed, the results given in Fig. 2 showed that the phenolic content was high in polar extracts. It seems clear that presence of polar phenolics is fundamental in the evaluation of free radical-scavenging. Moreover, radical-scavenging activity is one of various mechanisms contributing to overall activity, thereby creating synergistic effects. On the other hand, total antioxidant activities of the non-polar extracts (HE, CHE) could also be attributed to the volatile components, since they are still present in these extracts. Some mechanisms are available for the mode of action of phenolic compounds in antioxidant activity test systems. One of them has been put forward by Ramirez-Anguiano et al. (2007). According to this group, the oxidation of diphenols to quinines is a very fast reaction, which might occur in seconds. Even when only a few quinines are formed before the preparation of the extract, they become to the low molecular weight compounds and might react spontaneously with other phenols, generating molecules like dopachrome, indolic compounds, catechol dimers and other higher polymers yielding radical-scavenging degradation products. Therefore, it could be concluded that the phenolic compounds were highly involved in the antioxidant activity found in organic ex-
Fig. 1. Antioxidant activities of essential oil and extracts of C. aromatica. ( ), DPPH radical-scavenging activity; ( ), superoxide radical-scavenging activity. Data are given as mean ± S.D. (n = 3). EO, essential oil; MEE, methanol extract; EAE, ethyl acetate extract; CHE, chloroform extract; HAE, hexane extract; AA, ascorbic acid (control); BHA, butylated hydroxyanisole (control).
Fig. 2. The amount of total phenolics in C. aromatica. Data are given as mean ± S.D. (n = 3). MEE, methanol extract; EAE, ethyl acetate extract; CHE, chloroform extract: HAE, hexane extract.
3.2. Scavenging activity of DPPH radical The DPPH free radical is a stable free radical, which has been widely used as tool to estimate free radical-scavenging activity of antioxidants. Antioxidants, on interaction with DPPH, either transfer electrons or hydrogen atoms to DPPH, thus neutralizing the free radical character (Archana et al., 2005). The color of the reaction mixture changes from purple to yellow, and a decrease in absorbance. The DPPH radical-scavenging activity of the essential oil and the organic extracts are shown in Fig. 1. Lower IC50 value indicates higher antioxidant activity. Polar extracts exhibited stronger activity than non-polar extracts. Of all samples studied, the essential oil and methanol extract had the strongest free radical-scavenging activity with an IC50 value of 14.45 and 16.58 lg/ml, respectively. The ethyl acetate (IC50 = 30.70 lg/ml) and the chloroform extract (IC50 = 38.86 lg/ml) showed moderate DPPH radicalscavenging activity, while hexane extract showed little activity (IC50 = 45.97 lg/ml). 3.3. Superoxide radical-scavenging activity
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tracts of C. aromatica leaves and also able to enhance or complement their activity. 4. Conclusions This study concludes that the antioxidant and radical-scavenging activity of the essential oil and organic extracts of C. aromatica leaves indicate towards its strong protective role against oxidative diseases and possible use of C. aromatica leaves as a natural antioxidant, food supplement and potential pharmaceutical application. Further work is needed to fully understand the variables that can affect the evaluation of the antioxidant capacity by different methodologies. Conflict of Interest The authors declare that there are no conflicts of interest. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy, fourth ed. Allured Publishing Corporation, Carol stream, Illinois, USA. Archana, B., Dasgupta, N., De, B., 2005. In vitro study of antioxidant activity of Syzygium cumini fruit. Food Chem. 90, 727–733. Bordolol, A.K., Sperkova, J., Leclercq, P.A., 1999. Essential oils of C. Aromatica Salisb. from Northeast India. J. Essen. Oil Res. 11, 537–540. Bowles, E.J., 2004. The Chemistry of Aromatherapeutic Oils, third ed. Allen and Unwin Academic, Crows Nest, NSW. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in foods – a review. Int. J. Food Microbiol. 94, 223–253. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants, first ed. CSIR, New Delhi. p. 84. Choudhary, S.N., Ghosh, A.C., Saikia, M., 1996. Volatile constituents of the aerial and underground parts of C. Aromatica Salisb. from India. J. Essen. Oil Res. 8, 633–638.
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