Separation and Purication Technology 47 (2006) 119125

Supercritical carbon dioxide extraction of turmeric oil from Curcuma longa Linn and purication of turmerones
Li-Hsun Chang a , Ting-Ting Jong b , Ho-Shin Huang c , Yung-Feng Nien d , Chieh-Ming J. Chang a,
a

Department of Chemical Engineering, National Chung Hsing University, #250, KuoKuang Rd., Taichung 402, Taiwan, ROC b Department of Chemistry, National Chung Hsing University, #250, KuoKuang Rd., Taichung 402, Taiwan, ROC c Kaiser Pharmaceutical Co., Ltd, Tainan 710, Taiwan, ROC d Mechanical Industrial Research Laboratories/Industrial Technology Research Institute, Taichung 407, Taiwan, ROC Received 28 December 2004; received in revised form 23 June 2005; accepted 24 June 2005

Abstract This study investigates separation and purication of three turmerones from the turmeric oil extracted by supercritical carbon dioxide. Nearly, 800 g of supercritical carbon dioxide extraction of 75 g of 0.42 mm turmeric powders at 333 K and 300 bar obtained 6.98 wt% turmeric oil within 2.5 h. A response-surface methodology is adopted for optimizing supercritical carbon dioxide extraction to disclose that the combination of 320 K and 26 MPa gives an optimum production of turmeric oil containing 71 wt% purity of three turmerones. Subsequently, a normal phase silica gel 60 column is employed to separate and purify three major turmerones from the turmeric oil extracted by supercritical carbon dioxide. The 86 wt% purity of Ar-turmerone and 81 wt% purity of + -turmerone have been separated, collected, and identied by liquidsolid chromatography, NMR qualication, and HPLC quantication, respectively. 2005 Published by Elsevier B.V.
Keywords: Supercritical carbon dioxide; Turmerones; Turmeric oil; Extraction; Purication; Response-surface methodology

1. Introduction The popular nature rhizome of Curcuma longa Linn, i.e. turmeric, which belongs to the family of Zingiberaceae, is well known for all kinds of curry diets and has been widely used for food additives and condiments in most regions of southern Asia. The yellowish turmeric oil is quite stiff and commonly evolves a few slightly aromatic avors. These active compounds of turmeric are generally categorized into non-volatile and volatile parts. Many studies have already reported biological, physiological, and chemical properties of the turmeric. In summary, the major non-volatile curcuminoids are curcumin, demethoxycurcumin, and bisdemethoxycurcumin [1]. These curcuminoids have several biological properties, such as anti-oxidative effects [2], antiCorresponding author. E-mail address: cmchang@nchu.edu.tw (C.-M.J. Chang). URL: http://140.120.136.121. 1383-5866/$ see front matter 2005 Published by Elsevier B.V. doi:10.1016/j.seppur.2005.06.018

carcinogenic [3], anti-mutagenic [4], anti-inammatory [5], and anti-fungicidal [6]. Some researchers already indicate the functionality and utilization of volatile turmeric oil, such as insect repellent [7], anti-fungous [8], anti-bacterial [9], and anti-carcinogenic [10] properties. The content of turmeric oil [11] contains mainly ketones that are quite like turmerones. Turmerone is a kind of sesquiterpenoid cyclic ketones. Sesquiterpenoids reported by He et al. [1] are major constituents of turmeric oil and Ar-turmerone recognized by He et al. [1] is the major turmerone, identied by a GCMS analysis. The -turmerone and -turmerone are the second largest turmerones in the turmeric oil. Braga et al. [12] used different extraction techniques, such as supercritical carbon dioxide and co-solvents. The composition of the extracts was determined by gas chromatographyame ionization detection (GCFID) and UV. They found that Ar-turmerones reach 20 wt% in the turmeric oil, extracted by SC-CO2 with adding 14 wt% ethyl alcohol. The amount of the turmeric oil [13] extracted by distilled water indicates that the peak area per-

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centage of Ar-turmerone and + -turmerone in the GC chromatogram is relative to 18.5% and 7.7%, respectively. Supercritical CO2 extraction of turmeric oil from turmeric examined by Gopalan et al. [14,15] and Chassagnez-M endez et al. [16] indicates that the turmerone is abundant in turmeric oil. They suggested the use of pressure ranging from 20 to 40 MPa and temperature ranging from 313 to 333 K. A 5 wt% of total yield of turmeric oil reported by Gopalan et al. [14] can be obtained and nearly fully extracted. Their results indicate that a decreasing temperature and an increasing pressure lead to an increasing yield. They concluded that the optimum pressure for the extraction was 22.5 MPa. However, no quantication of turmerone has been done in milligram level so far in open literatures. Therefore, this study is mainly in purifying three turmerones of supercritical CO2 extracts by using a normal phase liquidsolid chromatography. In addition, a response-surface methodology (RSM) of turmerones is adopted to optimize SC-CO2 extraction conditions.

respectively, to obtain the corresponding size of particles. The moisture content of raw powder is 12 wt% determined by an infrared moisture meter (A-D4714, A&D Co. Ltd., Japan). De-ionized water was prepared using a Milli-Q purication system. N-hexane (H), acetone (A), acetonitrile, ethanol (EtOH), ethyl acetate (EA), methanol, and triuoroacetic acid (TFA) was purchased from a local supplier of Merck Company (Germany). 2.2. Extractions and procedures The steam distillation generated turmeric oil on collector of a distill heated from a 4 L boiling water pot, in which 500 g turmerics were placed on the top of a sieve plate. Fig. 1 depicts a schematic ow diagram of the SC-CO2 extraction. Chang et al. [17] previously reported more detailed procedure. The 75 g of turmeric powders were divided into seven layers, each containing 10 g mixed with 10 g glass beads (diameter of 3.9 mm), subsequently, packed inside a stainless steel tubular extractor (i.d. of 2.0 cm and length of 54.0 cm). Liquid CO2 from a siphon-tube-type cylinder went through a cooling bath (277 K) and then compressed to the desired working pressure by a syringe pump (Isco, 100DX, USA) and heated up to the supercritical condition by a coil-type heat exchanger. The supercritical CO2 maintained at a constant ow rate of 5.2 and 20.8 g/min, respectively, owed into an extractor, contacted with powders, and extracted turmeric oil. A thermostatic controller and a backpressure regulator

2. Experimental approaches 2.1. Reagents and materials Naturally air-dried turmerics (C. Longa L.) were donated by Kaiser Pharmaceutical Co. Ltd. (Tainan, Taiwan) and then ground down and screened through a series of Tyler 20, 40, and 80 mesh sieves (i.e. 0.177, 0.420, and 0.840 mm metric),

Fig. 1. Schematic ow diagram of SC-CO2 extraction [17]1: CO2 cylinder; 2: CO2 cleanup column; 3: cold liquid circulator; 4: temperature controller; 5: high-pressure pump; 6: pressure gauge; 7: hot liquid circulator; 8: preheater; 9: check valve; 10-1: extraction vessel; 10-2: reboiler; from 11-1 to 11-3: metering valve; 12-1 and 12-2: back pressure regulator; 13: needle valve; 14: separator; 15: wet gas valve; 16: thermocouple.

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Fig. 2. Information block diagram of separation of three turmerones by liquidsolid chromatography.

manipulated temperature and pressure. During the extraction, the oil-laden CO2 led into a separator and expanded through a spiro-type nozzle and collected at 5 MPa and 303 K. Finally, the expanded CO2 that came off from the extracted oil mixture passed through a wet gas ow meter and was released to the ambient condition. 2.3. Isolation of turmerones by liquid chromatography

decreased down to 39:1 (v/v). The ow rate for loading, washing, and elution was constantly kept at 0.5 mL/min. Nearly, 30 fractions were collected subsequently, and each fraction containing 10 mL was alternatively checked by a thin-layer chromatography (TLC) analysis. In summary, two washing eluents of N-hexane with acetone varying from 59:1 to 39:1 were used, respectively. 2.4. HPLC quantication of three turmerones

Past researches reported separating Ar-turmerone by using a liquidsolid column chromatography [10,18]. Liquid chromatography, a kind of liquidsolid phase extraction was utilized in this section to isolate three turmerones from the SC-CO2 -extracted oil. According to the polar difference of solutes partitioned in the normal phase resin, the extracted oil was rst diluted with N-hexane and subsequently loaded in a length of 300 mm and a diameter of 20 mm column packed with 40 g silica gel 60 resin. In the course of chromatography, two stepwise elution processes allowed to possibly separate three fat-afnity turmerones. In the rst, the mixture of N-hexane with ethyl acetate used as an eluting mobile phase continuously replaced the loaded materials and gradually eluted the samples out to give fractions. In this stepwise elution, most impurities were tightly adsorbed by the adsorbents but the total turmerones were eluted and collected in a digital fractional collector. Fig. 2 indicates the ow diagram of two stepwise chromatographic procedures. After the previous elution, the solvent was removed and the collected extract was diluted with pure N-hexane and reloaded again in the silica gel 60 column. The second elution was performed with the solvent mixture formed by N-hexane (H) added with acetone (A), starting fractions from 59:1 (v/v) and gradually

After Ar-turmerone and the mixture of + -turmerone were individually obtained from two-step liquidsolid chromatography, the HPLC analysis of three turmerones was performed on a Purospher RP-18 (5 m, 125 mm 4 mm) reverse-phase column with a Hitachi L-4200 UVvis detector, a Hitachi L-7100 pump, and a D-7000 control interface. The column temperature was maintained at 40 C. UV detection of samples was performed at a wavelength of 254 nm and the injection volume was 20 L. The gradient mobile phase used for analysis was solvent A: 0.0025% TFA solution and solvent B: acetonitrile. A ow rate of 1 mL/min under the initial condition of 80:20 (A:B) was held for 5 min, brought to 48% B in 5 min to 60% in 10 min, held for 10 min, and to 100% in 10 min. Ar-turmerone and + turmerone were completely separated by the above gradient elution. Fig. 3 shows HPLC chromatograms of Soxhlet 95% (v/v) ethanol-extracted sample, liquid-chromatography fractionated samples, and the SC-CO2 extracts. The turmerones content in the mixture were 121.89 g/g of Ar-turmerone and 124.03 g/g of + -turmerone, respectively, as showed in the chromatograms. The R-square (R2 ) of each calibration curve reached 0.999.

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Fig. 3. HPLC chromatograms of the typical samples: (I) Soxhlet 95% ethanol-extracted sample; (II) SC-CO2 extracts; (III) Ar-turmerone with 86 wt% purity; (IV) + -turmerone with 81 wt% purity.

3. Results and discussion 3.1. Analysis of turmerones Two unknown fractions were collected by the second elution process, which obtained from 6th to 14th parts at the ratio of 59 (H): 1 (A) and from 6th and 8th parts at the ratio of 39 (H): 1 (A), respectively. The unknown collected at the 39:1 (v/v) ratio is a more polar compound than that collected at the 59:1 (v/v) ratio, by viewing the distinctive and long retention time in the HPLC chromatogram. These two unknowns were individually qualied by EI-mass, 1 H NMR, and 13 C NMR spectra, respectively. The fraction collected from the parts 6th to 8th at 39:1 (v/v) ratio, was proved to be Ar-turmerone with 86 wt% purity, and the fraction collected from the 6th to 14th parts at 59:1 ratio, was proved to be + -turmerone with 81 wt% purity. Fig. 4 shows the EI-

Fig. 4. EIMS spectrum of the unknown fraction containing 86 wt% Arturmerone.

mass spectrum of the collected unknown containing 86 wt% Ar-turmerone. This EI-mass spectrum evidenced the results of Richard et al. [19]. The spectrum distinctively exhibited the molecular ion at 216 m/z, a loss ion of methyl group at 201 m/z, and a loss ion of 2-methyl-propylene moiety at 55 m/z, respectively. The cleavage of atom (C8) had disrupted to two ions group of aromatic (119 m/z) and carbonyl (83 m/z). There was an intra-molecular hydrogen transfer of McLafferty rearrangement also shown in the spectrum. The odd electron ion appeared at 132 m/z was formed by a transfer of -hydrogen (C14) to a double bond oxygen atom. This unique rearrangement had been represented as a ketone moiety existing in the major structures, described previously by David et al. [20]. Simultaneously, the unknown fraction containing Ar-turmerone was qualied by comparing the 1 H NMR (300 MHz, CD3 OD) description chemical shift (), i.e. it contained 1.21 (3H, d), 1.85 (3H, s), 2.04 (3H, s), 2.26 (3H, s), 2.65 (2H, m), 6.11 (1H, s), and 7.06 (4H, s). The 13 C NMR (300 MHz, CD3 OD) spectra contained 20.8, 21.0, 22.6, 27.6, 37.0, 53.5, 125.1, 127.7, 130.0, 136.7, 144.6, 157.1, and 202.6. Consequently, the unknown fraction containing + -turmerone was also identied by comparing the 1 H NMR (600 MHz, CD3 OD) description chemical shift (), i.e. -turmerone having 0.86 (3H, d), 1.37 (2H, m), 1.62 (2H, t), 1.86 (3H, s), 2.12 (3H, s), 2.18 (2H, m), 2.23 (1H, m), 2.41 (1H, m), 4.74 (2H, s), 5.63 (1H, t), 6.04 (1H, s), and 6.12 (1H, d); -turmerone having 0.86 (3H, d), 1.37 (2H, m), 1.86 (3H, s), 2.12 (3H, s), 2.18 (2H, m), 2.23 (1H, m), 2.41 (1H, m), 5.41 (1H, t), 5.63 (1H, t), 6.04 (1H, s), and 5.78 (1H, d). In the NMR spectra of the + -turmerone mixture, the area integral of that peak at the same position was twice as much. The formula was rechecked by HH correlation spectroscopy (COSY) spectra.

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Fig. 5. The comparison of extracted amount of turmeric oil and three turmerones by different methods (SC-CO2 extraction at 333 K, 30 MPa, and 104 min using 5.2 g/min; steam extraction at 373 K and 1 atm for 29 h).

Fig. 6. Effect of ow rate on extracting turmeric oil and three turmerones in SC-CO2 extraction at 333 K and 30 MPa.

3.2. SC-CO2 extraction of turmeric oil and three turmerones It was possible to use SC-CO2 extraction without the addition of co-solvents to completely extract the volatile oil from turmeric when comparing with previous study [16]. The inuence of particle size of turmeric powder had been initially investigated in this study. Fig. 5 indicates the amount of three turmerones and turmeric oil decreased when the particle size was larger than 0.420 mm. Fig. 5 also showed that the average amount of three turmerones per gram of turmeric was 39 mg. Fig. 6 shows that the total yield and three turmerones yield signicantly changed with the amount of CO2 used. The amount of three turmerones obtained at 333 K, 30 MPa, and 5.2 g/min, when the solvent to solid ratio reached 20.17 g CO2 /g turmeric, started to decrease. This result implied that turmerones might be decomposed at 333 K after a long extrac-

tion time (i.e. 252 min). Table 1 presents the operation conditions of SC-CO2 extraction of the turmeric oil in a RSM experimental design. Total yield of turmeric oil ranged from 0 to 6.47 wt%, three turmerones ranged from 0 to 40.50 mg/g turmeric, and purity of three turmerones in oil ranged from 0 to 71 wt%, respectively. All extractions were carried out at a solvent to solid ratio of 10 1 g CO2 /g turmeric with a CO2 ow rate of 20.8 g/min. Our HPLC quantication indicates the purity of three turmerones in SC-CO2 extracts can reach 71 wt% (runs 6 and 10). A three-level, two-factor RSM experimental design with a central composite scheme was used to nd the optimized extraction condition. Total yield of oil, three turmerones, and purity of three turmerones in oil were used as the response, respectively. A quadratic polynomial equation including an individual term of mean, liner, and cross-product coefcients was used as the regression model for each response. Figs. 79 individually exhibited three dimension plots showing the predicted response-surface of total yield, three turmerones, and purity of three turmerones

Table 1 Two variables RSM experimental design by using the central composite scheme Run 1 2 3 4 5 6 7a 8 9 10 11 12 S.D.b Standard deviation : S.D. = Temperature (K) 308 (1) 348 (+1) 308 (1) 348 (+1) 300 (1.414) 356 (+1.414) 328 (0) 328 (0) 328 (0) 328 (0) 328 (0) 328 (0) Pressure (MPa) 10 (1) 10 (1) 30 (+1) 30 (+1) 20 (0) 20 (0) 5.9 (1.414) 34.1 (+1.414) 20 (0) 20 (0) 20 (0) 20 (0) Total yield (wt%) 1.26 0.43 5.78 6.47 5.74 5.42 0.00 5.92 5.57 5.74 5.98 5.55 0.57 MSresidual ; residual mean of square : MSresidual =
SSresidual d.f. ;

Three turmerones (mg/g turmeric) 5.8 1.2 37.4 38.5 35.9 38.3 0.0 37.4 38.2 40.5 39.0 35.2 5.1
N

Purity (wt%) 46 27 65 60 63 71 0 63 69 71 65 63 8

residual sum of square : SSresidual =

i=1

i )2 ; actual value: Yi ; (Yi Y

i ; degrees of freedom: d.f. predicted value : Y a The CO was at the gaseous state. 2 b The standard deviation is estimated according to the RSM quadratic model by Design-Expert Version 6.0.11, http://www.statease.com.

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Fig. 7. RSM response-surface of total yield as a function of temperature and pressure (extractions at the solvent to solid ratio of 10 1 g CO2 /g turmeric with a ow rate of 20.8 g/min and the S.D. is 0.57 wt%).

Fig. 9. RSM response-surface of purity of three turmerones as a function of temperature and pressure (extractions at the solvent to solid ratio of 10 1 g CO2 /g turmeric with a ow rate of 20.8 g/min and S.D. is 8 wt%).

as a function of pressure and temperature. A quadratic regression model was determined from the F-testing and analysis of variance (i.e. Ftotal yield = 37.52 > 8.75 = F(0.01, 5, 6) , R2 = 0.97; Fthree turmerones = 20.99 > 8.75 = F(0.01, 5, 6) , R2 = 0.96; Fpurity = 14.67 > 8.75 = F(0.01, 5, 6) , R2 = 0.92). Clearly, there were signicant enhancements on turmeric oil, three turmerones, and the purity of three turmerones when pressure increased from 6 to 26 MPa. It might be due to the effect of increasing CO2 density. The RSM results disclosed the suitable extraction conditions were 320350 K and 2630 MPa for total yield, 310350 K and 2430 MPa

for three turmerones, and 2030 MPa for purity of three turmerones, respectively. Figs. 7 and 8 also demonstrated that the temperature effect was insignicant when pressure was lower than 14 MPa. Both predicted responses indicated that the temperature at under 320 K and pressure at least 26 MPa was suitable for the extraction of three turmerones and turmeric oil, simultaneously. It was realized that mass transfer resistance was overcome when CO2 molecules penetrated into the matrix of turmeric at elevated pressures. Fig. 9 also shows that temperature does not signicantly affect the purity of turmerones, but pressure does.

4. Conclusions This study demonstrated that SC-CO2 without co-solvent is a viable potential means of extracting turmeric oil and recovering three major turmerones from C. longa Linn. The sample extracted by SC-CO2 purication processes the least amount of impurities. Our experimental results showed that three forms turmerones from SC-CO2 extraction could be fractionated, puried, and separated by a normal phase silica gel liquidsolid chromatography. The NMR structure identication and HPLC quantication of three turmerones can be achieved for the SC-CO2 -extracted samples. In summary, 125.94 mg Ar-turmerone with 86 wt% purity and 16.14 mg + -turmerone with 81 wt% purity have been successfully obtained by two-step liquidsolid chromatography. Pressure is a signicant parameter for recovering turmeric oil with high purity of three turmerones in the SC-CO2 extraction. Our results indicate that decreasing temperature and increasing pressure lead to increasing total yield (similar to Refs. [14,15]) and increasing three turmerones. The temperature

Fig. 8. RSM response-surface of three turmerones as a function of temperature and pressure (extractions at the solvent to solid ratio of 10 1 g CO2 / g turmeric with a ow rate of 20.8 g/min and S.D. is 5.1 mg/g).

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does not signicantly affect the purity of three turmerones of the extracted oil. Furthermore, the RSM experimental design reveals that SC-CO2 extraction at under 320 K and at least 26 MPa is a suitable condition to optimally extract oil containing 71 wt% purity of three turmerones from turmeric.

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