Gopalakrishnan V.K et al. IRJP 2012, 3 (6) INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407 Research Article CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITY OF ESSENTIAL OIL FROM CURCUMA AMADA ROXB. Vishnupriya.M1, Nishaa.S1, Sasikumar.J.M1, Teepica Priya Darsini.D1, Hephzibah Christabel P 1, Gopalakrishnan V.K2* 1Department of Biotechnology, Karpagam University, Coimbatore, Tamilnadu, India 2Department of Biochemistry and Bioinformatics, Karpagam University, Coimbatore Tamilnadu, India Article Received on: 09/04/12 Revised on: 18/05/12 Approved for publication: 10/06/12 *Dr V.K Gopalakrishnan, Professor and Head, Department of Biochemistry and Bioinformatics, Karpagam University, Coimbatore – 641021 Email: [email protected] ABSTRACT This study was designed to examine the chemical composition and in vitro antioxidant activity of essential oil of Curcuma amada Roxb. The GC- MS analysis of the oil resulted in the identification of 12 compounds. β-myrcene (63.85%) and α-asarone (30.27%) were the two major components identified. The sample was subjected to screening for their possible antioxidant activity by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical, ABTS radical, Ferric reducing antioxidant power and β-Carotene bleaching assay. Results showed that the essential oil possessed a strong degree of antioxidant activity in terms of β- Carotene bleaching capacity followed by ABTS radical, ferric reducing power and a moderate DPPH radical scavenging activity. This study concludes that the essential oil from Curcuma amada Roxb could serve as an important bioresource of antioxidants for using in food and pharmaceutical industry. Key words: Curcuma amada Roxb, Essential oil, GC-MS Analysis, Antioxidant activity, β-myrcene, α-asarone INTRODUCTION against inflammations of mouth, ear, and gleets, ulcers on the It has long been recognized that naturally occurring male sex organs, scabies, lumbago and stomatatitis. It is still substances in higher plants have antioxidant activity. Plant used in post partum care; specifically to aid healing of kingdom is a good source of natural preparations containing wounds8. It has been reported to induce hypertriglyceridemic effective bioactive compounds which can be used for activity in Trition-induced hyperlipidemic rats. different applications particularly as food additives and health The present study was carried out to evaluate the constituents promoting ingredients in the formulations of functional foods of C. amada essential oil and to determine its in vitro and nutraceuticals1. Nowadays, the interest has considerably antioxidant activities. increased for the use in food, cosmetic and pharmaceutical MATERIALS AND METHODS products to replace synthetic antioxidants which are being The rhizomes of Mango ginger (Curcuma amada Roxb.) restricted due to their carcinogenicity2. were collected from the native farms of Coimbatore and were Essential oil and their components are gaining increasing authenticated by, Dr.C.Kunhikannan, Scientist E, Division of interest because of their relatively safe status, their wide Biodiversity, Institute of Forest Genetics and Tree Breeding, acceptance by consumers and their exploitation for potential Indian Council of Forestry Research & Education, multi-purpose functional use3. Antioxidants in the oils are Coimbatore. important in the stabilization of free fatty acids4. In this Extraction of Essential oil context, aromatic plants, particularly their essential oils, are The fresh rhizomes of plants were subjected for 3 hrs to water being evaluated for antioxidant activity since they find distillation using a Clevenger type apparatus. The obtained extensive use in food and beverage industry. Many studies essential oil was dried over anhydrous sodium sulphate have shown that the presence of natural antioxidants from (Na2SO4) and preserved in a sealed vial at 4°C until further various aromatic and medicinal plants is closely related to the analysis. reduction of chronic disease such as DNA damage, GC-MS analysis mutagenesis, and carcinogenesis5. Therefore, there has been a The analysis of the essential oil was performed using a growing interest in research concerning alternative Hewlett Packard 5890 II GC equipped with a FID detector antioxidant active compounds, including plant extracts and and HP-5 ms capillary column (30m´ 0.25m, film thickness essential oils that are relatively less damaging to the 0.25µm). For GC-MS detection, an electron ionization mammalian health and environment. system was used with ionization energy of 70 eV. Helium Mango ginger (Curcuma amada Roxb.) is a perennial herb, was the carrier gas, at a flow rate of 1ml/min. Injector and which morphologically resembles the ginger (Zingiber MS transfer line temperature were set at 220 and 290°C officinale) but, it imparts mango (Magnifera indica) flavour. respectively. Column temperature was initially at 50°C, and Mango flavour is mainly attributed to car-3-ene and cis- then gradually increased to 150°C at a 3°C/min rate, held for ocimene. It is widely used in making pickles in several parts 10 min and finally increased to 250Vc at 10Vc/min. Diluted of India. The mango ginger starch constitutes 43% of samples (1/100 in petroleum ether) of 1.0µl were injected amylose and resembles the characteristic of both Curcuma manually and split less. The components were identified longa and Zingiber officinale starch6. Difurocumenonol and based on the comparison of their relative retention time and amadannulen compounds, which are highly antioxidant and mass spectra with those of Wiley 7N Library data and antimicrobial in nature, have been isolated and characterized standards of the main components. The results were also by Policegoudra et.al, 20077. The rhizomes are used to treat confirmed by the comparison of the compounds elution order stomach and chest pains, fever and debility. It is also useful Page 99 Gopalakrishnan V.K et al. IRJP 2012, 3 (6) with their relative retention indices on non-polar phases The major components were β-myrcene and α-asarone, which reported in the literature9. represents 63.85% and 30.27% respectively. Of these 12 Antioxidant activity constituents, these two compounds accounts for about DPPH Radical Scavenging activity 94.12%. Additional notable components include β-pinene, a Radical scavenging using DPPH radical by the method of monoterpene of about 2.01% and 1-nitroguanidine of about Blois10. A solution of DPPH in methanol (24µg/ml) was 1.81%. Based on GC-MS analysis of the essential oil of prepared and 2ml of this solution was added to oil in Curcuma amada Roxb, the major compounds were found to methanol at different concentrations (100,200,300,400 and be β-myrcene followed by α-asarone. 500 µl). Initially 100 µl of the volatile oil was dissolved in Gas chromatography–mass spectrometry (GC–MS) is 1ml of methanol. Then, the absorbance was measured at certainly a useful and powerful tool in the essential oil 517nm. Radical-scavenging activity was calculated by % analysis. It is noteworthy that the composition of the essential inhibition. oils from a particular species of a plant can differ between ABTS•+ radical cation scavenging assay harvesting seasons, extraction methods, and geographical The ability to scavenge the ABTS radical cation (ABTS•+) sources, and that those from a different parts of the same was determined according to Re et al., 11. The stock solutions plant can also differ widely14. Maturation stages constitute an included 7 mM ABTS solution and 2.4 mM potassium important factor influencing essential oil composition in persulfate solution. The working solution was then prepared some plants15. by mixing two stock solutions in equal quantities and Antioxidant activity allowing them to react at dark for 12 hrs at room temperature. There are several methodological limitations for antioxidant The solution was then diluted by mixing 1 ml ABTS solution determinations16. The most widely used methods for with 60 ml of methanol to obtain an absorbance of measuring antioxidant activity are those that involve the 0.706±0.001 units at 734 nm. The essential oil at various generation of radical species, where the presence of concentrations was then allowed to react with 1 ml of ABTS antioxidants determines the disappearance of radicals17. It is solution and the absorbance was read at 734 nm after 7 min. essential to use different assay systems to assess the Radical-scavenging activity was calculated by % inhibition. comparative antioxidant capacity instead of relying on single Ferric reducing antioxidant power (FRAP) assay assay. From the chemical composition it was found that β- Reducing power of the essential oil was determined by the myrcene and α-asarone are the major components. The method prescribed by Oyaizu12.Various concentrations of the antioxidant nature of β-myrcene was reported by Koroch et diluted essential oil were added to 2.5 ml of (0.2 M) sodium al., 18. The synergistic role of various components present in phosphate buffer (pH 6.6) and .5 ml of 1% potassium the oil might also attributes to the antioxidant nature of oil. ferricyanide [K3Fe2(CN)6] solution. The contents were However it was also considered that minor components, vortexed well and incubated at 50°C for 20 min. After might also have possible interactions between the major incubation, 2.5 ml of 10% trichloroacetic acid was added to components which might also affect the antioxidant all the tubes and centrifugation was carried out at 3000g for activities. In that sense, for biological purpose, it is more 10 min. Thereafter, to 5 ml of the supernatant, 5 ml of informative to study the entire oil rather than its components. deionized water was added. To this, 1 ml of 1% ferric Figure 1 shows the DPPH radical scavenging (%) activity of chloride was added to each test tube and incubated at 35°C Curcuma amada Roxb., essential oil compared to BHT. The for 10 min. The formation of Perl’s Prussian colour was highest scavenging activity of the essential oil is about measured at 700 nm. 85.21% which is similar to that of BHT standard (87.26%).