Optimization Of Supercritical Carbon Dioxide Extraction Biology Essay Essays

Optimization Of Supercritical Carbon Dioxide Extraction Biology Essay Essays Optimization Of Supercritical Carbon Dioxide Extraction Biology Essay Essay Optimization Of Supercritical Carbon Dioxide Extraction Biology Essay Essay Optimization utilizing response surface methodological analysis for the extractions of phenoplasts from Citrus hystrix foliage was carried out by supercritical fluid extraction. The effects of CO2 rate, extraction force per unit area and extraction temperature on output, entire phenolic content and Diphenyl-picrylhydrazyl-IC50 were evaluated and compared with ethanol extraction. Ethanol infusions and optimal SFE conditions were analysed with HPLC. Among the three variables studied, extraction force per unit area had the most important influence on the output, TPC and DPPH-IC50 of the infusions, followed by CO2 rate and extraction temperature. The optimal conditions of force per unit area, CO2 rate and temperature were at 267 bars, 18 g/min and 50oC, severally. The output, TPC and DPPH-IC50 obtained were 5.06 % , 116.53 milligram GAE/g infusion and IC50 of 0.063 mg/ml, severally. These values were moderately near to their opposite number of predicted ( p gt ; 0.05 ) . Better suppress ion and TPC were obtained utilizing SFE method whereas higher output and phenolic acids were observed with ethanol extraction. The nerve-racking life manner and less balanced nutrient consumption globally partly due to high concentrations of free lipid groups, both in nutrient ( in vitro ) and in vivo after nutrient consumption has given to the demand to look at antioxidants as a functional ingredient in nutrient. Man-made antioxidants such as, butylated hydroxytoluene ( BHT ) , butylated hydroxyanisole ( BHA ) , tertiary- butyl hydro-quanone ( TBHQ ) and propyl gallate ( PG ) , are conventional nutrient antioxidants. Due to safety issues, consumer concerns and increasing regulative examination ( ( Jamilah et al. , 2009 ; Shahidi. , 1997 ) refering man-made antioxidants, the possibility of natural antioxidants as options is sharply researched. The foliages of Citrus hystrix, known locally as, Limau purut, is used in many Malayan and South-East Asiatic part local dishes and medicative readyings. C.hystrix as a possible new beginning of natural antioxidant was reported by Jamilah et Al. ( 1998 ) , Ching and Mo hamed ( 2001 ) , Jaswir et Al. ( 2004 ) , Idris et Al. ( 2008 ) , Chan et Al. ( 2009 ) and Butryee et Al. ( 2009 ) . All infusions were extracted utilizing the conventional dissolvers such as, ethyl alcohol, methyl alcohol, propanone and H2O. To bring forth infusions of high phenolic content and rich in antioxidants from C. hystrix foliages, requires high extraction efficiency influenced by factors such as atom size, extraction methods, solvent type, solvent concentration, solvent-to-solid ratio, extraction temperature, force per unit area and clip ( Banik et al, 2007 ; Lang et al. , 2001 ; Pinelo et al. , 2005 ; Silva et al. , 2007 ) . Steam distillment and organic dissolver extraction utilizing infiltration, maceration and Soxhlet techniques are conventionally used for the extraction of bioactive compounds from works beginnings. They are non efficient and economical and this can be overcome by utilizing the supercritical C dioxide ( SC-CO2 ) procedure ( Bimakr et al. , 2009 ) . Carbon dioxide ( critical temperature, force per unit area and denseness ~ 31.18 oC, 72.0 saloon ; 0.47 gcm-3, severally ) is safe, residue free, non-flammable, in expensive and environmentally- friendly ( Pyo and Oo, 2007 ) . The optimisation of supercritical fluids for the extraction of natural antioxidants and phenolic compounds from the foliages of C.hystrix has non been reported. Hence, this survey was carried out with the aim of optimising the extraction of the antioxidant and phenolic acids from the foliages of C. hystrix utilizing supercritical C dioxide ( SC-CO2 ) fluid extraction by changing and/or repairing known variables associated with the extraction techniques. 2 Materials and Methods 2.1 Reagents used Folin-Ciocalteu Reagent ( FCR ) and 1,1-Diphenyl-2-picryl-hydrazyl ( DPPH ) were purchased from Sigma ( St Louis MO USA ) . Carbone dioxide, ( pureness 99.99 % ) , incorporating in a Carbone dioxide dip tubing cylinder, was purchased from Malayan Oxygen ( MOX ) , Malaysia. Absolute ethyl alcohol ( 99.4 % , analytical class ) , the qualifier for SC-CO2 procedure, acetonitrile and methyl alcohol ( HPLC class ) as the nomadic stage for HPLC and phenolic acids criterions ( vanillic acid, syringic acid, p-coumaric acid, M-cumeric, trans cinnamic acid, benzoic acid, Gallic acid and sinapic acid ) were purchased from Fisher Scientific Chemical ( Loughborough, England ) . All other chemicals used were either analytical or HPLC class. 2.2 Preparation of Sample The foliages of C. hystrix were obtained from Pasar Borong, a whole sale market at Puchong, Selangor, Malaysia. Upon reaching at the research lab, foliages were sorted, washed under running tap H2O, oven dried at 40A °C for 24h and stored at ambient temperature off from the visible radiation. The dried foliages were land merely before extraction in a liquidizer ( MX-335, Panasonic, Malaysia ) for 10s to bring forth a pulverization with an approximative atom size of 0.5mm ( Bimak et al. , 2009 ) . 2.3 Solvent Extraction The phenolic compounds in the C. hystrix leaves powder were extracted harmonizing to Jamilah et Al. ( 1998 ) with little alterations. The first measure involved soaking the pulverization in 95 % ethyl alcohol for 24h at 50oC at an ethyl alcohol to flick ratio of 10:1 ( v/w ) . The petroleum infusion was so filtered and concentrated by vaporizing at 40oC in the rotary evaporator ( Eyela, A-1000S, Japan ) .When the ethyl alcohol was evaporated off the concentrated infusion was transferred into brown glass bottles, flushed with N and kept at 25oC until usage. The extraction was carried out in triplicate 2.4 Supercritical Carbon Dioxide ( SC-CO2 ) Extraction Supercritical C dioxide ( SC-CO2 ) fluid extraction utilizing the supercritical fluid extractor ( ABRP200, Pittsburgh, PA, USA ) , with a 500 milliliter extractor vas attached, was carried out harmonizing to Bimark et Al. ( 2009 ) with little alterations. The flow rate of CO2 and modifier, extraction temperature, force per unit area and clip were adjusted utilizing ICE package coupled with the supercritical fluid extractor. The liquid CO2 was pressurized and heated to the coveted force per unit area and temperature with the assistance of force per unit area pump ( P-50, Pittsburg, PA, USA ) to make the supercritical province prior to go throughing it into the extraction vas. Absolute ethyl alcohol was used as the qualifier to better the extraction of phenoplasts from C.hystrix foliages and fixed at a flow rate of 3 milliliters / min for all experimental processs. The continuance of the inactive extraction clip was fixed at 30 min, while the dynamic extraction clip was changeless at 9 0 min. Fifty gms of C. hystrix foliages ( pulverization ) was assorted with 150g glass beads ( 2.0 millimeter in diameters ) to systematize the flow rate and the mixture was placed in the extractor vas. The extraction was so performed under assorted experimental conditions as generated by the response surface methodological analysis ( RSM ) design. EtOH was removed from the infusions by vacuity vaporization utilizing a rotary evaporator ( Eyela, A-1000S, Japan ) at 40 A °C. The infusions were collected in the unit of ammunition bottle flask ( warped with aluminum foil to minimise light exposure and therefore oxidization ) and so placed in the oven at 40A °C for 30 min before being transferred into desiccators for concluding changeless weight. Infusions were transferred into brown glass bottles, flashed with N and stored in a deep-freeze of -25A °C until farther analysis. The extractions were carried out in extras. 2.5 Determination of Total Phenolic Content ( TPC ) The entire phenolic content of C.hystrix foliage infusions was determined utilizing the Folin-Ciocalteu reagent harmonizing to the method described by Singletone et Al. ( 1999 ) . An aliquot of the infusion ( 0.5mL ) was put in 0.5mL of Folin reagent, under dim visible radiation before 10mL ( 7 % ) of Na carbonate was added. The mixture was so left in the dark for 60A min. The optical density of the mixture was measured against EtOH ( space ) at 725A nanometers by utilizing a UV-Visible spectrophotometer ( UV-1650PC, Shimadzu, Kyoto, Japan ) . The standardization equation for Gallic acid, expressed as Gallic acid equivalent ( GAE ) in mg/g infusion, was y = 0.0064x + 0.0093 ( R2 = 0.9972 ) . 2.6 Determination of Free Radical Scavenging Activity Free extremist scavenging activity of C.hystrix foliage infusions was measured harmonizing to the process described by Ramadan et Al. ( 2006 ) with little alterations. A 0.1A milliliter aliquot of toluenic sample solution at different concentrations was added with 0.39A milliliters of fresh toluenic 1,1-Diphenyl-2-picryl-hydrazyl ( DPPH ) solution ( 0.1A millimeter ) . Triplicates were carried out for each concentration. The mixtures were shaken smartly and left in the dark for 60A min and optical density was read against pure methylbenzene ( clean ) at 515A nanometers utilizing a UV-Visible spectrophotometer ( UV-1650PC, Shimadzu, Kyoto, Japan ) . The free extremist scavenging activity of infusions was calculated as follows: % Inhibition = ( [ Acontrol-Asample ] /Acontrol ) *100 Where AcontrolA =A optical density of the control reaction ( incorporating all reagents except samples ) ; AsampleA =A optical density of the trial compound. Determination of IC50 in this trial was defined as the concentration of the infusion that was able to suppress 50 % of the entire DPPH groups. IC50 of the sample was expressed in mg/mL and calculated through the insertion of additive arrested development analysis ( Brand-Williams et al. , 1995 ) 2.7 Determination of Phenolic acids The phenolic acids of the C.hystrix foliage infusions that were obtained from the optimal SC-CO2 conditions ( optimum of output, TPC and DPPH-IC50 ) were analysed by a high-performance liquid chromatography ( HPLC ) , [ Agilent Technologies 1200 series theoretical account, 76337 Waldbronn, Germany ] equipped with Diode Array Detector ( DAD ) , and sensing at 254nm. The HPLC parametric quantities were modified from Anderson et Al. ( 1983 ) . The column temperature used was 30A °C at a maximal temperature 35A °C and the column used was Crespak RP C18S RP C18 ( 150mm L* 4.6mm ID, JASCO ) . The dissolvers were of HPLC class ( Fisher Scientific Chemical, Loughborough, England ) . All dissolvers were filtered through a cellulose nitrate membrane filters ( 0.45 A µm ) . Flow rate of nomadic stages used were 1.5ml/min for 25 % acetonitrile in formic acid-water ( 0.5:99.5 ) , run isocratically. The infusions were foremost filtered through 0.2 A µm nylon ( NYL ) filter, ( Whatman ) for the remotion of drosss and unwanted compounds. The injection volume used was 20A µL with extras for each of the SC-CO2 optimal conditions and ethanol infusions. The criterions used were vanillic acid, syringic acid, p-coumaric acid, M-cumeric, trans cinnamic acid, benzoic acid and sinapic acid ( Fisher Scientific Chemical Loughborough, England ) . Designation and quantification of phenolic acids in the infusions were based on the standard curves of the criterions every bit good as their extremums keeping times. 2.8 Experimental design and statistical analysis Response surface methodological analysis ( RSM ) was used to find the optimal conditions for the output, TPC and DPPH-IC50 in C.hystrix foliage infusions. The experimental design and statistical analysis were carried out utilizing the statistical package ( MINITAB let go of 14 ) . Central composite design was chosen to measure the joint consequence of three independent variables CO2 rate, extraction temperature and force per unit area, coded as X1, X2 and X3, severally. The minimal and maximal values for CO2 rate were set at 15 and 25 g/min, extraction temperature between 40 and 60 oC and force per unit area between 100 and 300 bars. The dependent values were yield, TPC and DPPH-IC50. For optimisation, output and TPC were maximized to accomplish highest values and loswest value for DPPH-IC50. The whole design consisted of 20 combinations including six replicates of the Centre point ( Table 1 ) ( Myers A ; Montgomery, 2002 ) . The ANOVA tabular arraies were generated and the consequence and arrested development coefficients of single linear, quadratic and interaction footings were determined. The significances of all footings in the multinomial were analyzed statistically by calculating the F-value at a chance ( P ) of 0.001, 0.01 or 0.05. The statistically found non-significant ( p gt ; 0.05 ) footings were removed from the initial theoretical accounts and merely important ( p lt ; 0.05 ) factors were involved in the concluding reduced theoretical account. It should be noted that non-significant additive footings were kept in the decreased theoretical account in instances where their quadratic or interaction footings were important ( p lt ; 0.05 ) ( Mirhosseini et al. , 2009 ) . Experimental informations were fitted to the undermentioned 2nd order multinomial theoreti cal account and arrested development coefficients were obtained harmonizing to the generalized second-order multinomial theoretical account proposed for the response surface analysis, given as follows Where I?0, I?i, I?ii, I?ij were arrested development coefficients for intercept, additive, quadratic and interaction footings, severally. Eleven and Xj were coded values of the independent variables, while K equaled to the figure of the tried factors ( k=3 ) . 3. Consequences and Discussion 3.1 Response Surface Methodology ( RSM ) Analysiss 3.1.1 Model Fitness Based on the scopes set for the identified parametric quantities, 20 trails of each parametric quantity, including six replicates of the Centre points that influence Yield, TPC and DPPH-IC50 were selected. In this survey, the lower and upper values for the variables were set at +alpha ( +I ±=1.633 ) and -alpha ( -I ±=1.633 ) and so all the factor degrees were chosen within the bounds that were practical with SFE ( above critical tempHYPERLINK hypertext transfer protocol: //en.wikipedia.org/wiki/Critical_temperature eratureA of 31A °C andA critical pressureA of 72A saloon ) and desirable. The experimental and predicted values for responses under the different combinations of extraction conditions via SC-CO2 extractions were as in Table ( 1 ) . The consequences indicated that output, TPC and DPPH-IC50 obtained, ranged from 0.4- 5 % , 15 128.9 milligram GAE/g infusion and 0.065 0.300 mg/ml, severally. By using multiple arrested development analysis, relationships between the tried parametric quantities and the responses were explained in equations 2, 3, and 4 for output, TPC and DPPH-IC50, severally. The fittingness of response map and experimental information was evaluated from the one-dimensionality, quadratic and arrested development coefficients of independent variables as shown in Table 2. The ANOVA of arrested development theoretical account showed that the theoretical accounts were perceptibly important due to the highly low chance value ( P lt ; 0.001 ) . The coefficient of finding ( R2 ) and significance of deficiency of fittingness was farther evaluated to look into the fittingness and theoretical account adequateness. The R2 equal to the integrity or a†°? 0.8, is desirable. R2 values for the arrested development theoretical account of output, TPC, and DPPH-IC50, were 0.935, 0.95, and 0.96, severally, which were close to 1 ( Table 2 ) . Therefore, bespeaking that t he predicted 2nd order multinomial theoretical accounts fitted good with the system. The values of adjusted R2 ( corrected value for R2 after the riddance of the unneeded theoretical account footings ) of output, TPC and DPPH-IC50 were besides really high, therefore proposing the high significance of the theoretical account ( 0.897, 0.92 and 0.93 ) . The coincident addition of both R2 and adjusted R2 plus the absence of any deficiency of tantrum ( p gt ; 0.05 ) in our information has proved its credibleness and theoretical account adequateness. The multiple arrested development consequences and the significance of arrested development coefficients yield, TPC and DPPH-IC50 theoretical accounts were as shown in Table 3. It could be observed that both the linear and quadratic term of all parametric quantities significantly ( p lt ; 0.05 ) effected the output, TPC and DPPH -IC50, nevertheless, CO2 rate did non significantly impact the DPPH-IC50 where temperature consequence on TPC was merely important in quadratic mode to stay in the theoretical account ( Table 3 ) . The undermentioned arrested development equations showed the concluding reduced theoretical accounts fitted for the parametric quantities and their responses. Yield= 3.33 + 0.142 X1 + 0.164X2 + 0.00735X3- 0.00669X12 0.00218 X22 0.000025 X32 Eq ( 2 ) TPC = 909 + 25.4 X1 + 25.6 X2 + 1.54 X3 0.668 X12 0.250 X22- 0.00278 X32 Eq ( 3 ) DPPH-IC50= 0.604 X2 0.0177 X3 + 0.00559 X22 + 0.000031 X32 Eq ( 4 ) 3.1.2 Verification of theoretical accounts The rightness of the response surface equation was tested by the rating of experimental and predicted values from the reduced response arrested development theoretical accounts. A close understanding between the experimental and predicted values ( Table1 ) was noted. No important ( p gt ; 0.05 ) difference was observed between those values, proposing the equal fittingness of the response equations. 3.2 Influence of Pressure, CO2 Rate and Temperature on SC-CO2 Extraction efficiency Figure 1 ( a ) showed the 3-dimensional response surface secret plans by showing the response in the map of two factors and maintaining the temperature at its in-between degree ( 50oC ) . It showed a higher output in the part of extraction force per unit area between 190 to 300 bars and at CO2 rate of 12 to 17g/min. Both extraction force per unit area and CO2 rate exhibited important additive and quadratic effects on output as shown Table ( 3 ) . The output was optimum at approximately 14.8g / min CO2 flow rate and at force per unit area of 320 bars. Eextraction force per unit area was more influential than CO2 rate as reflected by its higher linear and quadratic coefficients ( I?3=0.65819 ; I?33 = -0.25168 ) compared to the latter ( I?1= -0.35060 ; I?11=-0.16731 ) . In supercritical fluid extraction ( SFE ) , increased force per unit areas consequence in, increased solvent denseness and solvent power of fluid which may take to higher extraction outputs, on the other manus, increased pure CO2 rate under SFE is a good dissolver for lipotropic compounds ( non- polar ) but is hapless for phenoplasts ( polar ) ( Martinez, 2007 ) . Therefore, modifier ( ethyl alcohol ) was used to better the extraction of phenoplasts from C.hystrix foliages. Figure 1 ( B ) showed the effects of extraction force per unit area and extraction temperature on output at changeless CO2 rate of 20 g/min. Extraction force per unit area displayed a really important ( p lt ; 0.001 ) on the output in additive and quadratic mode as besides shown in Table ( 3 ) . At force per unit area of a†°?140 and temperature non transcending 47oC output increased, nevertheless with farther addition in the temperature the output showed a lessening which is most likely due to the reduced denseness of CO2. The relationship of CO2 rate and extraction temperature with output was plotted in Figure 1 ( degree Celsius ) . Both the parametric quantities exhibited important additive and quadratic consequence ( P lt ; 0.05 ) on output. The output increased quickly with diminishing CO2 rate up to 13 g/m and this followed by a little lessening thenceforth. By uniting all the consequences presented in Figure 1, it was obvious that extraction force per unit area had the most critical impact on output of the infusion followed by CO2 rate and extraction temperature. 3.3 Entire Phenolic Content ( TPC ) The TPC of the infusion was as shown in Figure 2. Depending on the force per unit area, temperature and CO2 rate, the TPC of the infusion ranged from 15.0 to 128.9 milligrams GAE/g infusion. No available literature study could be be used for comparing for the SC-CO2 extraction method ; nevertheless, Idris et Al. ( 2008 ) reported that TPC of the infusions was about 103.2 milligrams GAE/g infusion which was somewhat lower than our EtOH extracted TPC ( 112.7 milligram GAE per g infusion ) . Moderate degrees of the selected independent variables of SC-CO2 infusions ( run order 7, 10, 12, and 17, Table 1 ) reflected higher TPC of the C.hystrix foliage infusions than our EtOH extraction every bit good as Idris s ; this may hold something to make with possible partial debasement of the extracted compounds due to long extraction clip when conventional extraction methods are to be used. With SC-CO2 method the extraction clip ( 90 min ) was unusually shorter than that of EtOH extraction ( gt ; 20 H ) . 3.4 Free Radical Scavenging Activity Figure 3 demonstrated the consequence of temperature and force per unit area on the scavenging belongings of the C.hystrix leave infusions. The antioxidant activity of the infusions, determined by the IC50 of extremist scavenging belongingss of diphenylpicrylhydrazyl ( DPPH-IC50 ) , was found to be high at mean degree of temperature and comparatively increased phases of force per unit area i.e. DPPH-IC50 of the infusions bit by bit decreased with the addition of extraction temperature and force per unit area up to 50 A °C and 314 bars, severally to accomplish optimal value of IC50 at 0.0585 before it began to increase. The lesser the IC50, the stronger activity is the corresponding affair ( Mariod et al. , 2010 ) . Under the assay conditions employed here, the IC50 of BHA and I ±-tocopherol as a positive controls were 0.023mg/ml and 0.031mg/ml, severally, among the infusion run order 12, 9, and 16 ( table 1 ) possessed greater DPPH extremist scavenging activities with the lower I C50 values of 0.065, 0.08 and 0.085mg/ml, severally. This was in understanding to the findings of Idriss et Al. ( 2008 ) , where the activity of BHA was found to be higher than the sample. Compared to conventional solvent extraction method with the IC50 of 0.250 mg/ml ( Table 1 ) , it can be observed that SC-CO2 infusions demonstrated noteworthy DPPH radical-scavenging activity unusually greater than that of traditional extraction method. The IC50 values for CLE extracted by SC-CO2 ranged from 0.065 0.300 mg/ml depending on force per unit area and temperature where an addition in the force per unit area comparatively resulted in an addition in its antioxidant capacity. 3.5 Identification and Quantification of Phenolic Acids of the infusions. Out of seven standard phenolic acid solutions assorted, six have been detected in dissolver and supercritical C dioxide extraction of the infusions ( table 4 ) . By quantifying the sum of phenolic acids in the infusion, a considerable fluctuation between EtOH and SC-CO2 extraction was observed. Higher recovery of phenolic acids than that of SC-CO2 extraction was found utilizing 95 % EtOH as shown in Table ( 4 ) . The figure of polar map groups, e.g. hydroxyl groups, may hold influenced volatility of the solutes therefore finding their optimal extractability with SC-CO2 ( Lang and Wai, 2001 ) . For illustration, ( Stahl and Glatz, 1984 ) successfully extracted steroids with three hydroxyl groups below 300 bars but failed to pull out those steroids dwelling of four hydroxyl groups, or three hydroxyls and one acid group, or one phenolic hydroxyl with two other hydroxyl groups. Despite the difference in measure, the type of phenolic acids bing in the infusions for both EtOH and SC-CO2 ex traction methods remained changeless. Trans-cinnamic, M-coumeric and Vanillic acids represented as the prevailing phenolic acids, while P-coumeric, Benzoic and sinapic acids reasonably existed in the infusions ( Table 4 ) . 3.6 Decision The optimal conditions of force per unit area at 265 bars, temperature at 50oC and CO2 rate at 18 g/min was needed for higher SC-CO2 extraction of output, TPC and DPPH-IC50 of C.hystrix leave infusions. Of the three independent variables studied, extraction force per unit area was the most important factor act uponing on output, TPC and DPPH-IC50, flowed by CO2 rate and extraction temperature. Higher sums of output and phenolic acids than SC-CO2, was found in solvent extraction. Nevertheless, SC-CO2 extracts exhibited high quality in antioxidant activity measured by IC50 of 1,1-Diphenyl-picrylhydrazylA ( DPPH ) and entire phenolic content ( TPC ) . Even though some good consequences was achieved with the traditional EtOH extraction, supercritical CO2 extraction showed faster and better extraction of C.hystrix foliages. Therefore, the green engineering, reclaimable CO2 could be an alternate method of extraction for superior antioxidants from C.hystrix foliages. Recognition The writers appreciate and thankful for the fiscal support received from the RMC, the University Putra Malaysia for this survey.