An experimental design approach for the extraction of lemongrass (Cymbopogon citratus) oleoresin using pressurised liquid extraction (PLE)
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1 Interntionl Food Reserch Journl 20(1): (2013) Journl homepge: An experimentl design pproch for the extrction of lemongrss (Cymopogon citrtus) oleoresin using pressurised liquid extrction (PLE) * Nur Ain. A. H., Ziunnis, A. H., Hlimhton Zhrh, M. S. nd Norshikin, S. Fculty of Applied Sciences, Universiti Teknologi MARA Shh Alm Selngor Article history Received: 21 Octoer 2011 Received in revised form: 7 My 2012 Accepted: 7 My 2012 Keywords Pressurised liquid extrction lemongrss nerl gernil gerniol Astrct Extrction of lemongrss oleoresin ws successfully optimised using Pressurised Liquid Extrction (PLE). Chrcter impct compounds; nerl, gernil nd gerniol which constituted 72% oleoresin, were monitored during this optimistion study y using GCMSD. Bsed on mximum extrction of these compounds, the optimised operting conditions for PLE were temperture of 167 C, pressure of 1203 psi nd sttic time of min. The qulity of PLE extrct were compred with conventionl extrction methods, hydrodistilltion nd Soxhlet extrction. The proposed method ws found to e etter in term of quntity of the trgeted chrcter impct compounds. All Rights Reserved Introduction Lemongrss (Cymopogon citrtus) is plnt from the grss fmily nd contined out 1 2% essentil oil in dry weight sis. The essentil oil is chrcterised y high content of citrl (constituted y the isomers nerl nd gernil), which is used s rw mteril for the production of ionone, vitmin A nd β-crotene. The lemongrss essentil oil is very importnt in perfumery industry since it lends well with gret vriety of essentil oil (Crlson et l., 2006). The te mde from lemongrss leves is commonly used s ntispsmodic, nlgesic, ntiinflmmtory, ntipyretic, diuretic nd sedtive (Crlini et l., 1986). The current extrction methods of essentil oil from nturl products re tedious. These processes consumed lot of time nd mount of solvents. Therefore, the use of Pressurised Liquid Extrction (PLE) cn overcome these flws. Cristin et l. (2010) reported tht PLE is etter extrction technique in terms of quntifiction of ft-oil in red nd other derivtives products. PLE comines elevted temperture nd pressure with liquid solvents to chieve fst, efficient nd relile extrction. Moreover, PLE hs een shown to present sfe nd rpid technique for extrcting ntioxidnt compounds from plnts (Hossin et l., 2011 nd Mustf, 2010). Recently, Response Surfce Methodology (RSM) hs een used y mny reserchers s n effective tool to optimise processes (Wni, 2008; Firtgil-Durmus nd Evrnuz 2010). It helps in getting relevnt informtion in the shortest time with the minimum numer of experiments. A reserch hs een conducted in optimistion of lemongrss essentil oil extrction using Supercriticl Cron Dioxide (Huynh et l., 2007); ut none in PLE extrction. Therefore in this study, extrction of lemongrss mrker compounds is optimised with regrds to extrction temperture, pressure nd sttic time using PLE. Mterils nd Methods Smple preprtion Fresh lemongrsses were purchsed from locl supplier in Shh Alm, Selngor. Lemongrss stem ws cut 10 cm from root nd further cut to 2 mm dimeter prior to drying. The initil moisture level of lemongrss ws 11%. Smples were then dried in n oven t 40 C overnight nd kept in seled g. Chemicls n-hexne used ws of Ph. Eur grde (MERCK, Germny). Deionised wter used ws purified y Milli-Q purifiction system (Millipore) (Msschusetts, USA). Citrl nd gerniol stndrd re of MERCK, Germny. *Corresponding uthor. Emil: nu_rin@ymil.com
2 452 Nur Ain et l./ifrj 20(1): Pressurised liquid extrction (PLE) Extrction of lemongrss essentil oil ws done using n Accelerted Solvent Extrctor ASE 200 (Dionex Ltd. Cmerley, Surrey, UK). Aout 3 g of sliced lemongrss stem were ccurtely weighed nd plced into the 22 ml cells with cellulose filter t the ottom end. The smple cells were closed to finger tightness efore eing plced into the crousel of the ASE 200 system. Smple ws extrcted using n-hexne (Ph. Eur). The other prmeters ws stndrdised sed on: 1. Time = 5 min 2. Flush volume = 100% 3. Purge time = 60 s 4. Sttic cycle = 1 The extrcted nlytes were purged from the smple cell using pressurised nitrogen ( kp). Finlly the extrct ws evported to dryness to 1 ml using rotry evportor to clculte the yield of essentil oil (Ziunnis et l., 2009). Hydrodistilltion For this method, the Den Strk pprtus ws used. Aout 900 g smple of fresh lemongrss ws weighed in 500 ml flsk nd ws sumitted to hydrodistilltion for 12 hours. The distillte ws sturted with sodium chloride nd dded with n-hexne. Then, the ether lyer nd hydro lyer were seprted y funnel. After dehydrted y nhydrous sodium sulphte, the n-hexne lyer ws further dried t 40 C in rotry evportor to concentrte oil to 1 ml nd sujected to GC-MS nlysis (Gun et l., 2007). Soxhlet extrction Essentil oil of lemongrss ws extrcted from fresh plnt with n-hexne nd ethnol s solvent, for 16 hours using Soxhlet extrctor, following the AACC Method (AACC Interntionl, 2000). Smples were lter dried using rotry evportor to 1 ml nd nlysed y using GC-MS. Gs Chromtogrphy/Mss Spectrometry (GC/MS) Smple extrcted from vrious method of extrction mentioned were concentrted using rotry evportor to 1 ml in GC vil efore sujected to GC- MS nlysis. Anlyses of the smples were performed on Agilent Technologies Gs Chromtogrph-Mss Spectrometer (Model HP 5971 MSD, Hewlett- Pckrd, USA) using fused silic cpillry column DB-5 (20 m mm internl dimeter, 0.4 µm film thickness). The progrm strted t 100 C for 1 min. Then, the temperture ws incresed to 102 C t rte of 1 C/min. Injector nd detector tempertures were kept constnt t 250 nd 300 C, respectively. The smple volume injected ws 2 μl using utosmpler. Injections were done in triplictes. Finlly, compounds detected y GC-MS were referred to Flvour 2L nd NIST lirry from GC-MS softwre. Experimentl design pproch The optimised extrction condition ws pplied on PLE extrction. The effect of three independent vriles: temperture ( C), pressure (psi) nd sttic time (min) on response vriles nerl, gernil, gerniol nd yield of essentil oil were evluted using Response Surfce Methodology (RSM). The RSM experimentl design ws generted using Design-Expert version (Stt Ese Softwre). Ech of the vriles hd levels set t five coded levels: α, 1, 0, +1 nd +α. The results from centrl composite design (CCD) were ssessed using multiliner regression, using n eqution of the form: Y= A+ 2 B+ 3 C+ 4 A B C AB+ 8 AC+ 9 B C 0, 1 nd 2 = liner coefficients, 4, 5 nd 6 = qudrtic coefficients, 7, 8 nd 9 = cross-product coefficients. The dt otined ws nlysed using the SPSS 15.0 for Windows Evlution for nlysis of vrince nd Duncn s Multiple Rnge test. Sensory evlution The cceptility test conducted ws hedonic scle test (9 point scle). Aout 30 pnellists were involved in this test. Smples nlysed were lemongrss oleoresins extrcted from different methods; the optimised nd stndrd prmeters (100 C, 1000 psi, 30 minutes) of Pressurised Liquid Extrction (PLE) nd Soxhlet. The prmeters tested were lemongrss rom, chemicl rom, colour nd overll ttriutes. Results nd Discussions Comprison of PLE with other methods of extrction Tle 3 presents the mount of lemongrss mrker compounds (mg/100g) detected nd yield of totl voltile oil (%) from vrious extrction method. Ech method ws tested in triplicte to ensure reproduciility. Detection nd quntifiction of nerl nd gernil ws then determined y GC-MS s shown in Figure 1. For oth PLE nd Soxhlet extrction, n-hexne ws used s solvent. A preliminry study hd een done to select the est solvent for extrction of
3 Nur Ain et l./ifrj 20(1): Tle 1. Centrl composite design used for the PLE extrction of voltile compounds from lemongrss Temperture ( C) Pressure (psi) Extrction time (min) α α Tle 2. Comprison of voltile mrker compounds (mg/100g) extrcted from PLE stndrd method (100 C, 1000 psi, 30 minutes) using different type of solvents Solvents n-hexne Wter Mrker compounds (mg/100g) Nerl Gernil Gerniol ± ± ± ± ± ±6.3 Ethnol 6.23 c ± c ±0.7 n.d -c Sme letters within ech column indicte no significnt (p>0.05) difference ccording to Duncn s multiple rnge test. n.d = not detected Tle 3. Comprison of voltile mrker compounds (mg/100g) nd yield (%) using PLE, hydrodistilltion nd Soxhlet extrction from Cymopogon citrtus Extrction Methods Mrker compounds (mg/100g) Yield of Totl Nerl Gernil Voltile Oil(%) PLE ± ± ±0.44 Soxhlet extrction ± ± ±1.12 Hydrodistilltion c ± c ± ±0.00 -c Sme letters within ech column indicte no significnt (p>0.05) difference ccording to Duncn s multiple rnge test. 1 Pressurised liquid extrction conditions: smple, 3g; solvent, n-hexne; temperture, 100 C; pressure,1000 psi; sttic time, 30 min. 2 Soxhlet extrction conditions: smple, 2g (ir-dried); solvent, n-hexne; time,16 h. 3 Hydrodistilltion conditions: smple, 900g (fresh); time, 12 h. lemongrss mrker compounds (nerl nd gernil). From tle 2, it ws shown tht n-hexne gives significntly (p<0.05) highest mount of oth nerl nd gernil (mg/100g) s compred to wter nd ethnol. This is greele with Schneerg nd Khn (2002), where in their study revels tht n-hexne gives significntly (p<0.05) highest concentrtion of lemongrss mrker compound s compred to other solvents. Therefore, n-hexne ws selected s the extrction solvent in this study. As shown in Tle 3, percentge of totl voltile oil detected from Soxhlet extrction (3.81%) nd PLE (2.90%) were significntly (p<0.05) higher thn hydrodistilltion (0.01%). This ws due to the ility of solvent n-hexne to extrct lmost ll non-voltile nd voltile compound, s compred to hydrodistilltion which cn only extrct voltile Tle 4. Centrl composite design rrngement for independent vriles temperture ( C), pressure (psi) nd sttic time (min) nd their responses; nerl, gernil, gerniol nd yield (%, dry weight sis) Run Temperture ( C) Pressure (psi) Sttic time (min) Nerl (mg/10 0g) Gernil (mg/100 g) Gerniol (mg/100 g) Yield (%) Colour of extrc t Yellow Yellow Brown Brown Cler Brown Cler Cler Cler Yellow Brown Yellow Yellow Cler Yellow Yellow Yellow Yellow Yellow Brown compounds. PLE gve the significntly (p<0.05) highest mount of nerl nd gernil (78.61 ±0.1 mg/100g nd ±0.5 mg/100g, respectively) followed y Soxhlet (53.33 ±0.4 mg/100g nd ±0.3 mg/100g, respectively) nd hydrodistilltion (7.09 ±0.6 mg/100g nd ±0.3 mg/100g, respectively). This shows tht not only PLE gives rpid nd low cost extrction, ut it is lso the est lterntive for solvent solid/liquid extrction in terms of quntifiction of desired compounds from solid smple. Thus, extrction of lemongrss mrker compounds ws further optimised using PLE. The extrction prmeters optimised were temperture ( C), pressure (psi) nd sttic time (min). Experimentl design Effect of 3 independent vriles on response vriles nerl, gernil, gerniol nd yield of totl voltile oil were evluted using Response Surfce Methodology. The Centrl Composite Design (CCD) used in this study ws shown in Tle experiments were conducted sed on CCD. The prmeters nd responses otined were shown in Tle 3. As shown in Tle 4, effects of temperture ( C), pressure (psi) nd sttic time (min) on ll dependent responses (nerl, gernil, gerniol nd yield of totl voltile oil) were of second order (qudrtic). The R 2 vlues of ll interctions re ove 0.80, which ws suggested y Joglekr nd My (1987) s good fit of model. This shows tht interctions etween independent nd dependent vriles were very much relile. According to the significnt model terms, for ll equtions, temperture (A) nd sttic time (B) hs
4 454 Nur Ain et l./ifrj 20(1): Tle 4. ANOVA for response surfce for compounds nd yield Compounds Model Lck of Fit (F vlue) R 2 Eqution Significnt Model Terms Nerl Qudrtic Significnt 0.49 significnt Nerl = A B C A B C AB AC BC A, B Gernil Qudrtic Significnt 0.58 significnt Gernil = A B C A B C AB AC BC A, B Gerniol Qudrtic Significnt 4.07 significnt Gerniol = A B C A B C AB AC BC A Yield (%) Qudrtic Significnt A= Temperture, C; B= Pressure, psi; C= Sttic time, min 1.02 significnt Yield = A B C A B C AB AC BC A, A 2 Figure 1. GC chromtogrm otined using optimised PLE condition temperture: 167 C, pressure:1203 psi nd sttic time:20.43 mins the cceptnce of this oleoresin s compred to extrct from stndrd method (100 C, 1000psi, 30min) nd Soxhlet extrction method. According to Figure 2, oleoresin from optimised method ws uncceptle for ll prmeters tested. The oleoresin extrcted from this optimised prmeter ws significntly the lowest (p<0.5) s compred to stndrd method nd Soxhlet. The hedonic scle rnges from 3-4 which indicte dislike modertely. However, most pnellist prefer lemongrss oleoresin extrcted from stndrd method s it hs stronger lemongrss rom (7.33) nd cceptle colour (5.67). Conclusion Hedonic scle of lemongrss oleoresin from different extrction methods vs. sensory ttriutes Men hedonic scle c Lemongrss rom PLE 100 C, 1000psi, 30 min PLE C, 1203psi, min Soxhlet Chemicl (residue) rom Attriutes the profound effect on the mount of nerl (mg/100g) nd gernil (mg/100g) otined. Temperture is the only prmeter tht ffects mount of gerniol (mg/100g) nd yield of totl voltile oil (%). In this study, sttic time is the minor vrile in extrction efficiency. The optimised prmeters for lemongrss oleoresin PLE extrction were temperture of 167 C, pressure of 1203 psi nd sttic time of minutes. However, the ppernce of lemongrss oleorsin from this optimised condition ws undesirle, s it hs urnt smell nd drk rown in colour. Sensory evlution ws conducted to mesure Colour Overll ttriutes Figure 2. Br grph showing level of cceptnce of lemongrss oleoresin from different method of extrctions c The optimised operting conditions otined for PLE extrction of lemongrss oleoresin were, heting t 167 C, pressure of 1203 psi nd sttic time of min. However, sed on sensory evlution, stndrd method of PLE prmeters (100 C, 1000psi, 30min) ws selected for further lemongrss oleoresin extrction. Acknowledgement The uthors would like to cknowledge the finncil support of Universiti Teknologi MARA. References Crlini, E.A., J.D.P. Contr, A.R. nd Silv-Filho Phrmcology of lemongrss (Cymopogon citrtus Stpf) I. Effects of tes prepred from the leves on lortory nimls. J. Ethnophrmcology 17: Crlson, L. H. C., Mchdo C. B. S., Pereir L. K. nd Bolzn A Extrction of lemongrss essentil oil with dense cron dioxide. Journl of Supercriticl Fluids 21: Cristin Ruiz-Smlás, Luís Cudros-Rodríguez nd Antonio González-Cs Pressurised liquid extrction nd quntifiction of ft oil in red nd derivtives products. Tlnt 83: Firtligil- Durmus, E. nd Evrnuz O Response
5 surfce methodology for protein extrction optimiztion of red pepper seed (Cpsicum frutescens) LWT- Food Science nd Technology 43: Gun Wenqing, Li Shufen, Yn Ruixin, Tng Shokun, Qun Cn Comprison of essentil oils of clove uds extrcted with supercriticl cron dioxide nd other three trditionl extrction methods. Food Chemistry 101: Hossin, M.B., Brry-Ryn, C., Mrtin-Din, A.B. nd Brunton, N. P Optimistion of ccelerted solvent extrction of ntioxidnt compounds from rosemry (Rosmrinus officinlis L.), mrjorm (Orignum mjorn L.) nd oregno (Orignum vulgre L.) using response surfce methodology. Food Chemistry 126: Huynh K. P. H., Mridle J., Gspillo P., Hsik M., Mllun R. nd Kwski J Modeling nd optimiztion of supercriticl cron dioxide extrction on essentil oil from lemongrss using response surfce methodology. Journl of Reserch in Science, Computing, nd Engineering 4(3): Interntionl, 2000 AACC Interntionl, Approved methods of the Americn Assocition of Cerel Chemists. Approved method 08 01, 46 13, nd 30 25, The Assocition, St. Pul, MN (2000). Joglekr, A.M. nd My, A.T Product excellence through design of experiments. Cerel Foods World 32: Mustf Çm nd Ysr Hısıl Pressurised wter extrction of polyphenols from pomegrnte peels. Food Chemistry 123: Schnerg B. T. nd Khn I. A Comprison of extrction methods for mrker compounds in essentil oil of lemon grss y GC. Journl of Agriculturl nd Food Chemistry 50: Wni A. A., Kur D., Ahmd I. And Sogi D.S Extrction optimiztion of wtermelon seed protein using response surfce methodology. LWT- Food Science nd Technology 41: Ziunnis A.H., Norshikin S., Mmot S., nd Osmn H An experimentl design pproch for the extrction of voltile compounds from turmeric leves (Curcum domestic) using pressurised liquid extrction (PLE). LWT - Food Science nd Technology 42: Nur Ain et l./ifrj 20(1):
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