Separation of Curcuminoids Enriched Fraction from Spent Turmeric Oleoresin and Its Antioxidant Potential

S. Nagarajan, I. Rahath Kubra, and L. Jagan Mohan Rao

Abstract: The rhizomes of turmeric are processed to obtain oleoresin and subsequently curcuminoids are isolated. The

mother liquor, after partial isolation of curcuminoids, known as spent turmeric oleoresin (STO), is considered as industrial waste. Curcuminoids enriched spent turmeric oleoresin (CSTO) is prepared by removal of nonantioxidant constituents, and investigated for its antioxidant potential using in vitro methods, and also the total curcuminoids and phenolic contents were determined. CSTO has a total phenolic content of 267.27 5.75 mg GAE/g that is almost double the amount present in STO (118.3 3.0 mg GAE/g). The total amount of curcuminoids in CSTO is found to be 39 1.2%, whereas STO had 15 2.0%. CSTO possessed radical scavenging activity of 84% at 50 g/mL, antioxidant activity of 74% at 25 g/mL, high antioxidant capacity, and moderate total reducing power. These results provide scope for utilization of CSTO/STO as natural antioxidant/preservative as well as colorant in various foods.
Keywords: antioxidant and radical scavenging, Curcuma longa, curcuminoids, oleoresin, reducing power

Introduction

Turmeric (Curcuma longa) has been known for use as a spice in food from the very early days, mainly because of its yellow color in addition to its characteristic avor and associated medicinal properties (Govindarajan 1980). It is a tropical herb, belonging to the family of zingiberaceae, which is indigenous to South Asia. Turmeric is cultivated in India, Southeast Asia, China, North Australia, West Indies, and South America. Subsequently, its cultivation has spread to many African countries. It has been reported that 80% of the world production of turmeric is from India, and India is the largest exporter. India has 3.22 lakh hectares under turmeric cultivation with a total production of 14 lakh tonnes (Parthasarathy and others 2008). Rhizomes of Curcuma longa mainly are traded as a spice, food colorant, and source of industrial starch. Turmeric has many benecial properties and is used as a disinfectant, deodorizing agent, a cure for leach and insect bites, cold, cough, sneezing, purulent ophthalmia, various types of skin diseases, and related ailments, such as tinea versicolor, itching, patches, eruptions, scabies, acne, burn injuries, and skin boils. It is also used in dental diseases (Balakrishnan 2007) and digestive disorders such as dyspepsia and acidity, indigestion, atulence, affections of the liver, upper abdominal pain, asthma, gastric and duodenal ulcers, respiratory ailments, besides as a cure for the hallucinatory effects of hashish and other psychotropic drugs (Khanna 1999; Sasikumar 2001). The yellow-pigmented fraction of Curcuma longa contains curcuminoids, which are chemically related to its principal ingredient, curcumin; and turmeric has been found to be a rich source

of these phenolic compounds. The 3 main curcuminoids (2% to 9%) isolated from turmeric are curcumin, demethoxycurcumin, and bisdemethoxycurcumin, which are the important active ingredients responsible for many biological activities of turmeric. Pure curcumin, bisdemethoxycurcumin, and demethoxycurcumin are not available from commercial sources as individual constituents. Commercial curcumin contains curcumin (75% to 79%), demethoxycurcumin (16% to 18%), and bisdemethoxycurcumin (2% to 3%). Due to the discovery of new biological activities, there is an increasing demand for demethoxycurcumin and bisdemethoxycurcumin (Simon and others 1998; Ahsan and others 1999; Kim and others 2001). Curcumin shows a variety of physiological and pharmacological effects, and several studies indicate curcumin to be anticarcinogenic (Conney and others 1991) and anti-inammatory (Huang and others 1991). Curcumin acts as a superoxide radical scavenger (Reddy and Lokesh 1994; Ruby and others 1995) and as a singlet oxygen quencher (Das and Das 2002). Curcumin is reported to be a powerful antioxidant to restore both oxidative and reductive damage caused to proteins by radiation (Kapoor and Priyadarsini 2001). Some studies have pointed to the possible involvement of the -diketone moiety in the antioxidant action of curcumin and its derivatives (Schaich and others 1994; Masuda and others 1999). Jovanovic and others 1999 describe the H-atom donation from the -diketone moiety to a lipid alkyl or a lipid peroxyl radical as a potentially more important antioxidant action of curcumin. Turmeric rhizomes are processed to obtain turmeric powder initially, which on solvent extraction gives turmeric oleoresin. Curcuminoids, the yellow color pigments of turmeric are being isolated from the turmeric oleoresin subsequently. After the isolation of curcuminoids, the mother liquor of oleoresin (that is, MS 20090696 Submitted 7/21/2009, Accepted 5/16/2010. Authors are with spent turmeric oleoresin [STO]) is an industrial waste at present Plantation Products, Spices and Flavour Technology Dept., Central Food Technological and is being thrown into boilers. It has the composition of oil, Research Inst., Mysore 570 020, India. Direct inquiries to author Rao (E-mail: resin, and left over curcuminoids. This has no commercial value at ljnatpro@yahoo.com). present. Recently, pure curcuminoids were isolated from STO as
2010 Institute of Food Technologists doi: 10.1111/j.1750-3841.2010.01696.x
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Curcuminoids enriched fraction from spent turmeric oleoresin . . .

described in the previous studies ( Jayaprakasha and others 2004, 2006). However, the total phenolic content of STO and its antioxidant potential need to be determined in order to understand its health benets. This study was aimed at the enrichment of the left over curcuminoids in the STO and also to investigate its antioxidant potential using different assays to nd application in foods. made up to 10 mL by adding distilled water. After 2 h of incubation at room temperature, the absorbance was measured at 765 nm using a spectrophotometer. The experiment was carried out in triplicate and the mean results are presented.

Materials and Methods

Chemicals and reagents All solvents and reagents were of analytical grade and obtained from Merck (Mumbai, India). FolinCiocalteau reagent, sodium carbonate, potassium ferricyanide [K3 Fe(CN)6 ], Trichloroacetic acid, Ferric chloride (FeCl3 ), ammonium molybdate, sodium phosphate, and sulfuric acid were also procured from Merck. Standard curcuminoids were procured from Flavours and Essences Private Ltd. (Mysore, Karnataka, India). Gallic acid, DPPH (1,1diphenyl-2-picryl-hydrazyl >90%), -carotene, and Tween 40 (polyoxyethylene sorbitan monopalmitate) were procured from Sigma Chemicals Co. (St. Louis, Mo., U.S.A.). Absorbance was measured using a UV-visible spectrophotometer (Cintra 10, GBC, Victoria, Australia). Raw material The mother liquor (STO), after partial isolation of curcuminoids from turmeric oleoresin, was procured from the local turmeric processing industry from 3 different batches. It has the composition of oil (40% to 50%), resin (20% to 35%), and left over curcuminoids (14% to 20%). Method of extraction Preparation of Curcuminoids enriched spent turmeric oleoresin (CSTO). The oil from the mother liquor was extracted using petroleum ether. The residue was separated from the petroleum ether extract by ltration under vacuum. The clear petroleum ether ltrate was desolventized to get oil. The residue was the CSTO powder. Determination of total curcuminoids and phenolics Estimation of total curcuminoid content. American Spice Trade Assoc. (ASTA) analytical spectrophotometric method was used to measure curcuminoid content of the STO, STO oil, and the CSTO. Twenty-ve milligrams of the commercial curcumin (3 curcuminoids mixture) standard was dissolved in 100 mL of reagent grade acetone. From this stock, a 1 mL aliquot was removed and transferred to a 100 mL volumetric ask and made up to 100 mL with ethanol. The absorbance of the diluted standard solution was measured at max = 425 nm using UV-Visible spectrophotometer (Cintra 10, GBC). The other samples were diluted suitably and absorbance was measured at same wavelength. The total curcuminoids content is calculated by comparison of the absorbance of sample solution with that of standard solution (ASTA 1997). Estimation of total phenolic content. The concentration of total phenolic content in the samples was estimated spectrophotometrically by Folin-Ciocalteu method (Singleton and others 1999) with gallic acid as the reference standard, and the results are expressed as gallic acid equivalent (GAE) in milligrams per gram on dry weight basis. The samples were dissolved in methanol (0.1 mg/mL), and Folin-Ciocalteau reagent (0.5 mL) and 1.5 mL of 20% saturated sodium carbonate solution were added to all the tubes and the mixture was shaken well. Finally, the solution was

Measurement of antioxidant potential Evaluation of radical scavenging activity (RSA) using DPPH model system. RSA of the STO, STO oil, and CSTO was evaluated as per the method of Blois (1958). The samples and Butylated hydroxy anisole (BHA) solutions (each 1 mL containing 25, 50, 75, 100, 150, and 200 g) were taken in different test tubes. Four milliliters of 0.1 mM methanolic solution of DPPH was added to the tubes and shaken vigorously. The tubes were allowed to stand at 27 C for 20 min. The control was prepared as above without any extract and methanol was used for the baseline correction. Optical density (OD) of the samples was measured at 517 nm. All the determinations were carried out in triplicate and averaged. RSA was expressed as the inhibition percentage and was calculated using the following formula: % RSA = (Control OD Sample OD/Control OD) 100. Evaluation of antioxidant activity using -carotene linoleate model system. The antioxidant activity (AA) of the samples was evaluated as described by Jayaprakasha and Jaganmohan Rao (2000). -carotene (0.4 mg) in chloroform (0.4 mL), 40 mg of linoleic acid, and 400 mg of Tween 40 (polyoxyethylene sorbitan monopalmitate) were mixed in a 250 mL round bottom ask. Chloroform was removed at 40 C under vacuum using a rotavapour and the resulting solution was immediately diluted with 10 mL of triple-distilled water and the emulsion was mixed well for 2 min. To this emulsion, 90 mL of oxygenated water was added and mixed for 1 min. Aliquots of the emulsion (4 mL) were pipetted into stoppered test tubes containing samples (1 mL) in ethanol at different concentrations (50 and 125 g/mL). A control consisting of 1 mL of ethanol and 4 mL of the above emulsion was prepared. Optical density of all samples was measured immediately (t = 0) and 2nd reading after 15 min, followed by readings at 30 min interval, for 3 h (t = 180). The tubes were placed in water-bath at 50 C between the readings. Measurement of color was recorded until the color of -carotene disappeared. The AA of the extract was evaluated in terms of oxidation of -carotene using 0 the following expression: AA % = 100[1 ( A0 At / A0 0 At )], 0 where A0 and A0 are the absorbance values measured at zero time of the incubation for the test sample and the control, respectively. At and A0 t are the absorbance values measured in the test sample and the control, respectively, after incubation for 180 min. All the determinations were carried out in triplicate and averaged. Total reduction potential (reducing power). The reducing power of the samples was determined by the method of Oyaizu (1986). Different amounts of the extracts (25 to 200 g) in 1 mL of distilled water was mixed with phosphate buffer (2.5 mL, 0.2 mol/L, pH 6.6) and potassium ferricyanide [K3 Fe(CN)6 ] (2.5 mL, 1%). The mixture was incubated at 50 C for 20 min. Trichloroacetic acid (10%, 2.5 mL) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1%), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicates an increase in the reduction capability. Evaluation of antioxidant capacity by phosphomolybdenum method. The total antioxidant capacity of samples and the commercial standard antioxidants propyl gallate was evaluated by the method of Prieto and others (1999) with suitable
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modications. An aliquot of 0.01to 0.2 mL solutions (containing 10 to 200 g of sample, respectively) was made upto 4 mL using reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The tubes were capped and incubated in a boiling water bath at 95 C for 90 min. The samples cooled to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm against blank. A typical blank was prepared using reagent solution with appropriate volume of the solvent used for the sample and was incubated under same conditions. The antioxidant capacity of the STO was expressed as equivalents of ascorbic acid (mol/g of extract). has a total phenolic content of 267.27 5.75 mg GAE/g which is almost the double the amount present in STO (118.3 3.0 mg GAE/g). The amount of total curcuminoids enriched in CSTO is 39 1.2% as expected, whereas the STO had 15 2.0%. The quantities of individual curcuminoids (mg/g) present in STO are as follow: curcumin 29.6 1.13, demethoxycurcumin 40.8 1.46, and bisdemethoxycurcumin 86.1 1.72; and in CSTO: curcumin 75.9 1.57, demethoxycurcumin 106.6 5.93, and bisdemethoxycurcumin 221.01 10.27.

RSA Free radical scavenging potentials of STO and BHA at various Statistical analysis Statistical analysis was carried out as per Duncans Multiple concentrations (5 to 40 g/mL) were tested by DPPH method and the results are presented in the Figure 1. The method is based Comparison Test (Duncan 1955). on the reduction of alcoholic DPPH solution in the presence of Results and Discussion a hydrogen donating antioxidant because of the formation of the nonradical form DPPH-H by the reaction monitored by the deEnrichment of curcuminoids in STO crease in absorbance of solution at 517 nm. DPPH has been widely STO on treatment with apolar solvents, the turmeric oil es- used in the assessment of RSA because of ease and convenience. sentially a mixture of mono and sesqui terpenoids, got dissolved It has been found that the RSA of CSTO was 58% and 84% at along with other low polar components and separated by ltra- 20 and 40 g/mL concentrations, respectively, as compared with tion/centrifugation. The residue is enriched with curcuminoids STO (27% and 59% at similar concentrations). mixture and designated as CSTO. In CSTO, the contents of demethoxycurcumin and bisdemethoxycurcumin were found to be 10 1% and 20 2%, respectively ( Jayaprakasha and others Antioxidant activity The AA in this method is measured by the ability of the 2004; Jagan Mohan Rao and Nagarajan 2008). compound to minimize the coupled oxidation of linoleic acid Total phenolic content and curcuminoid content and -carotene in an emulsied aqueous system, which loses its It is known that the phenolic compounds contribute to overall characteristic orange color while reacting with the radicals. The antioxidant potential of plant materials. The total phenolic content presence of antioxidants can hinder the extent of -carotene and the total curcuminoids content of STO, STO Oil, and CSTO bleaching by neutralizing the linoleate-free radical and other free are presented in the Table 1. The results indicated that the CSTO radicals formed in the system ( Jayaprakasha and others 2001). The AA through -carotene linoleate model system of STO, Table 1 Total phenolic and curcuminoids content of STO, STO oil, and CSTO at 10 and 25 g/mL concentrations were CSTO, and STO oil. compared with BHA is presented in the Table 2. It has been seen Total phenolic content Total curcuminoids that the AA of CSTO and STO was 74.3 1.20% and 69.73 0.52%, respectively, at 10 g/mL concentration. But as the conSample (mg GAE per gram) content (%)a centration is increased to 50 g/mL, CSTO showed proxidant STO 118.38 3.05 15 2.05 activity. STO Oil 18.32 1.05 nd CSTO 267.27 5.75 39 1.2 In general, phenolic compounds show antioxidant behavior at nd = not detected; GAE = gallic acid equivalents; STO = spent turmeric oleoresin; low concentrations, but at higher concentrations, they show proxSTO oil = oil separated spent turmeric oleoresin; CSTO = curcuminoids enriched spent idant behavior, which may depend on the concentration of the turmeric oleoresin. a phenolic compound as well as the position and number of the ASTA method.
Figure 1Radical-scavenging activity (RSA) of STO, CSTO, STO oil, and BHA. Mean score with different letters differ signicantly at that particular level of concentration (P < 0.05); STO = spent turmeric oleoresin; STO oil = oil separated from STO; CSTO = curcuminoids enriched spent turmeric oleoresin; BHA = butylated hydroxy anisole.

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hydroxyl groups present in the molecule (Khan and others power of the CSTO and STO was determined using the potassium 2000). ferricyanide reduction method and compared to that of BHA (Figure 2). The experimental results indicated that the reducing power of the CSTO increased at 100 ug/mL compared with STO, Total reductive potential In this method, ferric (Fe3+ , ferricyanide) complex is reduced indicating that the antioxidants present in the CSTO possesses less 3+ to the ferrous (Fe2+ ) form in presence of antioxidants having Fe reducing property than the standard BHA. the reducing properties (Amarowicz and others 2004), which is monitored by measuring the change in yellow color of test solution Antioxidant capacity into various shades of green and blue colors at 700 nm. Reducing The total antioxidant capacity was quantitatively determined
Table 2Antioxidant activity (%) of CSTO, STO. Concentration Sample STO STO Oil CSTO BHA
a,b,c,d

10 g/mL 44.2 2.37a 2.25 0.37b 65.5 1.96c 91.3 0.64d

25 g/mL 69.73 0.52a 10.75 1.72b 74.3 1.20c 92.1 0.91d

Mean score with different letters in each column differ signicantly (P < 0.05). STO = spent turmeric oleoresin; STO oil = oil separated from STO; CSTO = curcuminoids enriched spent turmeric oleoresin; BHA = butylated hydroxy anisole.

spectrophotometrically by measuring the absorbance values because of phosphomolybdenum complex formed, and presented in Figure 3. It has been reported that the assay of phosphomolybdenum method has been used to quantify vitamin E in soyabean seeds (Prieto and others 1999). This being simple method and independent of other antioxidant measurements, its application was extended to plant polyphenols and related compounds. The order of antioxidant capacity as per absorbances is as follows: PG>CSTO>STO>STO oil. The antioxidant capacity was calculated in terms of equivalents of ascorbic acid and found to be 3954.3 61.6 and 3136.3 82.1 mole/g for CSTO and
Figure 2Reducing power of STO, CSTO, and BHA. Mean score with different letters differ signicantly at that particular level of concentration (P < 0.05). STO = spent turmeric oleoresin; STO oil = oil separated from STO; CSTO = curcuminoids enriched spent turmeric oleoresin; BHA = butylated hydroxy anisole.

Figure 3Antioxidant capacity of the STO, CSTO, and PG. Mean score with different letters differ signicantly at that particular level of concentration (P < 0.05). STO = spent turmeric oleoresin; STO oil = oil separated from STO; CSTO = curcuminoids enriched spent turmeric oleoresin; PG = propyl gallate.

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STO, respectively, when compared to propyl gallate (4454.2 122.6 mole/g).
Govindarajan VS. 1980. Turmeric chemistry, technology and quality. Crit Rev Food Sci 12:199301. Huang MT, Lysz T, Ferraro T, Abidi TF, Laskin JD, Conney AH. 1991. Inhibitory effects of curcumin on in vitro lipoxygenase and cycloxygenase activities in mouse epidermis. Cancer Res 51:8139. Jagan Mohan Rao L, Nagarajan S, inventors. 2008. Radical scavenging water soluble yellow colourant turmeric formulation. The Controller of Patents, The Patent Ofce, New Delhi, India. Patent application nr. 409/DEL/09. Jayaprakasha GK, Singh RP, Sakariah KK. 2001. Antioxidant activity of the grape seed (vitis vinifera) extracts on peroxidation models in vitro. Food Chem 73:28590. Jayaprakasha GK, Jaganmohan Rao L. 2000. Phenolic constituents from Lichen Parmotrema stuppeum (Nyl.) Hale and their antioxidant activity. Z Naturforschung C 55C:101822. Jayaprakasha GK, Jaganmohan Rao L, Sakariah KK, inventors. 2004 Nov 20. A process for the recovery of curcuminoids mixture from spent turmeric oleoresin. The Controller of Patents, The Patent Ofce, New Delhi, India. Indian patent nr. 194592. Jayaprakasha GK, Jaganmohan Rao L, Sakariah KK. 2006. Antioxidant activities of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Food Chem 98:72024. Jovanovic SV, Steenken S, Boone CW, Simic MG. 1999. H-Atom transfer is a preferred antioxidant mechanism of curcumin. J Am Chem Soc 121:967797. Kapoor S, Priyadarsini KI. 2001. Protection of radiation induced protein damage by curcumin. Biophys Chem 92:11926. Khanna NM. 1999. Turmeric Natures precious gift. Curr Sci 76:13516. Khan NS, Ahmed A, Hadi SM. 2000. Antioxidnt, proxidant properties of tannic acid and its binding to DNA. Chem-Biol Interact 125:17789. Kim DSHL, Park SY, Kim JY. 2001. Curcuminoids from Curcuma longa L. (Zingiberaceae) that protect PC12 rat pheochromocytoma and normal human umbilical vein endothelial cells from bA (1-42) insult. Neurosci Lett 303:5761. Masuda T, Hidaka K, Shimohara A, Maekawa T, Takeda Y, Yamaguchi H. 1999. Structural identication of new curcumin Dimmers and their contribution to antioxidant mechanism of curcumin. J Agr Food Chem 47:7175. Oyaizu M. 1986. Studies on the products of browning reaction prepared from glucose amine. Jpn J Nutr 44:30714. Parthasarathy VA, Chempakam B, Zachariah TJ. 2008. Chemistry of spices. Kingslynn, UK: Cabi Press, p 978. Prieto P, Pineda M, Aguliar M. 1999. Spectrophotometric quantitation of antioxidant capacity through the formation of phoshomolybdenum complex: specic application to the determination of vitamin E. Anal Biochem 269:33741. Reddy AC, Lokesh BR. 1994. Studies on the inhibitory effects of curcumin and eugenol on the formation of reactive oxygen species and the oxidation of ferrous iron. Mol Cell Biochem 137:18. Ruby AJ, Kuttan G, Babu KD, Rajasekharan KN, Kuttsn R. 1995. Anti-tumour and antioxidant activity of natural curcuminoids. Cancer Lett 94:7983. Sasikumar B. 2001. Turmeric. In: Peter K, editor. Handbook of herbs and spices. Boca Raton, FI: CRC Press. p 297310. Schaich KM, Fisher C, King R. 1994. In: Ho CT, Osawa T, Huang M-T, Rosen TR, editors. Food phytochemicals for cancer prevention II. Teas, spices, and herbs. ACS Symp. Ser. No. 547. Washington, D.C.: American Chemical Society. p 20421. Simon A, Allais DP, Duroux JL, Basly JP, Durand-Fontanier S, Delage C. 1998. Inhibitory effect of curcuminoids on MCF-7 cell proliferation and structureactivity relationship. Cancer Lett 129:1116. Singleton V, Orthofer R, Lamuela-Raventos R. 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteau reagent. In: Packer L, editor. Oxidants and antioxidants Part A. Methods in enzymology, Vol 299. New York: Academic Press. p 15278.

Conclusions
In the present study, the STO was enriched with curcuminoids by the removal of undesirable components and evaluated for its antioxidant potential. The study has provided wide scope for its application in various foods. The results obtained indicate that CSTO showed signicant potential of AA and radical-scavenging property due to the presence of higher quantity of phenolics compared to the STO. However, the STO/CSTO showed moderate reducing power on metal complexes. Hence, this may nd application as yellow colorant as well as preservative in the food products, since it possess antioxidant potential ( Jagan Mohan Rao and Nagarajan 2008) when used at optimum levels with suitable modications, which requires further study.

Acknowledgment
Authors are thankful to Director, Central Food Technological Research Inst., Mysore, India; and Head, Plantation Products Spices and Flavour Technology Dept. CFTRI, Mysore, India, for the support during the work.

References
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