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Curcuminoid Contents, Antioxidant and Anti-Inflammatory Activities of Curcuma Xanthorrhiza Roxb

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Curcuminoid Contents, Antioxidant and Anti-Inflammatory Activities of Curcuma Xanthorrhiza Roxb Prosiding Seminar Nasional Kimia Unesa 2012 – ISBN : 978-979-028-550-7 Surabaya, 25 Pebruari 2012 Curcuminoid Contents, Antioxidant and Anti-Inflammatory Activities of Curcuma xanthorrhiza RoxB. and Curcuma domestica Val. Promising Lines From Sukabumi of Indonesia Waras Nurcholis1, 2), Laksmi Ambarsari1, 2), Ni Luh Putu Eka Kartika Sari2, Latifah K Darusman1, 3) 1) Biopharmaca Research Center, Bogor Agricultural University, Indonesia 2) Department of Biochemistry, Bogor Agricultural University, Indonesia 3) Department of Chemistry, Bogor Agricultural University, Indonesia E-mail: [email protected] or [email protected] ABSTRACT The main bioactive substances in the rhizomes of Curcuma xanthorrhiza and Curcuma domestica that have eficacy as antioxidant and anti-inflamatory activities are curcuminoids. In this study, ethanol extracts of C. xanthorrhiza and C. domestica promising lines from Sukabumi of Indonesia were investigated for the presence of curcuminoids, antioxidant and anti-inflamatory activities. HPLC method was used to determined curcuminoids content. The antioxidant (radical scavenging) potential of the samples was evaluated using 2,2-diphenyl-1- picrylhydrazyl (DPPH) free radical method. While for the anti-inflamatory activity, the in vitro cyclooxygenase 2 (COX2) inhibition method was used. The curcuminoid content of C. xanthorrhiza and C. domestica were 31.27 and 66.32 mg/ g, respectively. IC50 values for DPPH radical scavenging activity were 81.99 and 73.31 µg/ mL, with C. domestica having lowest value and most potent than C. xanthorrhiza. Percent inhibition values for COX2 inhibitor activity were 74.84 and 67.96 %, with C. domestica having the highest value. In this study, the ethanol extracts of C. domestica promosing line from Sukabumi of Indonesia exhibited most in curcuminoids content, antioxidants properties and anti- inflamatory activity than C. xanthorrhiza promosing line. Keywords: Curcuminoid, Antioxidant, Anti-Inflamatory, Curcuma xanthorrhiza, Curcuma domestica, Promosing line INTRODUCTION diarrhea, dysentery, children’s Curcuma xanthorrhiza Roxb., fevers, hemorrhoids, and skin eruptions (Hwang et al., 2000). also know as “temulawak” in Indonesia, and Curcuma domestica Pharmacologically is has been Val., known in Indonesia as reported that C. xanthorrhiza has the antimicrobial (Hwang et al., 2000), “kunyit”, are a medicinal plant from the family Zingeberaceae distributed anti-metastatic (Choi et al., 2004), anti-cancer (Huang et al., 1998), anti- in Indonesia. Traditionally, C. xanthorrhiza rhizomes have been candidal (Rukayadi et al., 2006), anti-oxidant (Masuda et al., 1992) used to treat stomach diseases, liver disorders, constipation, bloody and hypolipidemic activities (Yasni et al., 1993). The rhizomes of C. C - 284 Prosiding Seminar Nasional Kimia Unesa 2012 – ISBN : 978-979-028-550-7 Surabaya, 25 Pebruari 2012 domestica are commonly used as a MATERIALS AND METHODS flavoring, coloring agent and Materials preservative (Thaikert & Paisooksantivatana, 2009). The The rhizomes of C. traditional Indian medicine claims xanthorrhiza and C. domestica the use of C. domestica powder promising lines were collected against biliary disorders, anorexia, during July 2011 from the area of coryza, cough, diabetic wounds, Sukabumi, West Java, Indonesia. hepatic disorder, rheumatism and The authentic chemical standards of sinusitis (Ammon et al., 1992). curcuminoid (curcumin, Pharmacolological research has demethoxycurcumin, and demonstrated that C. domestica bisdemethoxycurcumin) and 2,2- possesses anti-inflammatory diphenyl-1-picrylhydrazyl were (Mukophadhyay et al. 1982; Arora et obtained from Sigma-Aldrich, USA. al., 1971; Chandra and Gupta, 1972), The bioassay kit COX was purchased antioxidant (Unnikrishnan and Rao, from Cayman Chemicals. All the 1995; Pulla Reddy & Lokesh, 1992), chemicals and solvents used were anti-protozoal (Rasmussen et al., analytical grade. 2000), nematocidal (Kiuchi et al., 1993), anti-bacterial (Chopra et al., 1941; Bhavani Shankar & Murthy, Extraction of plans 1979), antivenom (Ferreira et al., Fresh rhizomes of plant 1992), anti-HIV (Mazumber et al., materials were washed with water, 1995; Eigner and Scholz 1999), and cut into small pieces and dried for 5 anti-tumor activities (Huang et al., days in the sun (the moisture: < 1988). 10%). They were then ground in a The main yellow bioactive grinder to be obtained in a powder substances in the rhizomes of C. form (the size: 100 mesh). One kilo xanthorrhiza and C. domestica are grams of the powder of plants were curcuminoid (Duke, 2002; Chainani- macerated using 1 x 10 L ethanol Wu, 2003). The pharmacological 70% in a tightly closed round bottom effect of curcuminoids has been flask at room temperature for a investigated, such as radical period of 24 h and filtered with scavenging (Sreejayan & Rao, 1996), Whatman filter paper (type 4). The the inhibition of nitric oxide (NO) whole process was repeated one (Pan et al., 2000; Onoda et al., 2000), times and the filtrate was anti-inflammation (Banerjee et al., concentrated under reduced pressure 2003), anti-tumor (Khar et al., 1999), on rotavapor (BUCHI, R-250, anti-allergy (Ram et al., 2003) and Switzerland) at 50 °C temperature. anti-dementia (Lim et al., 2001). This The concentrated extracts were then study was undertaken to verify the used for the experiments. variation C. xanthorrhiza and C. domestica promising lines from Sukabumi of Indonesia for their total Determination of curcuminoid curcuminoid, antioxidant and anti- content inflamatory activities. The curcuminoid content of ethanol extract of C. xanthorrhiza and C. domestica promising lines C - 285 Prosiding Seminar Nasional Kimia Unesa 2012 – ISBN : 978-979-028-550-7 Surabaya, 25 Pebruari 2012 from Sukabumi was carried out (Ac − Acb) − (As − Asb) = x 100 according to the principle and (Ac − Acb) protocol previously described by Jayaprakasha et al., (2002) using where Ac is the absorbance of high performance liquid water plus DPPH (in ethanol), Acb is chromatography (HPLC), with the absorbance of the blank (water modification. The systems condition plus ethanol without DPPH), As is of HPLC are C18 column, UV-Vis the absorbance of the sample plus detector, 10 µL volum injection, and DPPH (in ethanol) and Asb is the 48 °C temperature column. It’s used absorbance of the sample plus methanol as an additional mobile ethanol without DPPH. Different phase, , which included solvents A: sample concentrations were used in methanol; B: 2% acetic acid; and C: order to obtain calibration curves and acetonitrile. to calculate the IC50 values (IC50: concentration required to obtain a 50% radical scavenging activity). All Antioxidant activity through test samples were conducted in measurement of free radical triplicate (n = 3). scavenging capacity by DPPH assay Determination of anti- Free radical scavenging inflammatory activity capacity was investigated on the basis of the scavenging activity of The ethanol extracts of C. DPPH by measuring the reduction of xanthorrhiza and C. domestica absorbance at 517 nm. The method promising lines from Sukabumi was was carried out as described by evaluated as anti-inflammatory with Udenigwe et al., (2009) with a cyclooxygenase 2 (COX2) inhibitory modification. The different activities. COX2 inhibition activity concentrations of the plant extracts was measured using the Cayman (12.5–200 µg/ml; total volume of 40 Chemical Colorimetric COX (ovine) µL) in 96-well plates were mixed Inhibitor Screening Assay Kit. All with 160 µL of 100 mM DPPH in procedures were performed as ethanol and then incubated in the indicated in the assay kit instructions. dark for 30 min at room temperature The ethanol extracts were dissolved prior to reading the absorbance at in DMSO and added to the enzyme 517 nm in a micro plate reader. A reaction mixture. negative control, containing water instead of the sample and blank RESULTS AND DISCUSSION samples, using the same volume of ethanol only in place of the DPPH Curcuminoids content solution in ethanol, were all Curcuminoids compound are evaluated at the same time per micro commonly in Curcuma species and titre plate. have been reported to have several The percentage of radical biological activities including scavenging was calculated as antioxidant and anti-inflammatory follows; properties (Itokawa et al., 2008). The results of curcuminoids content on % radical scavenging C - 286 Prosiding Seminar Nasional Kimia Unesa 2012 – ISBN : 978-979-028-550-7 Surabaya, 25 Pebruari 2012 the ethanol extract of C. xanthorrhiza different successive extracts of C. and C. domestica promising lines xanthorrhiza and C. domestica. This from Sukabumi are shown in Figure activity was increased by increasing 1A. The curcuminoids content of C. the concentration of sample extracts. domestica (value, 66.32 mg/ g) most DPPH antioxidant assay is based on highest than C. xanthorrhiza with the ability of DPPH, a stable free value 31.27 mg/ g. The rhizomes of radical, to decolorize in the presence C. xanthorrhiza and C. domestica of antioxidants. The DPPH radical show different profiles in the three contains an odd electron, which is major curcuminoids. The responsible for the absorbance at 517 curcuminoids in C. domestica are nm and also for a visible deep purple mainly curcumin, demethoxy- color. When DPPH accepts an curcumin, and bisdemethoxy- electron donated by an antioxidant curcumin (Figure 1B) (Lechtenberg compound, the DPPH is decolorized, et al., 2004; Thaikert & which can be quantitatively
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