Kasetsart J. (Nat. Sci.) 43 : 358 - 369 (2009) Antioxidant Activities and Antioxidative Components in Extracts of Alpinia galanga (L.) Sw. Nopparat Mahae and Siree Chaiseri* ABSTRACT Galangal extracted using 50% ethanol in water was studied for its antioxidant activity and composition in comparison with two other samples based on a water extract and the essential oil. The antioxidant activities were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) methods. The ethanolic extract showed the highest DPPH free radical scavenging ability as well as the highest ORAC value when compared to the water extract and the essential oil. The IC50 values of the galangal ethanolic extract (10.66 mg/ml), water extract (55.48 mg/ml) and essential oil (455.43 mg/ml) were higher than those of α-tocopherol (1.45 mg/ml) and butylated hydroxyanisole (BHA; 0.41 mg/ml). The results indicated that the antioxidant activities of galangal extracts were lower than that of BHA, the commercial synthetic antioxidant generally used in food. The ethanolic extract contained the highest concentrations of total phenolic compounds (31.49 mgGAE/g) and flavonoids (13.78 mgCE/g). The water extract and the essential oil had a total phenolic content of 8.25 and 5.01 mgGAE/g and a total flavonoid content of 1.48 and 0.20 mgCE/g, respectively. Antioxidants in the galangal essential oil, namely, methyl eugenol (4,130.38 µg/g), chavicol (2,390.45 µg/g), and eugenol (728.30 µg/g) were found in its volatile fraction. The water extract contained mainly myricetin (14.60 mg/g extract) and an unknown phenolic compound. The major antioxidants in the ethanolic extract were 1´-acetoxycavichol acetate (10.56 mg/g extract), catechin (1.74 mg/g extract), and three unknown subtances. In addition to the phenolic compounds, 1´-acetoxycavichol acetate (ACA) could play an important role in the antioxidant activity of galangal. The ethanolic extract of galangal was also advantageous as an antioxidant in food due to its mild odor compared with the essential oil. Key words: Alpinia galanga, galangal extract, antioxidants, essential oil, phenolic compounds, 1´-acetoxycavichol acetate INTRODUCTION medicine (Yang and Eilerman, 1999), as the essential oil shows an antimicrobial activity Galangal (Alpinia galanga) is used to against gram-positive bacteria, yeast, and flavor foods throughout South and Southeast Asia. dermatophytes. The most active compound is Its aromatic characteristic is described as woody, terpinen-4-ol (Janssen and Scheffer, 1985). In minty and floral (Mori et al., 1995). Its rhizome addition, essential oil from galangal was reported has a wide range of applications in traditional as a potential anti-carcinogen (Zheng et al., 1993). Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand. * Corresponding author, e-mail: [email protected] Received date : 15/09/08 Accepted date : 07/01/09 Kasetsart J. (Nat. Sci.) 43(2) 359 Several researches reported that 1´- extract with regard to its antioxidant activity in acetoxychavicol acetate (ACA) and 1´- comparison with a water extract and the essential acetoxyeugenol acetate from galangal were anti- oil. tumor substances (Itokawa et al., 1987; Kondo et al., 1993). MATERIALS AND METHODS Natural antioxidants from plants have attracted significant interest because of their safety Chemicals and apparatus and potential nutritional and therapeutic effects. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Several plant materials have been investigated as fluorescein sodium salt (FL) and (+)-α-tocopherol a potent source of antioxidants. Antioxidants in were purchased from Sigma-Aldrich (Milwaukee, herbs and spices include: vitamins; phenolic WI, USA). 6-Hydroxy-2,5,7,8-tetramethyl- compounds including flavonoids and phenolic chroman-2-carboxylic acid (Trolox) and 2,2´- acids; and volatile compounds (Carrubba and azobis (2-methylpropionamidine) dihydrochloride Calabrese, 1998). The antioxidant activity of (AAPH) were obtained from Sigma-Aldrich phenolic compounds is mainly dependent on their (Munich, Germany), while the gallic acid was redox properties that allow them to act as reducing sourced from Fluka (Madrid, Spain) and Folin- agents, hydrogen donators and singlet oxygen Ciocalteu’s phenol reagent from Fluka (Buchs, quenchers. Additionally, they have a metal- Switzerland). Randomly methylated beta chelating potential (Rice-Evans et al., 1995). cyclodextrin (RMCD) for the ORAC test was Several studies have been conducted on the purchased from Cyclodextrin Technologies antioxidant activities of galangal extracts. The Development (Gainesville, FL, USA). Acetone, antioxidant activity of a 1% (w/v) acetone extract ethanol, and methanol came from Merck of galangal in 99.5% ethanol was stronger than (Darmstadt, Germany). A fluorescent microplate that of α-tocopherol (Jitoe et al., 1992). Zaeoung reader (FLUOstar OPTIMA, BMG Labtech, et al. (2005) found the new phenolic compound, Offenburg, Germany) and fluorescence filters with p-coumaryl-9-methyl ether, in the methanolic an excitation wavelength of 485 nm and emission extract of fresh galangal. Moreover, two phenolic wavelength of 520 were used. The 96-well black compounds, p-hydroxy cinnamaldehyde and [di- microplates were purchased from BMG Labtech. (p-hydroxy-cis-styryl)] methane, were isolated Volatile compounds were isolated with a high from the chloroform extract of dry galangal (Barik vacuum distillation unit (Edwards, West Sussex, et al., 1987). In food systems, research has mostly UK) and a spectrophotometer (Spectro 23, been carried out on the ethanolic extract (Cheah LaboMed, Inc., USA) was used for the and Abu Hasim, 2000; Juntachote et al., 2006). spectrophotometric assay. The ethanolic extract of galangal acted as a free radical scavenger, exhibiting strong superoxide, Sample preparation and extraction anion-scavenging activity, Fe2+ chelating activity Fresh rhizomes of galangal were and reducing power in a concentration-dependent purchased from local markets in Bangkok and manner (Juntachote and Berghofer, 2005). cleaned with tap water. The extracts were prepared Although, there were several studies on the from fresh and dried galangal. Dried galangal was antioxidant activities of galangal ethanolic prepared by freeze-drying the fresh rhizomes using extracts, the information related to their a freeze dryer (Heto Lab Equipment, Allerod, composition was limited. This study aimed to Denmark). The dried sample was ground to a fine investigate the composition of galangal ethanolic powder using a blender and then passed through a 360 Kasetsart J. (Nat. Sci.) 43(2) 60-mesh sieve. Antioxidant activity Three types of the extracts were The antioxidant activity of the water prepared: ethanolic extract, water extract and extract, ethanolic extract and essential oil from the essential oil. The ethanolic extract and water galangal rhizome were studied using DPPH and extract were prepared from galangal powder ORAC methods. The use of galangal extracts whereas the essential oil was steamed-distilled together with BHA in methanol was also from fresh galangal. investigated by the DPPH method to study the effects of using the extract with a commercial Ethanolic extract antioxidant. A comparative study on the Galangal powder was extracted using antioxidant activities of the samples, BHA and 50% ethanol (v/v) in water at a solid-to-solvent α-tocopherol was also conducted using the DPPH ratio of 1:10. The extraction was performed in a method and reported as IC50 . shaking water bath at 50°C for 1 h. The extract was filtered through a No.1 sinter glass funnel. DPPH assay The residue was re-extracted using the same The electron-donation abilities of the conditions. The two extracts were then combined extracts and commercial antioxidants were and concentrated in a rotary evaporator at 40°C. measured from the bleaching of purple DPPH in The concentrated extract was dried using a freeze methanol solution. dryer and kept in a glass vial at –40°C. The DPPH assay was carried out as described by Tepe et al. (2005). The ethanolic Water extract extract and the water extract were diluted with Ten grams of galangal powder were water and the essential oil was diluted with ethanol refluxed with 100 ml of distilled water for 3 h at to various concentrations. The concentrations of 80°C. The mixture was cooled to room temperature the ethanolic extract were from 0 to1.0% (w/v), and filtered through a No.1 sinter glass funnel. The the water extract samples were from 0 to 5.0% residue was then re-refluxed with 100 ml distilled (w/v), while the essential oil sample ranged from water under the same conditions. The extracts were 0 to 25% (w/v). The DPPH studies were conducted combined and concentrated in a rotary evaporator with and without the addition of BHA. The at 60°C. The concentrated extract was freeze dried ethanolic extract and water extract samples were and kept in a glass vial at – 40°C. supplemented with 0.01% BHA (w/v) in methanol, whereas the essential oil samples were Essential oil supplemented with 0.02% BHA (w/v) in methanol, The essential oil was obtained from fresh in order to be able to observe the antioxidant galangal rhizomes by steam distillation using a activities. glass essential oil extractor (Becthai Bangkok In the DPPH assay, 50 µl of samples at Equipment and Chemical, Bangkok, Thailand). various concentrations were added to 5 ml of 0.004 The fresh rhizomes were cut into small pieces % DPPH in methanol. After a 30 min incubation (1 cm × 1 cm × 0.3 cm) and 320 g of galangal was period at room temperature, the absorbance was comminuted with 400 ml water. The slurry was read at 517 nm. The purple color bleaching of the prepared in a one-liter, round-bottomed flask and DPPH reagent was evaluated as positive steam-distilled for 4 h. The essential oil was dried antioxidant activity. The inhibition of free radicals by filtering through anhydrous sodium sulfate and by DPPH in percent (I %) was calculated using kept in a glass vial at – 40°C.