Food Sci. Technol. Res., 19 (4), 655–659, 2013

Effective Extraction of Curcuminoids by Grinding (Curcuma longa) with

Medium-chain Triacylglycerols

1* 2 1 1 3 1,3 Makiko Takenaka , Takeshi Ohkubo , Hiroshi Okadome , Itaru Sotome , Toshihiro Itoh and Seiichiro Isobe

1 Food Engineering Division, National Food Research Institute, 2-1-12 Kan-nondai, Tsukuba, Ibaraki 305-8642, Japan 2 Functional Foods Research Laboratory, NOF Corporation, 3-3 Chidori-Cho, Kawasaki-ku, Kawasaki, Kanagawa 210-0865, Japan 3 Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan

Received November 5, 2012; Accepted March 23, 2013

Turmeric (Curcuma longa) rhizome contains abundant curcuminoids, which are highly hydrophobic bioactive compounds. This study aimed to extract curcuminoids using edible instead of chemical sol- vents, and a new extraction system using natural medium-chain triacylglycerols (MCTs) was constructed. After grinding turmeric with MCTs, the liquid and solid fractions were separated by pressing and filter- ing, with the resulting liquid subsequently clarified by centrifuging and heating. The recovery rate of curcuminoids from turmeric to the clarified MCT fraction was ≈ 10%, but the MCT fraction seemed to be practically saturated with curcuminoids. MCT extracts including curcuminoids can be directly used to add yellow color to many kinds of food, as well as curcuminoid functionality together with the physical and biological properties of MCTs.

Keywords: , curcuminoid, turmeric, medium-chain triacylglycerol

Introduction Turmeric (Curcuma longa) is a zingiberaceous perennial to suppress a hangover, and commercial beverages including plant originating from tropical Asia, with the rhizome con- turmeric extract are gaining popularity in Japan. taining abundant amounts of curcuminoids, such as curcum- Curcuminoids are used for foods and are obtained indus- in, demethoxycurcumin, and bis-demethoxycurcumin (Fig. trially by extraction from turmeric with chemical , 1). These curcuminoids are hydrophobic yellow pigments such as , which are, in some cases, removed after (Masuda et al., 1993) that have certain biological activities, extraction. Recently, technologies to enhance curcuminoid such as and anti-inflammatory effects, as well as in , by forming or gels, have been an inhibitory effect against accumulation of liver triacylg- developed (Ahmed et al., 2012; Modasiya and Patel, 2012). lycerols (Surh, 2002; Asai and Miyazawa, 2001). Recently, It is preferable to extract curcuminoids without using chemi- curcuminoids have attracted attention as having the ability cal solvents. Therefore, we attempted curcuminoid extraction from turmeric using edible oil as the medium. Curcuminoids in an edible medium can be used directly in foods, without the need to remove the extraction medium. Furthermore, ed- ible extraction media are operationally convenient and can decrease production costs. In this study, the solubility of curcumin in certain edible was examined, with medium- chain triacylglycerols (MCTs) exhibiting the highest degree of solubility. Fig. 1. Chemical structure of major curcuminoids in turmeric. MCTs are a component of triacylglycerols in natural products, such as coconut oil, cow’s milk and mother’s milk,

*To whom correspondence should be addressed. and have acyl groups with medium chain length (C5–12), E-mail: [email protected] while triacylglycerols in common edible oils have acyl 656 M. Takenaka et al. groups with long chains (mainly C16 and C18). In coconut oil, 120 whetstones. Grinding was conducted at an average feed the total abundance of MCTs in triacylglycerols is > 50%. rate of 30 – 40 g/s using a whetstone clearance of 1 mm MCTs are known to have higher stability against oxida- with the lower whetstone rotating at 2000 rpm. A number of tion and lower viscosity. Furthermore, MCTs are digested grinding cycles (1, 2 and 3) were used to compare extraction and metabolized faster, and are less likely to accumulate in efficiency as it relates to the number of grinding cycles used. the fatty tissue, compared with long-chain triacylglycerols Current intensity during grinding was monitored and sample (Marten et al., 2006). These properties of MCTs can be ad- temperature before and after grinding was measured. A por- vantageous in food processing or beneficial for patients with tion of the ground samples was placed in a filter cloth and fil- certain disorders of the digestive system. Other natural acyl- tered by pressing with a KT23-100 press machine (San Seiki, glycerol-type edible oils, in addition to long- and medium- Hagi, Japan) at a maximum pressure of 30 MPa to separate chain triacylglycerols, include short-chain triacylglycerols the solid (S1, S2, and S3 in Fig. 2) and liquid fractions (L1,

(C2–4), monoacyl and diacylglycerols with various acyl group L2, and L3 in Fig. 2). The solid and the liquid fractions were lengths. However, their natural abundance is very limited. weighed to calculate the solid-liquid separation ratio. This In this study, we aimed to establish a method for extracting series of extraction system was conducted as a single trial. curcuminoids from turmeric using MCTs. We found that Evaluation of particle size distribution in the solid frac- grinding turmeric in the presence of MCTs was effective for tions (S1, S2, and S3) A portion of each solid fraction was extraction, and determined optimal conditions for producing washed using 10 volumes (v/v) of ethanol to remove the oil, a clear MCT fraction that is rich in curcuminoids. and the remaining ethanol was air-dried. The dried S1 – 3 were screened with stainless steel sieves with apertures of 2, Material and methods 1, 0.5, 0.25 mm. Particles were classified into 5 groups (larger Turmeric The “Nettai ukon” strain of turmeric was than 2 mm, 1 – 2, 0.5 – 1, 0.25 – 0.5 mm, smaller than 0.25 harvested in February 2011 in Okinawa, Japan. The turmeric mm), and each group was weighed. The size distribution was rhizome was stored at 5℃ until use and the water content of calculated from the weight. the rhizome was 81%. Clarification of the liquid fractions (L1, L2, and L3) Oils The coconut-derived, edible oils Panacet 810® The liquid fractions were centrifuged at 5078 × g and subse- ® and Panacet 800 (NOF Corporation, Tokyo, Japan), which quently warmed in a water bath at 50℃. contain caprylic group (C8) and capric group (C10) at molar Measurement of curcuminoids The quantitative analysis ratios of 85:15 and 100:0, respectively as acyl groups of tria- of curcuminoids was performed using an HPLC system con- cylglycerols, were used as the MCTs. Other food grade oils sisting of a system controller CBM-20A, a delivery from rapeseed, soybean, corn and olive were purchased from unit LC-20AD, an auto-sampler SIL-20ACHT, a column a retail source. oven CTO-20A, and a UV-VIS detector SPD-20A (Shimadzu, Reagents Curcumin, demethoxycurcumin, and bis-de- Kyoto, Japan) equipped with a shim-pack VP-ODS column methoxycurcumin were purchased from Wako Pure Chemi- (ϕ2 × 150 mm, Shimadzu GLC, Tokyo, Japan). The column cal Industries (Osaka, Japan). oven and the UV detector were set at 40℃ and 420 nm, re- Measurement of curcumin solubility Excess curcumin spectively. Each oil sample was diluted 10 times with metha- was added to 2 g of MCTs, rapeseed oil, soybean oil, corn nol, passed through PTFE filters (pore size 0.5 μm), and oil and olive oil, and the mixtures were heated to 50℃ and subjected to HPLC. Curcumin, demethoxycurcumin, and bis- mixed well. After cooling to 25℃, the mixture was centri- demethoxycurcumin were eluted with 0.1% (v/v) TFA in wa- fuged at 5078 × g, and the supernatant was used as curcumin- ter/acetonitrile (60:40, v/v) at flow rate 1.0 mL/min, resulting saturated oil. The of curcumin in each curcum- in retention times of 11.2, 9.7, and 8.5 min, respectively. in-saturated oil was determined by measuring absorbance Standard curves were prepared using commercial curcumin, at the maximum absorption of curcumin (423 nm) using a demethoxycurcumin, and bis-demethoxycurcumin. spectrophotometer. This experiment was conducted at 25℃. Extraction of curcuminoids A flow chart of the extrac- Results and discussion tion protocol is shown in Fig. 2. The turmeric rhizome was The solubility of curcumin in 6 kinds of edible oil is sum- cut using a FC-27D food cutter (Aiho Tekko, Toyokawa, marized in Table 1. The solubility of curcumin in MCTs was Japan) for 2 min, resulting in cut-piece lengths < 5 mm. Cut significantly higher than in other common oils. This may be rhizome (2400 g) was mixed with MCTs (Panacet 810, 2400 due to MCTs having shorter acyl chains and higher polarity g) and fed to a Super Masscolloider MKZA10-15 grinder compared to common oils, and the polarity of MCTs being (Masuko Sangyo, Kawaguchi, Japan) equipped with GC10- more suited to interacting with curcumin. In this study, Pan- Extraction of Curcuminoids with MCTs 657

Fig. 2. Processing flow chart and material balance in the extraction system. Two hundred grams of the first-ground mixture, 250 g of the second-ground mixture, and 1350 g of the third-ground mixture were not recovered or not used for experiment. *Separation ratio (w/w).

Table 1. Solubility of curcumin in edible oils. acet 810, which is more widely used for foods than Panacet 800, was selected as a medium to extract curcuminoids. Oil Solubility of curcumin (mg/g)* The mixture of cut turmeric and MCTs was ground to smoothness. There was almost no load during grinder opera- Rapeseed oil 0.41 ± 0.00a tion, as current intensity during grinding was practically con- Soybean oil 0.47 ± 0.02a stant at ≈ 13 A both with and without sample. Furthermore, Corn oil 0.53 ± 0.01a sample temperatures before and after grinding were 23℃ Olive oil 0.45 ± 0.01a and 25℃, respectively. In pressing, solid-liquid separation Panacet 810 1.85 ± 0.01b was easily achieved with the separation ratios shown in Fig. Panacet 800 b 2.02 ± 0.03 2. All pressings resulted in separation ratios of ≈ 20:80. Solid *Mean ± standard error, n = 3. Values with different letters particle sizes decreased with grinding cycles (Fig. 3). are significantly different at p < 0.01 (Tukey’s multiple Figure 4 shows images of samples during the clarifica- comparison test). tion process. Just after pressing, the liquid fractions were 658 M. Takenaka et al.

cuminoids from turmeric to the MCT fractions was around 6% – 10% for 1-cycle grinding and 9% – 15% for 2- and 3-cycle grindings (Fig. 6). The recovery rate with 2-cycle grinding was higher than 1-cycle grinding for each curcumi- noid, but there was no difference between 2- and 3-cycle grindings. It was suggested that the higher recovery rate of curcuminoids in 2- and 3-cycle grindings was due to a higher extraction efficiency resulting from smaller particle sizes of ground turmeric. Therefore the optimal number of grinding cycles in this extraction system was 2. The observed recov- ery rate for curcuminoids was curcumin > demethoxycur- cumin > bis-demethoxycurcumin, which is likely to be due to differences in the solubility of each curcuminoid in MCTs. It is assumed that increasing the volume of MCT used in tur- Fig. 3. Particle size distribution in the solid fractions. meric extraction will result in increased curcuminoid yield. S1 – 3 are defined in Fig. 2. We have to consider which aspects require optimization to effectively maximize curcuminoid extraction, such as ease not always clear, due to the presence of small particles and of handling, energy costs, labor costs, etc., which will be ad- water. After centrifugation, the clarity of the liquid fractions dressed in future studies. It appears that a weight ratio of 1:1 improved but was not complete. The samples were then (turmeric/MCTs) was appropriate for grinding, and the cost warmed in a water bath (50℃) and the samples became al- per gram of MCTs is much higher than that of turmeric. most completely clear. The number of grinding cycles did There are some publications relevant to our work, such as not affect the appearance of the MCT fractions. After warm- the extraction of curcumin from dried turmeric powder using ing the MCT fractions, clearness was maintained for more an oil primarily composed of diacylglycerol at 95℃ (Ohshiro, than 3 months at room temperature. It was suggested that 2002) and the extraction of flavor components from spices cloudiness of the MCT fractions before warming was not or nuts in MCTs with heating (Sugiura and Watanabe, 2007). solely due to the solubility of curcuminoids and other com- However, our work is novel due to the use of fresh material ponents, but also to certain substances in suspension or in a and direct grinding with MCTs at room temperature. The state of association, aggregation, etc., that were dissociated present protocol is very simple, with drastically reduced pro- by warming and thereby became soluble in MCTs. When the duction costs compared to other methods, because this proto- liquid fractions were warmed before centrifuging, they did col does not require drying of the material or heating during not become clear. extraction. This system also does not use organic solvents Figure 5 shows the concentration of curcuminoids in and associated evaporation systems, which are usually re- the MCT fractions (MF1 – 3). of curcumin quired in traditional extraction of curcuminoids from turmer- in MF2 and MF3 were similar to the solubility of curcumin ic. Moreover, it is assumed that curcuminoids dissolved in alone in MCTs, which indicates that curcuminoids in MF2 edible oil have the advantage of higher stability in food ma- and MF3 were almost saturated. The recovery rate of cur- trices than those prepared by traditional methods, in which

Fig. 4. Images of the liquid fraction clarification process. A: Three hours after pressing; B: After centrifugation of A; C: After warming of B. Only for C, characters on the test tubes and lattices of a test tube stand are visible through the liquid parts. L1 – 3 are defined in Fig. 2. Extraction of Curcuminoids with MCTs 659

many kinds of foods, such as liquid seasonings, soups, meat or fish products, dairy products, and confectionaries, to add yellow color and curcuminoid functionality together with the physical and biological properties of MCTs.

Acknowledgments The authors would like to thank Mr. Norihiro Yoshizaki (NOF Corporation, Tsukuba, Japan) for suggestions about the analysis of curcuminoids.

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