Antioxidant Activity and Chemical Constituents of Essential Oil And
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Food Chemistry 125 (2011) 456–463 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Antioxidant activity and chemical constituents of essential oil and extracts of Rhizoma Homalomenae ⇑ Ling-Bin Zeng a, Zhong-Rong Zhang a, Zhu-Hua Luo b, Ji-Xiao Zhu c, a Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong SAR, China b Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, Fujian, China c College of Pharmaceutical Science, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, Jiangxi, China article info abstract Article history: Antioxidant activity and composition of essential oil and extracts of Rhizoma Homalomenae were deter- Received 25 February 2010 mined. The extracts, especially the ethyl acetate extract (QJ4 fraction) of the aqueous residue after oil dis- Received in revised form 28 July 2010 tillation, had considerable antioxidant potency which was significantly associated with their total Accepted 7 September 2010 phenolic and flavonoid contents, but the essential oil showed only weak or moderate activity. GC–MS analysis of the essential oil (yield: 0.82%, v/w) resulted in the identification of 77 compounds, accounting for 96.5% of the content of the oil. The major components, epi-a-cadinol (14.8%), a-cadinol (14.8%), a-ter- Keywords: pineol (13.8%), linalool (11.1%), terpinen-4-ol (4.92%), and d-cadinene (4.91%) constituted 64.3% of it. LC– Homalomena occulta MS/MS and HPLC analyses showed seven phenolic compounds (protocatechuic acid, vanillic acid, syringic Rhizoma Homalomenae Antioxidant activity acid, caffeic acid, p-coumaric acid, ferulic acid and apigenin) with a great amount in the ethyl acetate Essential oil extract (QJ4 fraction). The strong antioxidant properties of the plant extracts may be attributed to the GC–MS presence of these phenolics. LC–MS/MS Ó 2010 Elsevier Ltd. All rights reserved. HPLC 1. Introduction muscles and bones, relieving stomachache, and relief from rheu- matoid arthritis, healing pain and swelling due to traumatic injury Reactive oxygen species (ROS) include both oxygen radicals, (Zhong Hua Ben Cao Editorial Committee, 1999). Traditionally, it is such as superoxide, hydroxyl, peroxyl, and hydroperoxyl radicals, mostly used as an aqueous decoction, alcoholic beverage or for and some non-radical oxidising agents, such as hydrogen peroxide, external application (Zhong Hua Ben Cao Editorial Committee, hypochlorous acid, and ozone and the non-radical ROS can convert 1999). Its essential oils showed anti-virus and antibiotic activities easily into radicals (Bayr, 2005). Excessive ROS levels damage lip- and the aqueous or alcohol extracts had anti-histamine, anti-coag- ids, proteins and nucleic acids through oxidation and thus are asso- ulant, anti-inflammatory and antialgesic activities (Zhong Hua Ben ciated with various diseases, such as atherosclerosis, arthritis, Cao Editorial Committee, 1999). A series of sesquiterpenoids, phe- neurodegenerative disorders, and cancers (Balsano & Alisi, 2009). nolic acids and other compounds (Elbandy, Lerche, Wagner, & Regular supplement of antioxidants can assist the endogenous de- Lacaille-Dubois, 2004; Hu, Yang, Ye, & Cheng, 2003; Hu et al., fence systems to counterbalance the harmful effects of excessive 2008; Hu, Yang, Wang, & Ye, 2009; Tian, Zhao, Yu, & Fang, 2010) ROS (Kaur & Geetha, 2006). But, synthetic antioxidants, such as was obtained from organic extracts of dry rhizome or aerial parts butylated hydroxytoluene (BHT) and butylated hydroxyanisole of H. occulta. The phenolic acids exhibited BACE1 (b-secretase) (BHA) are suspected of being responsible for some severe toxic ef- inhibitory activity (Tian et al., 2010) and four of the sesquiterpe- fects. Thus, natural antioxidants, from medicinal plants, vegetables, noids had a stimulative effect on proliferation and differentiation and fruits, are considered to be better alternatives and receive of cultured osteoblasts (Hu et al., 2008). increasing attention. To the best of our knowledge, no study on the antioxidant activ- Rhizoma Homalomenae is the dry rhizome of Homalomena occ- ity of essential oil and organic or aqueous extracts from Rhizoma ulta (Lour.) Schott. It is a famous traditional Chinese medicine Homalomenae has been reported so far. Thus, in the present study, (called as Qiannianjian in Chinese). Its main medical functions in- the antioxidant activity of the essential oil and various solvent ex- clude invigoration of the kidney and liver, strengthening of the tracts of Rhizoma Homalomenae were evaluated. Total phenolic and flavonoid contents of the extracts, chemical composition of ⇑ Corresponding author. Tel.: +86 791 7118873. the essential oil and the phenolic constituents of the extract with E-mail address: [email protected] (J.-X. Zhu). most potent antioxidant ability were also determined. 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.09.029 L.-B. Zeng et al. / Food Chemistry 125 (2011) 456–463 457 2. Materials and methods (10 ll) was mixed with 900 ll of 100 mM Tris–HCl buffer (pH 7.4), 40 ll of methanol and 50 ll of 0.5% (w/w) Tween 20 solution 2.1. Plant materials and chemicals (Bruni et al., 2004). And ABTS radical was generated by mixing 7 mM ABTS and 2.45 mM potassium persulphate via incubation Rhizoma Homalomenae was purchased from Hong Kong in Jan at 23 °C in the dark for 12 h. Then, 0.1 ml of sample solution was 17, 2009 (originally produced in Guangxi Province, China). The mixed with 2.6 ml of diluted ABTS radical solution. After incuba- dry materials were ground to powder and passed through a 20- tion at 23 °C for 6 min, absorbance of the mixture solution was mesh sieve for the preparation of essential oil and extracts. measured at 734 nm in a microplate reader (PowerWave XS, Bio- 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,20-azinobis(3-ethyl- Tek Instruments Inc.). The ABTS radical-scavenging activity (%) benzothiazoline-6-sulphonic acid) (ABTS), potassium persulphate, was calculated by the following equation: scavenging activity 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), (%) = (1 À Asample/Acontrol)  100, where Asample is the absorbance nitroblue tetrazolium (NBT), b-nicotinamide adenine dinucleotide in the presence of extracts and Acontrol is the absorbance of control. (NADH), phenazin methosulphate (PMS), b-carotene, linoleic acid, BHT was used as the reference compound. potassium ferricyanide, trichloroacetic acid, ferric chloride, BHT, Folin–Ciocalteau reagent, gallic acid, rutin, protocatechuic acid, vanillic acid, syringic acid, caffeic acid, p-coumaric acid, ferulic 2.5. Antioxidant assay using the b-carotene bleaching method acid, and apigenin were purchased from Sigma–Aldrich (St. Louis, MO). All other reagents were of analytical or HPLC grades. The antioxidant activity of the extracts and essential oil were also evaluated by the b-carotene bleaching test (Conforti, Statti, b 2.2. Preparation of essential oil and extracts Uzunov, & Menichini, 2006). Briefly, 1 ml of -carotene solution (0.2 mg/ml in chloroform) was added to 0.02 ml of linoleic acid The sieved powder was subjected to water distillation for 5 h and 0.2 ml of 100% Tween 20. The mixture was then evaporated at 40 C for 10 min in a rotary evaporator to remove chloroform. using a distillation apparatus. The essential oil obtained was dried ° After evaporation, 60 ml of distilled water were slowly added to over anhydrous sodium sulphate and stored at 4 °C in the dark un- the mixture and agitated vigorously to form an emulsion. 0.2 ml til used. of sample solution was transferred to the emulsion (5 ml) and The aqueous phase, after the essential oil distillation, was 0.2 ml of solvent instead of sample solution in 5 ml of the above extracted exhaustively by dichloromethane, ethyl acetate, and emulsion was used as a control. BHT was used as the reference n-butanol, successively, at a ratio of 1:1 (v/v, aqueous phase/ compound. The mixture obtained was then gently shaken and solvent) and the extracts obtained were numbered as QJ3, QJ4, placed at 50 C for 60 min. The absorbance of the mixture was and QJ5, respectively. After each extraction, the organic solvents ° measured at 470 nm, using a microplate reader (PowerWave XS, were removed under vacuum at 50 °C with a rotary evaporator. BioTek Instruments Inc.) against a blank, consisting of an emulsion One part of the aqueous phase after the solvent extraction above without b-carotene. The antioxidant activity was measured using was lyophilised and numbered as QJ1, while the other part was the following equation: antioxidant activity = [1 (S S )/ treated with ethanol to precipitate carbohydrates and the superna- À 0 À t (C C )] 100, where S and C are the absorbances measured tant after the removal of ethanol with a rotary evaporator was 0 À t  0 0 at the initial incubation time for samples and control, respectively, lyophilised and numbered as QJ2. The solid residue after the essen- while S and C are the absorbances measured in the samples and tial oil distillation was lyophilised and then extracted exhaustively t t control at t = 60 min, respectively. with ethyl acetate and ethanol, successively, at a ratio of 1:20 (w/v, lyophilised solid residue/solvent) and the extracts obtained were numbered as QJ6 and QJ7, respectively. After each extraction, the 2.6. Reducing power assay organic solvents were removed. All the extracts were lyophilised and kept in the dark at 4 °C until used. The preparation procedure Reducing power of the extracts was also determined (Lee et al., of the essential oil and extracts is schematically represented in 2007). Extract solution (80 l), sodium phosphate buffer (200 l, Fig. 1. l l 0.2 M, pH 6.6) and potassium ferricyanide (200 ll, 10 mg/ml) were mixed and incubated at 50 °C for 20 min.