Curcuminoid Contents, Antioxidant and Anti-Inflammatory Activities

Prosiding Seminar Nasional Kimia Unesa 2012 – ISBN : 978-979-028-550-7 Surabaya, 25 Pebruari 2012

C - 284

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

Curcuma xanthorrhiza Roxb., also know as “temulawak” in Indonesia, and Curcuma domestica Val., known in Indonesia as “kunyit”, are a medicinal plant from the family Zingeberaceae distributed in Indonesia. Traditionally, C. xanthorrhiza rhizomes have been used to treat stomach diseases, liver disorders, constipation, bloody

diarrhea, dysentery, children’s fevers, hemorrhoids, and skin eruptions (Hwang et al., 2000). Pharmacologically is has been reported that C. xanthorrhiza has the antimicrobial (Hwang et al., 2000), anti-metastatic (Choi et al., 2004), anti-cancer (Huang et al., 1998), anti-candidal (Rukayadi et al., 2006), anti-oxidant (Masuda et al., 1992) and hypolipidemic activities (Yasni et al., 1993). The rhizomes of C.


C - 285

domestica are commonly used as a flavoring, coloring agent and preservative (Thaikert & Paisooksantivatana, 2009). The traditional Indian medicine claims the use of C. domestica powder against biliary disorders, anorexia, coryza, cough, diabetic wounds, hepatic disorder, rheumatism and sinusitis (Ammon et al., 1992). Pharmacolological research has demonstrated that C. domestica possesses anti-inflammatory (Mukophadhyay et al. 1982; Arora et al., 1971; Chandra and Gupta, 1972), antioxidant (Unnikrishnan and Rao, 1995; Pulla Reddy & Lokesh, 1992), anti-protozoal (Rasmussen et al., 2000), nematocidal (Kiuchi et al., 1993), anti-bacterial (Chopra et al., 1941; Bhavani Shankar & Murthy, 1979), antivenom (Ferreira et al., 1992), anti-HIV (Mazumber et al., 1995; Eigner and Scholz 1999), and anti-tumor activities (Huang et al., 1988).

The main yellow bioactive substances in the rhizomes of C. xanthorrhiza and C. domestica are curcuminoid (Duke, 2002; Chainani-Wu, 2003). The pharmacological effect of curcuminoids has been investigated, such as radical scavenging (Sreejayan & Rao, 1996), the inhibition of nitric oxide (NO) (Pan et al., 2000; Onoda et al., 2000), anti-inflammation (Banerjee et al., 2003), anti-tumor (Khar et al., 1999), anti-allergy (Ram et al., 2003) and anti-dementia (Lim et al., 2001). This study was undertaken to verify the variation C. xanthorrhiza and C. domestica promising lines from Sukabumi of Indonesia for their total curcuminoid, antioxidant and anti-inflamatory activities.

MATERIALS AND METHODS Materials

The rhizomes of C. xanthorrhiza and C. domestica promising lines were collected during July 2011 from the area of Sukabumi, West Java, Indonesia. The authentic chemical standards of curcuminoid (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) and 2,2-diphenyl-1-picrylhydrazyl were obtained from Sigma-Aldrich, USA. The bioassay kit COX was purchased from Cayman Chemicals. All the chemicals and solvents used were analytical grade.

Extraction of plans Fresh rhizomes of plant

materials were washed with water, cut into small pieces and dried for 5 days in the sun (the moisture: < 10%). They were then ground in a grinder to be obtained in a powder form (the size: 100 mesh). One kilo grams of the powder of plants were macerated using 1 x 10 L ethanol 70% in a tightly closed round bottom flask at room temperature for a period of 24 h and filtered with Whatman filter paper (type 4). The whole process was repeated one times and the filtrate was concentrated under reduced pressure on rotavapor (BUCHI, R-250, Switzerland) at 50 °C temperature. The concentrated extracts were then used for the experiments.

Determination of curcuminoid content

The curcuminoid content of ethanol extract of C. xanthorrhiza and C. domestica promising lines


C - 286

from Sukabumi was carried out according to the principle and protocol previously described by Jayaprakasha et al., (2002) using high performance liquid chromatography (HPLC), with modification. The systems condition of HPLC are C18 column, UV-Vis detector, 10 µL volum injection, and 48 °C temperature column. It’s used methanol as an additional mobile phase, , which included solvents A: methanol; B: 2% acetic acid; and C: acetonitrile.

Antioxidant activity through measurement of free radical scavenging capacity by DPPH assay

Free radical scavenging capacity was investigated on the basis of the scavenging activity of DPPH by measuring the reduction of absorbance at 517 nm. The method was carried out as described by Udenigwe et al., (2009) with a modification. The different concentrations of the plant extracts (12.5–200 µg/ml; total volume of 40 µL) in 96-well plates were mixed with 160 µL of 100 mM DPPH in ethanol and then incubated in the dark for 30 min at room temperature prior to reading the absorbance at 517 nm in a micro plate reader. A negative control, containing water instead of the sample and blank samples, using the same volume of ethanol only in place of the DPPH solution in ethanol, were all evaluated at the same time per micro titre plate.

The percentage of radical scavenging was calculated as follows;

% radical scavenging

=(Ac − Acb)− (As− Asb)

(Ac − Acb) x 100

where Ac is the absorbance of water plus DPPH (in ethanol), Acb is the absorbance of the blank (water plus ethanol without DPPH), As is the absorbance of the sample plus DPPH (in ethanol) and Asb is the absorbance of the sample plus ethanol without DPPH. Different sample concentrations were used in order to obtain calibration curves and to calculate the IC50 values (IC50: concentration required to obtain a 50% radical scavenging activity). All test samples were conducted in triplicate (n = 3).

Determination of anti-inflammatory activity

The ethanol extracts of C. xanthorrhiza and C. domestica promising lines from Sukabumi was evaluated as anti-inflammatory with cyclooxygenase 2 (COX2) inhibitory activities. COX2 inhibition activity was measured using the Cayman Chemical Colorimetric COX (ovine) Inhibitor Screening Assay Kit. All procedures were performed as indicated in the assay kit instructions. The ethanol extracts were dissolved in DMSO and added to the enzyme reaction mixture.

RESULTS AND DISCUSSION Curcuminoids content Curcuminoids compound are commonly in Curcuma species and have been reported to have several biological activities including antioxidant and anti-inflammatory properties (Itokawa et al., 2008). The results of curcuminoids content on


C - 287

the ethanol extract of C. xanthorrhiza and C. domestica promising lines from Sukabumi are shown in Figure 1A. The curcuminoids content of C. domestica (value, 66.32 mg/ g) most highest than C. xanthorrhiza with value 31.27 mg/ g. The rhizomes of C. xanthorrhiza and C. domestica show different profiles in the three major curcuminoids. The curcuminoids in C. domestica are mainly curcumin, demethoxy-curcumin, and bisdemethoxy-curcumin (Figure 1B) (Lechtenberg et al., 2004; Thaikert & Paisooksantivatana, 2009). While for the curcuminoids in C. xanthorrhiza are curcumin and demethoxy-curcumin (Figure 1C) (Lechtenberg et al., 2004).

Antioxidant activity The measures of antioxidant

activity were obtained using DPPH (2,2-diphenyl-1-picrylhydrazyl) method. Figure 2 shows the DPPH radical scavenging activity of the

different successive extracts of C. xanthorrhiza and C. domestica. This activity was increased by increasing the concentration of sample extracts. DPPH antioxidant assay is based on the ability of DPPH, a stable free radical, to decolorize in the presence of antioxidants. The DPPH radical contains an odd electron, which is responsible for the absorbance at 517 nm and also for a visible deep purple color. When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized, which can be quantitatively measured from the changes in absorbance. Thus, the DPPH radicals were widely used to investigate the scavenging activity of some natural compounds. DPPH is widely used to evaluate the antioxidant activity of natural compounds (Udenigwe et al., 2009). However, DPPH’s scavenging activity indicates the ability of the antioxidant compound to donate electrons or hydrogen, thereby converting the radical to a more stable species (Bougatef et al., 2009).

Figure 1. (A). Curcuminoids content of C. xanthorrhiza and C. domestica promising lines from Sukabumi, West Java, Indonesia. (B). the curcuminoids component of C. domestica: curcumin, demethoxycurcumin, and bisdemethoxycurcumin. (C). the curcuminoids component of C. xanthorrhiza: curcumin and demethoxycurcumin.

0.

10.

20.

30.

40.

50.

60.

70.

Curcuma xanthorrhiza Curcuma domestica

31.27

66.32

Curc

umin

oids

con

tent

(mg/

g)

Promising lines from Sukabumi

A

Curcumin

Demethoxycurcumin

Bisdemethoxycurcumin

B

C


C - 288

Figure 2. Scavenging effect of C. xanthorrhiza and C. domestica promising lines from Sukabumi, West Java, Indonesia on DPPH (2,2-diphenyl-1-picrylhydrazyl), as measured by changes in absorbance at 517 nm.

Antioxidant activity using

DPPH radical scavenging assay reported with IC50 value is show in Figure 3. The lower the IC50 is the higher the antioxidant activity of the Curcuma promising lines.

Figure 3. DPPH free radical scavenging activity of C. xanthorrhiza and C. domestica promising lines from Sukabumi, West Java, Indonesia.

The results showed that the ethanol extracts of C. xanthorrhiza and C. domestica have highest of antioxidant activity (with IC50 value < 200 µg/ mL, Blois, 1958). 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. Curcuminoids compound are the major C. xanthorrhiza and C. domestica compounds with antioxidant activity (Jayaprakasha et al., 2006; Masuda et al., 1992). This study, we used ethanol crude extract of C. xanthorrhiza and C. domestica, so it’s possible has pure compound most stronger as DPPH free radical scavenging activity than that extracts.

Anti-inflammatory activity Anti-inflammatory activity of C. xanthorrhiza and C. domestica promising lines from Sukabumi was evaluated through the percentages of

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

12.5 25 50 100 200

Abs

orba

nce

mea

sure

d at

517

nm

Concentration (µg/ mL)

C. xanthorrhiza

C. domestica

73.3181.99

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

C. domestica C. xanthorrhiza

Ant

ioxi

dant

act

ivit

y, I

C50

valu

e (µ

g/m

L)



C - 289

cyclooxygenase 2 (COX2) inhibition. Results are given in Figure 4. Percent inhibition values for COX2 inhibitor activity of C. domestica and C. xanthorrhiza were 74.84 and 67.96 %, with C. domestica having the highest value. Curcuminoids compound are the major C. xanthorrhiza and C. domestica compounds with anti-inflammatory properties (Chainani-Wu, 2003; Tohda et al., 2006; Ozaki, 1990). This study, we used ethanol crude extract of C. xanthorrhiza and C. domestica, so it’s possible has pure compound most stronger as anti-inflammatory activity than that extracts.

Figure 4. Anti-inflammatory activity of C. xanthorrhiza and C. domestica promising lines from Sukabumi, West Java, Indonesia on cyclooxigenase 2 (COX2) inhibition.

CONCLUSION

Curcuma domestica promising line from Sukabumi, West Java, Indonesia is the highest

curcuminoids content compared to Curcuma xanthorrhiza, whic are also known to have antioxidant and anti-inflammatory activities.

ACKNOWLEDGEMENTS We express our gratitude to Dr.

Min Rahminiwati, dr. Lina Sulimin, and Dr. Eka Intan Kumala Putri for excellence technical support. This research was supported by grants from the Directorate General of Higher Education, Ministry of Education and Culture Republic of Indonesia (Hibah Kompetitif Penelitian Strategis Unggulan Nasional Nomor: 476/SP2H/PL/ Dit.Litabmas/V/2011).

REFERENCES Ammon HPT, Anazodo MI, Safayhi

H, Dhawan BN, Srimal RC. 1992. Curcumin: a potent inhibitor of Leukotriene B4 formation in rat peritoneal polymorphonuclear neutrophils (PMNL). Planta Med. 58: 26.

Arora RB, Basu N, Kapoor V, Jain AP. 1971. Anti-inflammatory studies on Curcuma longa (Turmeric). Indian J. Med. Res. 59: 1289-1295.

Banerjee M, Tripathi LM, Srivastava VM, Puri A, Shukla R. 2003. Modulation of inflammatory mediators by ibuprofen and curcumin treatment during chronic inflammation in rat. Immunopharmacol. Immunotoxicol. 25: 213–24.

Bhavani Sankar TN, Murthy S. 1979. Effect of Turmeric (Curcuma longa) fractions on the growth of some intestinal and pathogenic bacteria in vitro.

74.84

67.96

64

66

68

70

72

74

76

C. domestica C. xanthorrhiza

COX2

inhi

biti

on (

%)



C - 290

Indian J. Exp. Biol. 17: 1363-1366.

Blois MS. 1958. Antioxidant determinations by the use of a stable free radical. Nature 181: 1199-1200.

Bougatef A, Hajji M, Balti R, Lassoued I, Triki-Ellouz Y, Nasri M. 2009. Antioxidant and free radical-scavenging activities of smooth hound (Mustelus) muscle protein hydrolysates obtained by gastrointestinal proteases. Food Chem. 114: 1198–1205.

Chainani-Wu N. 2003. Safety and anti-inflammatory activity of curcumin: a component of turmeric (Curcuma longa). J. Alter. Complement Med. 9: 161-168.

Chainani-Wu N. 2003. Safety and anti-inflammatory activity of curcumin: a component of turmeric (Curcuma longa). J. Altern. Complement Med. 9: 161-8.

Chandra D, Gupta SS. 1972. Anti-inflammatory and antiarthritic activity of volatile oil of Curcuma longa (Haldi). Indian J. Med. Res. 60: 138-142.

Choi M, Kim S, Chung W, Hwang J, Park K. 2004. Xanthorrhizol, a natural sesquiterpenoid from Curcuma xanthorrhiza, has an anti-metastatic potential in experimental mouse lung metastasis model. Biochem. Biophys. Res. Commun. 326: 210-217.

Chopra RN, Gupta JC, Chopra GS. 1941. Pharmacological action of the essential oil of Curcuma longa. Indian J. Med. Res. 29: 769-772.

Duke JA. 2002. CRC Handbook of Medicinal Spices. CRC Press: Washington DC, pp. 137–144.

Eigner D, Scholz D. 1999. Ferula asa-foetida and Curcuma longa in traditional medical treatment and diet in Nepal. J. Ethnopharmacol 67: 1-6.

Ferreira LAF, Henriques OB, Andreoni AAS, Vital GRF, Campos MMC, Habermehl GG, Moraes VLG. 1992. Antivenom and biological effects of ar-turmerone isolated from Curcuma longa (Zingiberaceae). Toxicon 30: 1211-1218.

Huang M, Lou Y, Xie J, Ma W, Lu Y, Yen P, Zhu B, Newmark H, Ho C. 1998. Effect of dietary curcumin and dibenzoylmethane on formation of 7, 12-dimethylbenz [a] anthracene-induced mammary tumors and lymphomas/leukemias in Sencar mice. Carcinogenesis. 19: 1697-1700.

Huang MT, Smart RC, Wong CQ, Conney AH. 1988. Inhibitory effect of curcumin, chlorogenic acid, caffeic acid and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 48: 5941-5946.

Hwang J, Shim, J, Pyun, Y. 2000. Antibacterial activity of xanthorrhizol from Curcuma xanthorrhiza against oral pathogens. Fitoterapia 71: 321-323.

Itokawa H, Shi Q, Akiyama T, Morris-Natschke SL, Lee KH. 2008. Recent advances in the investigation of


C - 291

curcuminoids. Chinese Medicine 3 (11): 1-13.

Jayaprakasha GK, Rao LJ, Sakariah KK. 2002. Improved HPLC method for determination of curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Agric. Food. Chem. 50: 3668-3672.

Jayaprakasha GK, Rao LJ, Sakariah KK. 2006. Antioxidant activities of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Food Chem. 98(4): 720-724.

Khar A, Ali AM, Pardhasaradhi BV, Begum Z, Anjum R. 1999. Antitumor activity of curcumin is mediated through the induction of apoptosis in AK-5 tumor cells. FEBS lett 19:165–8.

Kiuchi F, Goto Y, Sugimoto N, Akao N, Kondo K, Tsuda Y. 1993. Nematocidal activity of Turmeric: synergistic action of curcuminoids. Chem. Pharm. Bul. 41: 1640-1643.

Lechtenberg M, Quandt B, Nahrstedt A. 2004. Quantitative determination of curcuminoids in Curcuma rhizomes and rapid differentiation of Curcuma domestica Val. and Curcuma xanthorrhiza Roxb. by capillary electrophoresis. Phytochem. Anal. 15, 152–158.

Lim GP, Chu T, Yang F, Beech W, Frautschy SA, Cole GM. 2001. The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse. J. Neurosci. 21:8370–7.

Masuda T, Isobe J, Jitoe A, Nakatani N. 1992. Antioxidative curcuminoids from rhizomes of Curcuma xanthorrhiza. Phytochemistry. 31: 3645-3647

Masuda T, Isoke J, Jitoe A, Nakatani N. 1992. Antioxidative curcuminoids from rhizomes of Curcuma xanthorrhiza. Phytochemistry 31 (10): 3645-3647.

Mazumber A, Raghavan K, Weinstein J, Kohn KW, Pommer Y. 1995. Inhibition of human immunodeficiency virus type-1 integrase by curcumin. Biochem. Pharmacol. 49: 1165-1170.

Mukophadhyay A, Basu N, Ghatak N, Gujral PK. 1982. Anti-inflammatory and irritant activities of curcumin analogues in rats. Agents and Actions 12: 508-515

Onoda M, Inano H. 2000. Effect of curcumin on the production of nitric oxide by cultured rat mammary gland. Nitric Oxide 4:505–15.

Ozaki Y. 1990. Antiinflammatory effect of Curcuma xanthorrhiza Roxb. and its active principles. Chem. & Pharmaceutic. Bulletin 38(4):1045-8.

Pan MH, Lin-Shiau SY, Lin JK. 2000. Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IkB kinase and NFkB activation in macrophages. Biochem. Pharmacol. 60:1665–76.

Pulla Reddy Ach, Lokesh BR. 1992. Studies on spice principles as


C - 292

antioxidants in the inhibition of lipid peroxidation of rat liver microsomes. Mol. Cell. Biochem. 111: 117-124.

Ram A, Das M, Ghosh B. 2003. Curcumin attenuates allergen-induced airway hyperresponsiveness in sensitized guinea pigs. Biol Pharm. Bull. 26: 1021–4.

Rasmussen HB, Christensen SB, Kvist LP, Karazmi A. 2000. A simple and efficient separation of the curcumins, the antiprotozoal constituents of Curcuma longa. Planta Med 66: 396-398.

Rukayadi Y, Yong D, Hwang JK. 2006. In vitro anticandidal activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb. J. Antimicrob. Chemother. 132: 1-4.

Sreejayan N, Rao MN. 1996. Free radical scavenging activity of curcuminoids. Arzneimittelforschung 46:169–71.

Thaikert R, Paisooksantivatana Y. 2009. Variation of total curcuminoids contents, antioxidant activity and genetic diversity in turmeric (Curcuma longa L.) collections. Kasetsart J. (Nat. Sci.) 43: 507-518.

Tohda C, Nakayama N, Hatanaka F, Komatsu K. 2006. Comparison of anti-inflammatory activities of six Curcuma rhizomes: a possible curcuminoid-independent pathway mediated by Curcuma phaeocaulis extract. Evid Based Complement Alternat Med. 3(2): 255–260.

Udenigwe CC, Lu YL, Han CH, Hou WC, Aluko RE. 2009. Flaxseed protein-derived peptide fractions: Antioxidant properties and inhibition of lipopolysaccharide-induced nitric oxide production in murine macrophages. Food. Chem. 116(1): 277–284.

Unnikrishnan MK, Rao MN. 1995. Inhibition of nitrite induced oxidation of hemoglobin by curcuminoids. Pharmazie 50: 490-492.

Yasni S, Imaizumi KT, Nakamura M, Aimoto J, Sugano M. 1993. Effects of Curcuma xanthorrhiza Roxb. And curcuminoids on the level of serum and liver lipids, serum apolipoprotein A-I and lipogenic enzymes in rats. Food Chem. Toxic. 31: 213-218.

Documents

Curcuminoid Contents, Antioxidant and Anti-Inflammatory Activities