A comparative account of extraction of oleoresin from Curcuma aromatica Salisb by solvent and supercritical carbon dioxide: Characterization and bioactivities

LWT - Food Science and Technology 116 (2019) 108564

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A comparative account of extraction of oleoresin from Curcuma aromatica Salisb by solvent and supercritical carbon dioxide: Characterization and bioactivities

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Umesh Kannamangalam Vijayan, Sadineni Varakumar, Rekha S. Singhal∗ Department of Food Engineering and Technology, Institute of Chemical Technology, Matunga (E), Mumbai, 400 019, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Curcuma aromatica Salisb Supercritical carbon dioxide extraction LC-MS/MS Antibacterial activity Antioxidant activity

Extracts of Curcuma aromatica Salisb rhizomes are traditionally used in the Indian sub-continent in herbal and cosmetic applications. Composition of extracts and their resulting bioactivities is dependent on the extraction technique. The present study compares yield, composition, and bioactivities of extract from C. aromatica by Soxhlet and supercritical carbon dioxide (SC–CO2) extraction. Extraction using ethyl acetate for 240 min gave maximum yield (8.34 ± 0.2% w/w) among the solvents while SC-CO2 extraction at 40 MPa/45 °C/60 min with 20% (v/w) isopropanol as entrainer gave a maximum yield of 8.94 ± 0.1% (w/w). Isoborneol, curdione and vellaral were found only in SC-CO2 extract, while procurcumadiol, germacrone 4,5-epoxide, 3,7-epoxycaryophyllan-6-one and curcumadione were some prominent compounds seen in both extracts as determined by LCQ-TOF-MS and GC-MS analysis. Ethyl acetate extract showed better antioxidant (DPPH, FRAP, ABTS) and antiinflammatory activities while SC-CO2 extract (with 20% v/w iso-propanol) showed better antibacterial action against S. aureus (Gram-positive) and P. aeruginosa (Gram-negative).

1. Introduction Spices are widely used as a source of natural colour, flavour, antioxidants, and antimicrobial agents in cosmetic and perfumery industry (Peter & Babu, 2012). The use of spices loaded with naturally occurring bioactives and other antioxidants have gained attention of the industry since the past few decades. Bioactives extracted from spices such as curcuminoids from turmeric and piperine from pepper are used as functional food additives. Curcuma aromatica Salisb (Zingiberaceae), also called as wild turmeric, is rich in bioactives and found majorly in India, China and Japan. The rhizome is a good appetiser and carminative agent, and has found applications in health drinks in Japan (Kojima, Yanai, & Toyota, 1998). It possesses immunomodulatory (Al-Reza, Rahman, Sattar, Rahman, & Fida, 2010), ulcer protective, anti-age related macular degeneration, and in-vivo hepatoprotective activities (Khare, 2008). Oil from C. aromatica Salisb has been reported to have in-vitro antitumor activity (Li, Wo, Liu, Li, & Martin, 2009). The extract from these rhizomes can suppress the ultraviolet A (UVA) irradiation induced melanin production and tyrosinase activity, and has been exploited in cosmetic formulations (Panich et al., 2010). The extracts have shown anticancer,

antimicrobial, anti-fungal, and antioxidant activities (Dosoky & Setzer, 2018). The relative abundance of bioactives in extracts from botanical sources is dependent on the technique employed for its extraction (Pereira & Meireles, 2010). The conventional Soxhlet extraction of oils and oleoresins is widely used commercially due to process economics, simple setup, and good yield due to the constant change in transfer equilibrium. However, this technique requires longer extraction times and relatively larger volume of organic solvents. An alternative greener technique for extraction of oleoresin and bioactives is supercritical carbon dioxide extraction (SC–CO2). SC-CO2 selectively extracts nonpolar components like oils and lipophilic bioactives at specific operational pressure and temperature. The low viscosity and high diffusivity of SC-CO2 enables faster extraction rates. There are reports on selective and higher extraction of nonpolar molecules using SC-CO2 extraction. One such example is selective extraction of cis-lycopene from tomato seeds and peels (Vallecilla-Yepez & Ciftci, 2018). The present work aimed at a comparative analysis of extracts by solvent extraction vis-à-vis SC-CO2 in terms of composition and bioactivities. To the best of our knowledge, data on the composition and bioactivities of C. aromatica Salisb extracted by solvent and SC-CO2 is

∗ Corresponding author. Food Engineering and Technology Department, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai 400 019, India. E-mail address: [email protected] (R.S. Singhal).

https://doi.org/10.1016/j.lwt.2019.108564 Received 3 March 2019; Received in revised form 24 August 2019; Accepted 26 August 2019 Available online 27 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

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2.4. Antioxidant activity

scant. The composition of the extracts were analysed by liquid chromatography–quadrupole time-of-flight spectrometry (LC-Q-TOF-MS) and gas chromatography mass spectrophotometry (GC-Q-TOF-MS) while the bioactivities were evaluated in terms of in-vitro antioxidant activity, in-vitro anti-inflammatory activity by lipoxygenase (LOX) inhibition assay, and total phenolic content (TPC). The optimised extracts were examined for antibacterial activities against model Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) organisms, and further, an effort was made to understand the mechanism of antimicrobial action at the cellular level.

Antioxidant activity of different extracts were determined using DPPH, FRAP, and ABTS assays. The results of the antioxidant activities were expressed as μmol Trolox equivalent (TE) per gram of oleoresin. 2.4.1. DPPH The antioxidant activity of the extracts was estimated by slightly modified DPPH method (Lee, Weng, & Mau, 2007). The DPPH assay was carried out by mixing 0.1 mL of appropriate diluted oleoresin in methanol with 3 mL of DPPH radical (100 μmoL/L prepared in methanol) solution and incubating in dark for 30 min. The absorbance was measured at 517 nm on a UV-VIS spectrophotometer (Shimadzu UV 1800; Tokyo, Japan) using methanol as blank. A standard graph of Trolox was plotted in the range 0–32 μmoL/L. The regression equation correlating the percentage inhibition of DPPH radicals (Y) with the concentration of Trolox (X) was Y = 2.955X (R2 = 0.995).

2. Materials and methods 2.1. Materials and chemicals C. aromatica Salisb rhizomes were procured from Aromatic and Medicinal Plants Research Station - Kerala Agricultural University, Ernakulum, Kerala, India. Linoleic acid and lipoxygenase (LOX) enzyme (100000 IU/g) were procured from SRL Pvt. Ltd. Mumbai, India. Acetonitrile (LC-MS grade) was purchased from Merck, Mumbai, India. Supelco37 component FAME mix was purchased from Sigma Aldrich, Mumbai, India. All other chemicals and solvents were of analytical grade and purchased from reliable sources.

2.4.2. FRAP The ferric reducing ability of the extracts was evaluated using the FRAP assay (Pavlić et al., 2018). The assay was performed by mixing 0.1 mL of appropriate diluted oleoresin in methanol with 3 mL of FRAP reagent and incubating at 27 ± 2 °C for 8 min after which the absorbance was measured at 593 nm against a distilled water blank. A standard graph of Trolox was plotted in the range 0–700 μmoL/L. The regression equation correlating the absorbance (Y) with the concentration of Trolox (X) was Y = 0.0013X (R2 = 0.995).

2.2. Soxhlet extraction The rhizomes were powdered and passed through 40 mesh sieve to obtain uniform particle size (0.42 mm). The moisture content of powder was approximately 10% w/w. The powder was stored in airtight container under refrigerated conditions (4 ± 2 °C) until further use. Oleoresin was extracted by using various solvents in Soxhlet apparatus until maximum extraction was achieved (~240 min). The extract was then concentrated by rotary vacuum evaporator (IKA RV 10 digital, Germany) under reduced pressure at 50 °C. The oleoresins so obtained were stored under refrigerated conditions until further use. The yield of the oleoresins was expressed as percent w/w of dried rhizome powder.

2.4.3. ABTS ABTS assay was performed as per the procedure reported by Duque, Pinto, and Macias (2011). The ABTS reagent (7 mmoL/L) was mixed with potassium persulfate (2.45 mmoL/L in the final mixture) and allowed to stand in the dark for at least 16 h prior to use. The ABTS*+ solution was prepared by mixing previously prepared ABTS reagent with ethanol until the absorbance at 734 nm was 0.70 ± 0.02. The reaction mixture was prepared by mixing 0.1 mL of appropriate diluted oleoresin in methanol with 3.9 mL of ABTS*+ solution and incubating for 20 min. The absorbance was measured against ethanol as blank. A standard graph of Trolox was plotted in the range 0–10 μmoL/L. The regression equation correlating the percentage inhibition of ABTS*+ (Y) with the concentration of Trolox (X) was Y = 1.708X (R2 = 0.993).

2.3. Supercritical fluid CO2 (SC–CO2) extraction Oleoresin extraction was carried out using lab scale supercritical fluid extractor (Speed SFE of Applied separations, USA) in semi-batch mode by monitoring the set parameters of pressure, oven temperature, and extraction time. The extraction vessel was packed by filling the rhizome powder along with polypropylene wool and frits at both ends. The flow rate of CO2 (> 99% purity) and time of extraction were kept constant at 2 L/min and 60 min, respectively. The batches were optimised by varying one factor at a time while keeping the other two constant, on the basis of yield of oleoresin, in-vitro antioxidant activities [2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH), ferric reducing antioxidant power activity (FRAP), and 2, 2′-azino-bis3-ethylbenzothiazoline-6-sulphonic acid radical cation scavenging activity (ABTS)], total polyphenol content (TPC), and lipoxygenase (LOX) inhibition assay.

2.5. Total phenolic content (TPC) Total phenolic content was estimated by Folin-Ciocalteu method as reported by Al-Reza et al. (2010). An aliquot (0.2 mL) of the extract along with 46 mL distilled water and 1.0 mL Folin–Ciocalteu reagent was taken in a 50 mL volumetric flask. Sodium carbonate (7.5% w/v) solution (3 mL) was added after 3 min and incubated at 30 ± 2 °C for 2 h with intermittent shaking. The absorbance was measured at 760 nm, and results were expressed as milligrams of gallic acid equivalents (mg GAE) per gram of extract. A standard graph of gallic acid was plotted in the range 0–1.0 mg/mL. The regression equation correlating the absorbance (Y) with the concentration (X) was Y = 0.6311X + 0.0774 (R2 = 0.993).

2.3.1. Effect of process conditions on the yield of oleoresins Effect of pressure on the extraction yield and its bioactivity was evaluated by varying the pressure from 10 to 40 MPa, keeping other parameters constant. Temperatures were varied from 40 to 65 °C by keeping pressure and time constant. The effect of time on the extraction yield and its bioactivity was evaluated by varying the time from 30 to 120 min while keeping the optimised pressure and temperature constant. The polarity of the SC-CO2 was modified by various organic solvents (entrainers). The biomass was wetted with specific concentration of solvents (10, 20, 30% v/w) prior to SC-CO2 extraction. Ethanol, acetone, methanol and iso-propanol were screened and evaluated for oleoresin yield.

2.6. Lipoxygenase (LOX) inhibition assay LOX inhibition assay was carried out as per the modified procedure reported by Chaubey et al. (2018). In brief, the reaction mixture was prepared by mixing 0.025 mL of oleoresin (3 mg/mL in DMSO) with 0.475 mL enzyme solution (400 IU/mL) and incubated for 2 min. Linoleic acid [0.5 mL (250 μmoL/L, prepared in borate buffer (pH-9 and 0.2 moL/L)] was later added to the mixture and incubated at room temperature (ca. 30 ± 2 °C) for 10 min. A blank solution was prepared by adding 0.025 mL of DMSO instead of oleoresin. Indomethacin (1 mg/ 2

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tubes. To this mixture, 7.5 mL of methanol/chloroform (2:1, v/v) was added and kept at room temperature for 2 h under shaking conditions. The bacterial cells were centrifuged at 5000 x g for 10 min to obtain supernatant 1. The cell pellet was further extracted using 9.5 mL of methanol/chloroform/water (2:1:0.8 v/v) for 2 h under shaking conditions and then centrifuged to collect supernatant 2. The supernatants after the two extractions were pooled and then extracted with 4.5 mL chloroform followed by addition of same volume of water. This mixture was mixed properly and allowed to settle to get two clear phases. The lower chloroform fraction having bacterial lipids was collected used for the preparation of FAME as follows. The chloroform fraction was evaporated to dryness under a stream of nitrogen gas. To the dried extract, 2 mL of 2.5% (v/v) H2SO4 in anhydrous methanol (stored over anhydrous sodium sulphate) was added and heated at 70 °C for 2 h under sealed conditions after which the reaction was stopped by addition of 3 mL of 9% (w/v) NaCl solution. The FAMEs so obtained was extracted using pet ether (60–80 °C bp) and analysed by gas chromatography Agilent GC 7820A using capillary column (DB-23, 30 m × 0.25 mm internal diameter, film thickness 0.25 μm, Agilent J & W, USA) (David, Sandra, & Vickers, 2005). Inlet temperature was set at 210 °C and oven temperature was programmed so as to start at 60 °C with a hold time of 2 min, and then increased to 230 °C at ramping of 10 °C and then held for 10 min. Peak area percentage and retention time of total fatty acids were determined using Supelco37 component FAME mix standard.

mL) was used as positive control (standard inhibitor of LOX). The absorbance was measured at 234 nm, and inhibitory activity was calculated by using following formula,

% LOX inhibitory activit y=

OD Control − OD Sample × 100 OD Control

2.7. Characterization of oleoresin using LC-Q-TOF-MS Bioactives were characterized by LC-Q-TOF-MS using an Agilent 6200 liquid chromatography system equipped with G6550A ultra high definition accurate-mass quadrupole time of flight mass spectrophotometer (Mass Hunter Software version B.05.01) and connected with Luna C18 column (5 μm, 150 mm × 2 mm, Phenomenex, USA). A flow rate of 0.2 mL/min and column temperature of 25 °C was used for entire analysis. An injection volume of 3 μL of appropriately diluted oleoresin with a total run time of 30 min was taken for each analysis. The mobile phase comprised of water 95% v/v solvent (A): acetonitrile 5% v/v solvent (B) with a gradient solvent programme for initial 15 min. The proportion of solvent B was then increased to 95% over next 10 min, then returned to the initial composition (5%) over next 5 min. Sample ionization was achieved using an electrospray ionization (ESI) interface in positive ion mode. The mass spectrophotometer was operated at 2 GHz and the full scan mass covered the range of mass/ charge (m/z) from 130 to 1000.

2.10. Statistical analysis 2.8. Characterization of oleoresin using GC-MS All the experiments were carried out in triplicates and data was analysed using Statistical Package for Social Sciences (SPSS®, version 23). The results were expressed as mean ± SD of three determinations. One way ANOVA was applied to check the mean and statistical significance amongst the values obtained with the Duncan's New Multiple Range test at P < 5%.

The volatile composition of oleoresin was analysed on a GC-MS system (Agilent 7890-GC with Jeol AccuTOF GCV mass detector) employing capillary column (DB-5MS, 60 m × 0.25 mm internal diameter, film thickness 0.25 μm, Agilent J & W, USA) and helium as carrier gas (flow rate, 1 mL/min). The inlet temperature was set at 250 °C with a split ratio of 50:1 and gradient oven temperature was programmed with an initial temperature of 40 °C and held for 1 min, and then increased at a rate of 3 °C/min up to 280 °C. The electron-impact mode with ionization energy of 70 eV was used. The component analysis was performed by comparing the recorded mass spectra of each compound with NIST library.

3. Results and discussion 3.1. Soxhlet extraction Different organic solvents viz. hexane, iso-propanol, ethanol, methanol and ethyl acetate were screened for extraction of oleoresin from the rhizome powder using Soxhlet extraction. The highest yield of oleoresin was obtained from ethyl acetate (8.34 ± 0.20% w/w) (supplementary file Fig. S1). An earlier report on extraction of oleoresin from C. aromatica Salisb using ethyl acetate had shown a yield of 6.4% w/w (Al-Reza et al., 2010). Since ethyl acetate gave maximum yield of the oleoresin from C. aromatica Salisb, it was selected for further studies and comparison with SC-CO2 extraction.

2.9. Antibacterial activity of extracts Gram-positive Staphylococcus aureus (NCIM 5021) and Gram-negative Pseudomonas aeruginosa (NCIM 5029) were procured from National Collection of Industrial Microorganisms (NCIM, Pune, India) to test the antimicrobial potential of oleoresin. Broth dilution method was performed for determining the antimicrobial activity and minimum inhibitory concentration (MIC) of the extract (Chen et al., 2017). After determination of MIC of both ethyl acetate and SC-CO2 extracts, the action of the oleoresin at bacterial cellular level was determined by analyzing bacterial lipid profile. Cells were collected by centrifuging the broth at 5000 x g for 10 min at 4 °C. The pellets so obtained were dispersed in 0.02 moL/L PBS saline (pH 7.0) under aseptic conditions to obtain an optical density of ~1.0 (OD600 nm). The oleoresins above their MIC were added to each suspension at 30 °C for 4 h and immediately processed for membrane fatty acid analysis using gas chromatography (Di Pasqua et al., 2007). Cells incubated without oleoresin were used as control.

3.2. Extraction using supercritical fluid CO2 (SC–CO2) The process variables have a significant impact on the extraction yield, and hence the influence of process variables like pressure, temperature, time, and entrainers on the extraction yield and bioactivities was evaluated. 3.2.1. Effect of process variables on the yield of oleoresin Pressure is a critical parameter for SC-CO2 that enables selective separation of the bioactive components from the plant matrix. An increase in pressure from 10 to 35 MPa resulted in a significant increase in yield from 3.13 ± 0.12% to 4.96 ± 0.18% w/w (Fig. 1A). Although further increase in pressure from 35 to 40 MPa did not increase the yield significantly (5.13 ± 0.11% w/w), it showed an enhanced antioxidant and LOX inhibitory activity. Hence 40 MPa was considered as optimum pressure for further study. A previous report on the yield of oleoresin from Piper nigrum also showed an increase from 1.1% w/w to 5.8% w/w on increasing the pressure from 10 to 25 MPa (Nagavekar &

2.9.1. Gas chromatography analysis of bacterial fatty acid methyl esters (FAME) Bacterial cells were recovered by centrifugation for 15 min at 5000 x g and bacterial lipids were extracted from the cell pellets and used for the preparation of FAME as per the method reported by Evans, McClure, Gould, and Russell (1998). The wet bacterial pellet was suspended in 2 mL of sterile water and transferred to 20 mL stoppered glass 3

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Fig. 1. Yield of oleoresin by SC-CO2 extraction due to the effect of (A) pressure (40 °C/60 min), (B) temperature (40 MPa/60 min), (C) time (45 °C/40 MPa) and (D) ); different letters above the bars indicate significantly different (p < 0.05) values; entrainers (45 °C/40 MPa/60 min) in comparison to Soxhlet extraction ( methanol, acetone, ethanol, iso-propanol.

depends on the mode of extraction, and nature of the biomass and its morphological characteristics.

Singhal, 2018). Temperature plays a critical role in extraction of oleoresins and many phytochemicals in SC-CO2. In this study, an increase in the oven temperature from 40 to 45 °C resulted in the increase of oleoresin yield from 5.13 ± 0.11 to 5.76 ± 0.09% w/w (Fig. 1B), beyond which there was no improvement. This is in accordance with an earlier report on C. zedoaria extraction where maximum yield was obtained at 50 °C and did not improve further with increasing temperature (Ma, Yu, & Han, 1995). A previous report on the extraction of carotenoids showed higher yield at 50 °C compared to 80 °C (Shi et al., 2013). Earlier reports on the extraction of bioactives within the temperature range of 40–60 °C showed higher yield at 60 °C for a traditional medicinal plant Dracocephalum kotschyi Boiss (Nejad-Sadeghi, Taji, & Goodarznia, 2015), and better yield at 40 °C for milk thistle seeds (Rahal, Barba, Barth, & Chevalot, 2015). Thus the effect of temperature on extraction varies with the nature of biomass. A significant improvement in the yield of oleoresin was observed on increasing the time of extraction from 30 (3.75% w/w) to 60 min (5.76% w/w), and further increase up to 120 min did not increase the yield further (Fig. 1C). Different solvents viz. acetone, ethanol, methanol and isopropanol were tested as entrainers at 10–30% (v/w of feed) for modifying the polarity of the SC-CO2 fluid. Among the solvents screened, the highest yield of oleoresin was obtained for iso-propanol. The increase of iso-propanol from 10% to 20% v/w increased the yield significantly from 7.45 ± 1.2 to 8.94 ± 0.12% w/w, and further increase to 30% v/w did not improve the yield significantly (Fig. 1D). For comparison, methanol (30% v/w), acetone (30% v/w) and ethanol (30% v/w) as entrainers gave yields of 7.27 ± 0.14% w/w, 7.54 ± 0.16% w/w and 7.47 ± 0.12% w/w, respectively. Iso-propanol as entrainer (20% v/w) gave the highest yield of volatile oil fraction (1.05 ± 0.03% w/w) compared to other entrainers and Soxhlet extraction (0.36 ± 0.02% w/w) (Fig. S2). Iso-propanol and ethanol are preferred solvents in food applications owing to their GRAS status. The extraction yield and percentage of bioactives in the oil

3.3. Antioxidant activity of the extracts The process parameters used for extraction showed varied response on the antioxidant activities of the C. aromatica Salisb extracts. An increase in pressure from 10 to 40 MPa significantly increased the antioxidant activity from 28.6 to 101.5, 30.8 to 105.9, 20.9–52.8 μmol TE/g for DPPH, FRAP, and ABTS respectively. A similar trend of increase in the antioxidant activity of the Piper nigum oil on increasing the pressure has been reported (Bagheri, Manap, & Solati, 2014). Ethyl acetate extract using Soxhlet extraction showed highest activity of 131.1 ± 2.2, 142.3 ± 1.9 and 68.2 ± 1.6 μmol TE/g by DPPH, FRAP, and ABTS assays respectively (Fig. 2A). There was a marginal increase in the antioxidant activity when the temperature of extraction was increased from 40 to 45 °C. Further rise in temperature to 65 °C showed no significant improvement in the activities (Fig. 2B). Mild conditions of 45 °C/16 MPa are known to be favourable for higher antioxidant activity of grape seeds (Ghafoor, Park, & Choi, 2010). The C. aromatica Salisb extract showed highest activity at 60 min, and further increase in time did not improve the activities (Fig. 2C). The antioxidant activity of C. aromatica Salisb extract significantly improved to 119.9 ± 2.6, 128.9 ± 2.4, 64.2 ± 1.2 μmol TE/g for DPPH, FRAP, and ABTS respectively, on addition of iso-propanol (20% v/w) as an entrainer (Fig. 2D). A previous report has shown the antioxidant-rich extracts from sumac, thyme, and mint to retard the oxidation of corn oil (Baştürk, Ceylan, Çavuş, Boran, & Javidipour, 2018). On similar lines, the antioxidant rich C. aromatica Salisb oleoresin obtained by using conventional ethyl acetate extraction and/or SC-CO2 extraction using iso-propanol (20% v/w) as an entrainer can be used in food products for improving oxidative stability of vulnerable constituents in foods. 4

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Fig. 2. Antioxidant activity (μmol TE/g) of SC-CO2 extracts due to the effect of (A) pressure (45 °C/60 min), (B) temperature (40 MPa/60 min), (C) time (45 °C/ FRAP, DPPH, ABTS, Soxhlet FRAP, Soxhlet 40 MPa), and (D) entrainer (45 °C/40 MPa/60 min) in comparison to Soxhlet extraction; Soxhlet ABTS; different letters above the bars indicate significantly different (p < 0.05) values. DPPH,

62.5 ± 2.20% to 86.55 ± 2.3%, suggesting better anti-inflammatory activities of the C. aromatica Salisb extract obtained on addition of entrainer (Fig. 3D). These results are in accordance with the earlier reports which showed the inhibition of LOX to be directly proportional to the polyphenol content in peanut sprout extracts (Limmongkon et al., 2018), red wine (Duque et al., 2011), and spice extracts from tarragon and oregano (Gawlik-Dziki, 2012). The oleoresin fractions of C. aromatica Salisb which showed good anti-inflammatory activity can be explored further to improve life-style disorders as well as to avert food allergies and improve nutraceutical aspects of food.

3.4. Total phenolic content (TPC) An increase in pressure from 10 to 40 MPa increased the TPC of the extract from 10.79 ± 0.80 to 20.32 ± 1.20 mg GAE/g, respectively (Fig. 3A). Soxhlet extract (ethyl acetate) showed TPC of 36.8 ± 1.60 mg GAE/g and was significantly higher than the SC-CO2 extracts. The maximum extraction of TPC from C. aromatica Salisb was at 45 °C (23.2 ± 0.65 mg GAE/g) (Fig. 3B). Da Porto, Decorti, and Natolino (2014) found a similar reduction in the TPC from 6.28 to 5.76 mg GAE/g with grape marc on increasing the temperature from 40 to 60 °C, and concluded the reduction of the density of SC-CO2 at higher temperature to be the major reason for the lower yield. An increase in extraction time from 30 to 60 min significantly increased the TPC from 10.1 to 23.2 mg GAE/g, beyond which it did not increase further (Fig. 3C). Iso-propanol as an entrainer at 20% v/w (45 °C/40 MPa/ 60 min) increased the total phenolic content to 29.24 mg GAE/g as shown in Fig. 3D. Earlier reports have shown iso-propanol to selectively increase the yield of terpenoids and phenolic ketones (Zancan, Marques, Petenate, & Meireles, 2002).

3.6. Characterisation of optimised extract using LC-MS/MS LC-MS/MS showed various compounds in the Soxhlet and SC-CO2 extracts. The ethyl acetate extract and SC-CO2 extract showing the best bioactivities (45 °C/40 MPa/20% v/w iso-propanol) were selected for LC-MS/MS identification. The compounds were identified by the mass spectra and fragmentation patterns based on the m/z ratio compared to reference compounds suggested by the mass library and published data. Thirty one most probable compounds were identified, out of which 26 compounds were common in both SC-CO2 and Soxhlet extracted C. aromatica Salisb oleoresin samples (Table 1). Most of the bioactives which were identified from genus Curcuma belongs to the class of terpenoids especially menthane type of monoterpenes, eudesmane and furanoeudesmane, bisabolane, curcumane, bisabolane, elemane, germacrane and guaiane type of sesquiterpenes (Nahar & Sarker, 2007) . p-Cymene, curcolone, ar-turmerone, β-turmerone, curcumadione, curzerene, curdione, dehydrocurdione, germacrone, gemacrone-4,5-epoxide, germacrone-13-al, furanodienone, zederone, curcumenol, zedoarolide B, procurcumadiol and 3,7-epoxycaryophyllan-6-one were prominent bioactives observed in both extracts. p-Cymene exhibited a molecular ion at m/z-133.10 and a fragment

3.5. Lipoxygenase (LOX) inhibition assay An increase in pressure from 10 to 40 MPa increased the inhibition of LOX from 35.4% to 55.3% as against an inhibition of 91.2% and 95.6% for Soxhlet and drug indomethacin (positive control), respectively (Fig. 3A). A rise in temperature from 40 to 45 °C increased the inhibition of LOX enzyme to 62.9% (Fig. 3B), beyond which there was no further improvement in LOX inhibition. An increase in the time of extraction from 30 to 60 min increased the LOX inhibition from 29.6 to 62.9% under SC-CO2 conditions (45 °C/40 MPa). Further rise in time did not significantly improve the enzyme inhibition (Fig. 3C). The addition of entrainer (iso-propanol, 20% v/w) to the sample extracted by SC-CO2 (45 °C/40 MPa/60 min) increased the inhibition of LOX from 5

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Fig. 3. Anti-inflammatory activity ( ) and total phenolic content ( ) of SC-CO2 extracts due to the effect of (A) pressure (40 °C/60 min), (B) temperature (40 MPa/60 min), (C) time (45 °C/40 MPa), and (D) entrainer (45 °C/40 MPa/60 min) in comparison to Soxhlet extraction ( lipoxygenase inhibition and TPC) and indomethacin ( ); different letters above the bars indicate significantly different (p < 0.05) values.

inflammatory, anticancer, insecticidal, hemagglutination and antimicrobial properties (Raj et al., 2008).

ion at m/z-134.10. It belongs to menthane type of monoterpenoids, and is a known antibacterial agent and precursor of carvacol (Goñi et al., 2009). Turmerones, especially β-turmerone, was seen with the molecular peak at m/z-219.14 and the fragmentation ion at 220.14, whereas ar-turmerone showed an m/z of 217.16 and fragmentation ions at 218.16 and 219.17. Ar-turmerone and β-turmerone belong to bisabolane type sesquiterpenes. Bioactives like curdione (m/z-237.18), dehydrocurdione (m/z235.17), germacrone (m/z-219.17), gemacrone-4,5-epoxide (m/z235.17), germacrone-13-al (m/z-233.15), furanodienone (m/z-230.14) and zederone (m/z-247.14) belong to germacrane type of sesquiterpenes. Caryophyllane-type sesquiterpenes like 3,7-epoxycaryophyllan-6-one (m/z-237.18) was also present in both extracts. Aerugidiol, curcumenol, curcumapentadecanol and curlone were absent in SC-CO2 extracts. It is interesting to note that curcumin was not detected in both the extracts. The rhizome itself was white in colour (Fig. S4) and the extracts were brownish.

3.8. Antibacterial activity of extracts The antibacterial activity of ethyl acetate and SC-CO2 extract obtained under optimised conditions was tested against Gram-positive bacteria S. auerus and Gram-negative P. aeruginosa. The oleoresin showed varied antibacterial activity with different strains. For S. aureus, minimum inhibitory concentration (MIC) was found to be 580 ± 5.0 and 520 ± 10.0 μg/mL for ethyl acetate and SC-CO2 extracts, respectively (Table 3). For P. aeruginosa, the MIC was found to be 690 ± 5.0 and 660 ± 10.0 μg/mL for ethyl acetate and SC-CO2 extracts, respectively. Based on MIC values, the SC-CO2 extract showed higher antibacterial activity against both Gram-positive and Gram-negative bacteria than the ethyl acetate extract. It is known that exposure of microbes to essential oils alter their membrane permeability by changes in the fatty acid composition of the cell membrane (Liang, Yuan, Vriesekoop, & Lv, 2012). To the best of our knowledge, there are no reports on comparative antimicrobial activity and its impact on the fatty acid composition of the bacterial cells using solvent and SC-CO2 extracts of C. aromatica Salisb. The microbial cells were treated with both ethyl acetate and SC-CO2 oleoresins above MIC. The control S. aureus group contained 31.90 ± 2.90% unsaturated fatty acids, while that treated with ethyl acetate and SC-CO2 extract of C. aromatica Salisb showed 27.5 ± 1.44% and 21.6 ± 1.70% of unsaturated fatty acids, respectively. The oleic acid (C18:1) percentage was found to be 16.57 ± 1.12%, 9.67 ± 0.98% and 5.69 ± 1.11% in control, ethyl acetate and SC-CO2 extract, respectively. A previous study on S. aureus treated with cinnamaldehyde and carvacrol showed a decrease in unsaturated fatty acids (myristoleic and oleic acid) in the cell membrane (Di Pasqua et al., 2007 & Liang et al., 2012). In case of P. aeruginosa, a

3.7. Characterisation of optimised extract using GC-MS The GC-MS analysis showed bioactives, especially sesquiterpenes like curdione and monoterpene molecule like iso-camphol which were present in both Soxhlet and SC-CO2 extracts. Bioactive compounds such as velleral, camphor (labdane type diterpenoid) and 4-oxo-β-isodamascol were detected in the SC-CO2 extracts only (Table 2). Specific detection of velleral in the essential oils of Curcuma varieties like C. attenuate and C. kwangsiensis rhizomes (Raj et al., 2008), and of camphor and borneol (Peter & Babu, 2012) as well as curcumol and curdione in oil of C. aromatica Kuroyanagi, Ueno, Ujiie, & Sato, 1987) are reported. Curdione is also formed as an intermediate in the biosynthesis of curcuma lactone and curcumenol (Jia, Xiao Chi, Xiu Lan, Hong Wei, & Dean, 2008), and both these constituents possess analgesic, anti6

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Table 1 LC-Q-TOF-MS profile of C. aromatica Salisb extract of SC-CO2 (CA1) and ethyl acetate extract (CA2). Rt (min)

Mass (Da)

Compounds

CA1

CA2

Observed M+/(M + H)+

MS/MS2 ions

1

7.15

280.13

Aromaticane F

+

+

281.14

2

7.28

246.31

Curcolone (Nehipetol)

+

+

247.13

3 4

7.49 8.33

170.01 234.34

Unidentified compound-1 Dehydrocurdione [1(10),7(11) Germacradiene-5,8-dione]

+ +

+ +

171.06 235.17

5

8.40

230.13

Furanodienone

+

+

230.14

6 7

8.53 9.47

493.01 229.24

Unidentified compound-2 Ermanthin

– +

+ +

494.01 230.25

8

9.57

232.32

Germacrone-13-al

+

+

233.15

9 10 11 12

9.80 10.61 10.94 11.16

218.34 250.33 245.11 234.0

β-Turmerone Aerugidiol (4S)-4-hydroxy-gweicurculactone Germacrone-4,5-epoxide

+ – + +

+ + + +

219.14 251.16 245.12 235.17

13

11.96

216.10

Aromatic (ar-) turmerone (1,3,5,10-Bisabolatetraen-9-one)

+

+

217.16

14

12.22

234.30

Curcumenol

–

+

235.17

15 16 17 18 19 20

12.61 12.74 13.15 13.22 13.44 13.96

246.30 132.10 226.10 216.10 250.10 234.33

Zederone p-Cymene Curcumapentadecanol α-Turmerone 4,5-Dihydroxy-7(11),9-guaiadien-8-one (Procurcumadiol) Aromaticane C

+ + – + + +

+ + + + + +

247.14 133.10 227.10 217.16 251.17 235.17

21 22 23 24 25 26 27 28

13.97 14.48 14.49 14.51 14.79 14.81 14.82 15.20

216.15 236.18 236.33 134.11 259.0 236.18 218.10 233.30

Curzerene Curdione 3,7-Epoxycaryophyllan-6-one Unidentified compound-7 Unidentified compound-8 Neocurdione Germacrone Curcumadione

+ + + + – + + +

+ + + + + + + +

217.15 237.18 237.18 135.16 259.17 237.18 219.17 233.15

29

16.38

236.18

Unidentified compound-9

–

+

237.18

30 31

16.47 26.15

235.17 136.10

Procurcumenol Camphene

+ +

+ +

235.17 136.11

282.14 283.14 248.14 249.14 172.06 236.17 237.17 231.14 232.14 493.01 231.25 232.25 234.15 235.15 220.14 252.16 246.12 236.17 237.17 218.16 219.17 236.17 253.17 248.14 134.10 228.17 218.16 252.17 236.17 237.17 218.16 238.19 238.18 135.16 260.17 238.18 220.17 234.16 235.16 237.18 238.18 236.17 136.11 137.11

“+” denotes present and “-” denotes absent; Rt denotes retention time of the compound.

the presence of higher antibacterial rich components (velleral, pcymene) and presence of higher volatiles fractions in SC-CO2 extract (1.05 ± 0.12% w/w) compared to the ethyl acetate extract (0.35 ± 0.09% w/w) (Fig. S2). A previous study reported a combination of carvacrol with potassium sorbate at sub-inhibitory concentrations to show antimicrobial activity in tomato paste during its storage (Batista et al., 2019). This gives an insight towards the use of antibacterial rich oleoresins which can be potentially used in improving the shelf life of food products. Further evaluation of the antimicrobial

reduction in unsaturated fatty acids was observed in both ethyl acetate and SC-CO2 extracts (Table 3). The content of unsaturated fatty acids in the control group of P. aeruginosa was 48.77 ± 1.90% which reduced to 44.41 ± 3.10% and 43.28 ± 2.48%, respectively, on exposure to ethyl acetate and SC-CO2 extract (Table 3). An earlier report on the antibacterial activity of essential oils like thymol, eugenol, and carvacrol on P. fluorescens has documented a reduction of 36.52%, 65.80% and 24.82% in unsaturated fatty acids, respectively (Di Pasqua et al., 2007). The higher antibacterial activity of the SC-CO2 extract is due to

Table 2 GC-Q-TOF MS profile of C. aromatica Salisb extract of SC-CO2 (CA1) and ethyl acetate extract (CA2). No.

Rt (min)

1 2 3 4 5 6 7 8 9

6.69 22.60 23.07 23.29 28.44 31.21 32.06 32.49 32.63

Mass (Da)

Compounds

CA1

CA2

Molecular formula

154 368 216 236 232 234 208

2-pentanone Camphor Isoborneol/isocamphol Piperazline,1-[15fluro-2-methoxyphenyl)sulfonyl-4-92-furanylcarbonyl Benzofuran, 6-ethenyl-4,5,6,7-tetrahydro-3,6-dimethyl-5-isopropenyl-trans Curdione Velleral/5,6-Azulenedicarboxaldehyde, 1,2,3,3a,8,8a-hexahydro-2,2,8-trimethyl,(3aα, 8aα) 2-(4a,8-Dimethyl-6-oxo-1,2,3,4,4a,5,6,8a-octahydro-naphthalen-2yl)-propioaldehyde 4-Oxo-β-isodamascol

+ + + + + + + + +

+ – + – – + – – –

C6H12O2 C10H16O C10H8O C16H17FN2O5S C15H20O C15H24O2 C15H20O2 C15H22O2 C13H20O2

“+” denotes present and “-” denotes absent, Rt denotes retention time of the compound. 7

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Table 3 Effect of ethyl acetate and SC-CO2 extract of Curcuma aromatica Salisb on the MIC and FAME analysis of the bacterial cells. Staphylococcus aureus Control MIC (μg/mL) Fatty acids (%) C8:0 C10:0 C12:0 C13:0 C14:0 C14:1 C15:1 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 (n9) C20:2 C21:0 C20:3 C20:4 (n6) C22:0 C22:1 C24:0 UFAs

6.08 ± 0.90a 0.35 ± 0.11a 2.10 ± 0.23a 1.38 ± 0.30b 1.20 ± 0.06c 1.64 ± 0.30a ND 21.26 ± 1.12 3.95 ± 0.09a 2.34 ± 0.19a 6.55 ± 0.14c 16.57 ± 1.12a 1.30 ± 0.40c 4.97 ± 0.12b 2.73 ± 0.38b 0.72 ± 0.20a 1.18 ± 0.08c 1.13 ± 0.01c 1.57 ± 0.50b ND 2.79 ± 0.08c ND 1.52 ± 0.13c 31.9 ± 2.9a

Pseudomonas aeruginosa Control

Ethyl acetate

SC-CO2

580 ± 5.0

520 ± 10.0

1.56 ± 0.09b 0.30 ± 0.11a 1.18 ± 0.01c 1.18 ± 0.04b 19.44 ± 1.56a 0.67 ± 0.03c ND ND 2.53 ± 0.03b 1.51 ± 0.45b 10.81 ± 0.12a 9.67 ± 0.98b 3.86 ± 0.17a 4.37 ± 0.12a 2.75 ± 0.18b 0.95 ± 0.01a 2.24 ± 0.10b 2.99 ± 0.02a 3.21 ± 0.12a ND 4.23 ± 0.02a ND 3.53 ± 0.08a 27.5 ± 1.44b

6.05 ± 0.12a 0.34 ± 0.20a 1.85 ± 0.03b 1.24 ± 0.30a 12.64 ± 0.21b 1.09 ± 0.10b ND ND 2.03 ± 0.01c 2.17 ± 0.04a 9.09 ± 0.16b 5.69 ± 1.11c 2.08 ± 0.29b 5.48 ± 0.08a 3.41 ± 0.15a 0.94 ± 0.03a 2.80 ± 0.05a 2.27 ± 0.12b 1.51 ± 0.01b ND 3.39 ± 0.01b ND 2.77 ± 0.10b 21.62 ± 1.70c

4.47 ± 0.10b 0.34 ± 0.12a 0.99 ± 0.11b 0.58 ± 0.01 5.25 ± 0.10b 0.75 ± 0.21a 31.61 ± 1.32c ND 4.67 ± 0.11a 0.48 ± 0.03b 1.86 ± 0.02b 6.57 ± 0.01b 0.31 ± 0.02c 1.34 ± 0.01b 0.90 ± 0.07c 2.86 ± 0.13a ND 0.38 ± 0.01c ND 0.27 ± 0.03c 1.04 ± 0.03c 0.38 ± 0.02c ND 48.77 ± 1.90a

Ethyl acetate

SC-CO2

690 ± 5.0

660 ± 10.0

7.06 ± 0.11a 0.52 ± 0.03a 2.56 ± 0.12a 0.77 ± 0.12 6.65 ± 0.21a 0.95 ± 0.11a 23.79 ± 2.21b ND 2.77 ± 0.12c 1.17 ± 0.13a 0.53 ± 0.10c 6.38 ± 0.13b 5.95 ± 0.15a 0.75 ± 0.13c 2.72 ± 0.01a 1.31 ± 0.05b ND 1.19 ± 0.17a ND 1.01 ± 0.06b 2.21 ± 0.05a 1.50 ± 0.16b ND 44.41 ± 3.10ab

4.57 ± 0.13b 0.37 ± 0.12a 1.16 ± 0.10b ND 5.44 ± 0.10b 0.85 ± 0.11a 19.63 ± 1.44a ND 3.06 ± 0.13b 1.03 ± 0.14a 3.92 ± 0.22a 8.53 ± 0.17a 3.12 ± 0.04b 3.36 ± 0.32a 1.82 ± 0.08b 1.35 ± 0.01b ND 0.82 ± 0.03b ND 1.38 ± 0.04a 1.41 ± 0.01b 2.00 ± 0.18a ND 43.28 ± 2.48b

UFAs-unsaturated fatty acids, ND-not detected; all values are mean ± standard deviation of three or more determinations. Values with same superscript in a row for the two cultures independently do not vary significantly (p < 0.05).

activity of the individual components from both the extracts could give deeper insight into the reasons for the observations reported in this paper. A thorough study on the exact mechanism of biological action of the extracts using various techniques, their stability in food systems as well as dose-response studies need further investigations.

[Grant no. F.4-1/2006/(BSR)/5-52/2007(BSR)], New Delhi, Government of India, for providing financial assistance during the course of this study.

4. Conclusion

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.108564.

Appendix A. Supplementary data

Among the organic solvents evaluated for conventional Soxhlet extraction, ethyl acetate gave the highest yield (8.34% w/w) of oleoresin from Curcuma aromatica Salisb. Under optimised conditions of SCCO2 extraction (40 MPa/45 °C/60 min), iso-propanol (20% v/w) as an entrainer gave maximum yield of oleoresin (8.94% w/w). The Soxhlet extract demonstrated 10% higher antioxidant activities as assayed by DPPH, FRAP and 6% for ABTS over the optimised SC-CO2 extract. The anti-inflammatory activity of the Soxhlet extract was also found to be higher by 26% in the SC-CO2 extract. These effects may be attributed to higher content of phenolics in the Soxhlet extract. Both the extracts showed the presence of anti-inflammatory compounds such as curcumenol, furanodienone, curdione, and germacrones. In contrast, the SCCO2 extract showed better antibacterial activities against S. aureus and P. aeruginosa strains than the Soxhlet extract, which could be due to the presence of p-cymene, turmerones, germacrone and germacrane type sesquiterpenes. Thus, ethyl acetate extract of C. aromatica Salisb can be used for averting inflammatory reactions and improving oxidative stability of vulnerable constituents of food products, while its SC-CO2 extract can be used as an anti-bacterial agent for food preservation.

References Al-Reza, S. M., Rahman, A., Sattar, M., Rahman, M. O., & Fida, H. M. (2010). Essential oil composition and antioxidant activities of Curcuma aromatica Salisb. Food and Chemical Toxicology, 48(6), 1757–1760. Bagheri, H., Manap, M. Y. B. A., & Solati, Z. (2014). Antioxidant activity of Piper nigrum L. essential oil extracted by supercritical CO2 extraction and hydro-distillation. Talanta, 121, 220–228. Baştürk, A., Ceylan, M. M., Çavuş, M., Boran, G., & Javidipour, I. (2018). Effects of some herbal extracts on oxidative stability of corn oil under accelerated oxidation conditions in comparison with some commonly used antioxidants. LWT-Food Science and Technology, 89, 358–364. Batista, A. F. P., dos Santos, A. R., da Silva, A. F., Trevisan, D. A. C., Ribeiro, L. H., Campanerut-Sá, P. A. Z., et al. (2019). Inhibition of Salmonella enterica serovar Typhimurium by combined carvacrol and potassium sorbate in vitro and in tomato paste. LWT-Food Science and Technology, 100, 92–98. Chaubey, P. S., Somani, G., Kanchan, D., Sathaye, S., Varakumar, S., & Singhal, R. S. (2018). Evaluation of debittered and germinated fenugreek (Trigonella foenum graecum L.) seed flour on the chemical characteristics, biological activities, and sensory profile of fortified bread. Journal of Food Processing and Preservation, 42(1). Chen, C. H., Chang, T. C., Chen, S. Y., Hsu, S. J., Huang, H. W., & Lee, C. K. (2017). Chemical composition and antioxidant, bactericidal, and matrix metalloproteinase inhibition activity of food-related plant. LWT-Food Science and Technology, 82, 411–419. Da Porto, C., Decorti, D., & Natolino, A. (2014). Water and ethanol as co-solvent in supercritical fluid extraction of proanthocyanidins from grape marc: A comparison and a proposal. The Journal of Supercritical Fluids, 87, 1–8. David, F., Sandra, P., & Vickers, A. K. (2005). Column selection for the analysis of fatty acid methyl esters. Food analysis application. Palo Alto, CA: Agilent Technologies. Di Pasqua, R., Betts, G., Hoskins, N., Edwards, M., Ercolini, D., & Mauriello, G. (2007). Membrane toxicity of antimicrobial compounds from essential oils. Journal of Agricultural and Food Chemistry, 55(12), 4863–4870. Dosoky, N., & Setzer, W. (2018). Chemical composition and biological activities of

Conflicts of interest There is no conflict of interest with any individual or organization. Acknowledgements The authors are grateful to the University Grants Commission 8

LWT - Food Science and Technology 116 (2019) 108564

U.K. Vijayan, et al.

Nagavekar, N., & Singhal, R. S. (2018). Enhanced extraction of oleoresin from Piper nigrum by supercritical carbon dioxide using ethanol as a co‐solvent and its bioactivity profile. Journal of Food Process Engineering, 41(1), e12670. Nahar, L., & Sarker, S. D. (2007). Phytochemistry of the genus Curcuma. In P. N. Ravindran, K. Nirmal Babu, & K. Sivaraman (Eds.). Turmeric – The genus Curcuma (pp. 71–106). Boca Raton: CRC Press. Nejad-Sadeghi, M., Taji, S., & Goodarznia, I. (2015). Optimization of supercritical carbon dioxide extraction of essential oil from Dracocephalum kotschyi Boiss: An endangered medicinal plant in Iran. Journal of Chromatography A, 1422, 73–81. Panich, U., Kongtaphan, K., Onkoksoong, T., Jaemsak, K., Phadungrakwittaya, R., Thaworn, A., et al. (2010). Modulation of antioxidant defense by Alpinia galanga and Curcuma aromatica extracts correlates with their inhibition of UVA-induced melanogenesis. Cell Biology and Toxicology, 26(2), 103–116. Pavlić, B., Bera, O., Teslić, N., Vidović, S., Parpinello, G., & Zeković, Z. (2018). Chemical profile and antioxidant activity of sage herbal dust extracts obtained by supercritical fluid extraction. Industrial Crops and Products, 120, 305–312. Pereira, C. G., & Meireles, M. A. A. (2010). Supercritical fluid extraction of bioactive compounds: Fundamentals, applications and economic perspectives. Food and Bioprocess Technology, 3(3), 340–372. Peter, K., & Babu, K. N. (2012). Introduction to herbs and spices: Medicinal uses and sustainable production. (2nd ed.). Handbook of Herbs and Spices: Vol. 2, (pp. 1–16). Elsevier. Rahal, N. B., Barba, F. J., Barth, D., & Chevalot, I. (2015). Supercritical CO2 extraction of oil, fatty acids and flavonolignans from milk thistle seeds: Evaluation of their antioxidant and cytotoxic activities in Caco-2 cells. Food and Chemical Toxicology, 83, 275–282. Raj, G., Baby, S., Dan, M., Thaha, A. R. M., Sethuraman, M. G., & George, V. (2008). Volatile constituents from the rhizomes of Curcuma haritha Mangaly and Sabu from southern India. Flavour and Fragrance Journal, 23(5), 348–352. Shi, X., Wu, H., Shi, J., Xue, S. J., Wang, D., Wang, W., et al. (2013). Effect of modifier on the composition and antioxidant activity of carotenoid extracts from pumpkin (Cucurbita maxima) by supercritical CO2. LWT-Food Science and Technology, 51(2), 433–440. Vallecilla-Yepez, L., & Ciftci, O. N. (2018). Increasing cis-lycopene content of the oleoresin from tomato processing byproducts using supercritical carbon dioxide. LWT-Food Science and Technology, 95, 354–360. Zancan, K. C., Marques, M. O., Petenate, A. J., & Meireles, M. A. A. (2002). Extraction of ginger (Zingiber officinale roscoe) oleoresin with CO2 and co-solvents: A study of the antioxidant action of the extracts. The Journal of Supercritical Fluids, 24(1), 57–76.

essential oils of Curcuma species. Nutrients, 10(9), 1196. Duque, A. L., Pinto, M. D. C., & Macias, P. (2011). Lipoxygenase inhibition by red wine phenolics compounds. Journal of Food Biochemistry, 35(2), 542–555. Evans, R., McClure, P., Gould, G., & Russell, N. (1998). The effect of growth temperature on the phospholipid and fatty acyl compositions of non-proteolytic Clostridium botulinum. International Journal of Food Microbiology, 40(3), 159–167. Gawlik-Dziki, U. (2012). Dietary spices as a natural effectors of lipoxygenase, xanthine oxidase, peroxidase and antioxidant agents. LWT-Food Science and Technology, 47(1), 138–146. Ghafoor, K., Park, J., & Choi, Y. H. (2010). Optimization of supercritical fluid extraction of bioactive compounds from grape (Vitis labrusca B.) peel by using response surface methodology. Innovative Food Science & Emerging Technologies, 11(3), 485–490. Goñi, P., López, P., Sánchez, C., Gómez-Lus, R., Becerril, R., & Nerín, C. (2009). Antimicrobial activity in the vapour phase of a combination of cinnamon and clove essential oils. Food Chemistry, 116(4), 982–989. Jia, N., Xiao Chi, M., Xiu Lan, X., Hong Wei, J., & Dean, G. (2008). Structural determination of two new sesquiterpenes biotransformed from germacrone by Mucor alternata. Magnetic Resonance in Chemistry, 46(2), 178–181. Khare, C. P. (2008). Indian medicinal plants: An illustrated dictionary. Springer Science & Business Media. Kojima, H., Yanai, T., & Toyota, A. (1998). Essential oil constituents from Japanese and Indian Curcuma aromatica rhizomes. Planta Medica, 64(04), 380–381. Kuroyanagi, M., Ueno, A., Ujiie, K., & Sato, S. (1987). Structures of sesquiterpenes from Curcuma aromatica Salisb. Chemical and Pharmaceutical Bulletin, 35(1), 53–59. Lee, Y. L., Weng, C. C., & Mau, J. L. (2007). Antioxidant properties of ethanolic and hot water extracts from the rhizome of Curcuma aromatica. Journal of Food Biochemistry, 31(6), 757–771. Liang, H., Yuan, Q., Vriesekoop, F., & Lv, F. (2012). Effects of cyclodextrins on the antimicrobial activity of plant-derived essential oil compounds. Food Chemistry, 135(3), 1020–1027. Limmongkon, A., Nopprang, P., Chaikeandee, P., Somboon, T., Wongshaya, P., & Pilaisangsuree, V. (2018). LC-MS/MS profiles and interrelationships between the anti-inflammatory activity, total phenolic content and antioxidant potential of Kalasin 2 cultivar peanut sprout crude extract. Food Chemistry, 239, 569–578. Li, Y., Wo, J. M., Liu, Q., Li, X., & Martin, R. C. (2009). Chemoprotective effects of Curcuma aromatica on esophageal carcinogenesis. Annals of Surgical Oncology, 16(2), 515. Ma, X., Yu, X., & Han, J. (1995). Application of off‐line supercritical fluid extraction-gas chromatography for the investigation of chemical constituents in Curcuma zedoaria. Phytochemical Analysis, 6(6), 292–296.

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