
 o. ficus indica is a species of cactus that belongs to the cactaceae family , the order centrospermae , and the genus opuntia .
this plant is associated with the semiarid zones of the world mainly in africa , mediterranean countries , southwestern united states , northern mexico , and other areas . in tunisia ,
the main studies on the opuntia fruits were the chemical analysis of pulp , skin , and seeds .
the proximate composition of o. ficus indica l. has been investigated [ 1 , 3 ] .
seeds constitute about 1015% of edible pulp and are usually discarded in the food industry as waste after pulp extraction .
edible oil can be obtained from prickly pear seeds in yield of 5.813.6% [ 1 , 46 ] .
the oil has a high degree of unsaturation in fatty acids ( 82% ) with a linoleic acid content of 73.4% . according to the investigation of ramadan and mrsel   using a chloroform / methanol as extraction solvent procedure of total lipids in seeds oil of cactus pear ( o. ficus indica l. ) , linoleic acid was the dominating fatty acid ( 53.5% ) , followed by palmitic ( 20.1% ) and oleic acids ( 18.3% ) .
two spices of prickly pear from tunisia , o. ficus indica and o. stricta , were investigated for fatty acid composition seeds by ennouri et al .   with an exceptional level of linoleic acid ( up to 70% ) ; the oils were extracted with hexane in a soxhlet extractor . moreover
, no data has been mentioned about the difference of fatty acids composition in the two spines ( wild ) and thornless ( cultivated ) forms of tunisian o. ficus indica seeds .
the oil obtained by mechanical separation processes was of high quality , but , in most cases , the yield was lower .
hexane extraction achieved almost complete recovery of the oil , and the resulting oil contains traces of solvent ; however , the solvent was dangerous and unacceptable as it was quite harmful to human health and the environment , which may restrict its use in food , cosmetic and pharmaceutical industries .
supercritical fluid extraction ( sfe ) with sc - co2 represents an alternative method for the extraction of oils from natural products and has received considerable attention .
sfes have been used as solvents for a wide varieties of applications such as essential oil extraction .
sfe use increases in the last years because of legal limitations of solvent residues , and solvents make this process more economical .
the co2 remained the most commonly used fluid for sfe applications thanks to its low critical parameters ( tc = 31.1c ; pc = 74 bar ) , it has non - toxic , nonflammable properties , and it was available in high purity with low cost .
the oils obtained by sc - co2 extraction were of outstanding quality , and the yields were comparable with those obtained with organic solvent extraction method .
the co2 can be used in many extractions and reactions and has been recognized as earth - compatible solvent . therefore , sfe may serve as a promising technology in food and pharmaceutical processing .
the objective of the present research was to illustrate and to compare between the sc - co2 and the soxhlet n - hexane profiles fatty acids of two forms of tunisian o. ficus indica seeds .
research on oil extracted from o. ficus indica seeds showed that this material was a potential source of food and feed .
moreover , these oils have several applications in pharmacology , medicinal , and cosmetic fields .
the fact that extraction with sc - co2 enables to have pure oil free of solvent makes it possible to better enhance these oils safety .
fresh and mature pears fruits of two forms of tunisian o. ficus indica were collected in summer 2009 .
the spiny wild form was from the region of al - ala in the centre of tunisia located at 35 36  n ( north ) latitude , 9 34  e ( east ) longitude , and 450  m ( meter ) altitude .
the thornless cultivated form was from local pilot cultivar of bou argoub region in the north east of tunisia located at 36 32  n latitude , 10 33  e longitude , and 62  m altitude .
the seeds were recovered , after mixing the pulp and filtration through a 2  mm sieve .
the seeds were washed thoroughly with bidistilled water , dried in an oven at 35c for 48  h. the seeds were ground by a domestic coffee grinder ( seb prep line ) just before extraction .
the moisture content of the seeds was determined by drying 50  g of seeds at 70c to a constant weight in an oven for 72  h. reference standards of caprylic acid ( c8  :  0 ) , capric acid ( c10  :  0 ) , lauric acid ( c12  :  0 ) , myristic acid ( c14  :  0 ) , palmitic acid ( c16  :  0 ) , palmitoleic acid ( c16  :  1 ) , heptadecanoic acid ( c17  :  0 ) , stearic acid ( c18  :  0 ) , oleic acid ( c18  :  1 ) , linoleic acid ( c18  :  2 ) , linolenic acid ( c18  :  3 ) , arachidic acid ( c20  :  0 ) , eicosenoic acid ( c20  :  1 ) , eicosadienoic acid ( c20  :  2 ) and behenic acid ( c22  :  0 ) that had a minimum purity of 99% , sodium methoxide , methanol ( hplc grade ) , hexane , and sulfuric acid ( 1n ) were purchased from sigma aldrich ( usa ) . the sc - co2 extractions were carried out with a high purity carbon dioxide ( 99.95% ) supplied by messer ( france ) . the extractions were conducted in a cylindrical stainless steel extractor ( 300  mm  23  mm , internal volume : 125  cm ) and a three cyclonic separators type . the ground seeds ( 50  g ) of o. ficus indica ( spiny : 92.49%thornless : 93.64% dry matter ) were loaded into steel cylinder , filled into the extractor vessel and extracted at temperatures of 35 , 40 , 45 , 50 , 60 , and 70c ( spiny ) and 40c ( thornless ) , 180 bar and the co2 flow rate of 15  mls for 135  min .
the desired extraction temperature was achieved by heating the extraction vessel , and the temperature was monitored by a thermocouple and regulated by a controller .
the extraction pressure was controlled by a back pressure regulator , and the flow rate of co2 was controlled by manual adjustment of a needle - metering valve .
the extracts were collected in one tube throughout the 135  min , and the yields were calculated ( for each temperature ) .
the final volume of extract was analyzed using gc - fid to determine the fatty acids profile as described below .
soxhlet n - hexane extraction was carried in triplicate for 30  g of ground seed of o. ficus indica by 250  ml of n - hexane ( 99% ) for 8  h. a rotary evaporator was used for the efficient removal of solvent ( n - hexane ) from samples by evaporation at 68c .
the extracts were stored in a glass jars in the deep freeze ( 20c ) pending for further analysis .
the extract samples were methylated by sodium methoxide according to the method of carreau and dubacq .
the methylation of fatty acids occurred in the presence of 0.5  ml sodium methoxide in methanol 3% and 2  ml of hexane . a known amount of methyl ester heptadecanoic acid ( c17  :  0 ) was used as internal standard to quantify the fatty acids .
after stirring during 60 seconds and decantation for 2  min , the reaction was neutralized by the addition of 0.2  ml of h2so4 ( 1n ) .
finally , 1.5  ml salt water ( 10% nacl ) was added to allow the separation of two phases .
the organic higher phase containing dissolved methyl esters in hexane was retained . after a concentration under nitrogen current ,
the methyl - esterified fatty acids were analyzed by gc - fid to determine fatty acid constituents .
the gc analysis was performed by using a hewlett - packard ( hp ) 6890 series equipped with a flame ionization detector ( fid ) and a polar capillary column : hp innowax ( 30  m length  0.25  mm internal diameter and 0.25  m film thickness).the column temperature was initially at 120c ( held for 1  min ) and then increased to 200c at 15c  min and finally increased to 250c at 2c  min .
the operational conditions were injector temperature 250c ; detector temperature 275c ; carrier gas was nitrogen at a flow of 1.7  ml  min with a split ratio of 40  :  1 .
the fatty acids were identified by comparing their chromatograms to those of pure standards analyzed under the same conditions .
the hp chemstation software allowed the end of each analysis to provide the surface of each peak and its percentage .
the standard deviations of the extractions yields and the fatty acid constituents were based on triplicate measurements .
in this study , we intend to compare the efficiency of the sfe by sc - co2 extract process with the solvent extraction by soxhlet n - hexane .
efficiency is based on the extraction yield ( quantity ) with its relationship to composition of fatty acids ( quality ) .
various parameters potentially influenced the sc - co2 extraction process , and the first step of sc - co2 extract knowledge was to optimize the temperature at fixed conditions : pressure = 180 bar , co2 flow rate = 15  ml  s , and time = 135  min , to obtain a better yield of extract .
the experimental results of sc - co2 extract yield of the spiny form were calculated and illustrated in figure 1 .
the yields ( y ) of oil defined on 100  g dry seeds of o. ficus indica basis and the total extraction yield were calculated as follow : 
 ( 1)y%=[(m2m1)100]m , 
 where m2 was the mass of glass trap with extract , m1 was the mass of empty glass trap , and m was the initial mass of seeds in the extractor .
the extract collected in the cold trap at atmospheric pressure consisted of a yellow solid mass and water . according to figure 1 ,
the extraction yields first increased from 1114.8  mg  34.5/50  g of ground seeds at t = 35c , reached a maximum value at 40c ( 1702.4  mg  40.1/50  g of ground seeds ) , and then decreased at higher temperatures 70c ( 122.3  mg  6.6/50  g of ground seeds ) .
extraction with sc - co2 at t = 40c versus time of spiny and thornless forms of o. ficus indica seeds showed , respectively , an extraction percentage of 3.4% ( 1702.4  mg  40.1/50  g of ground seeds ) and 1.94% ( 970.7  mg  49/50  g of ground seeds ) ( figure 2 ) . in the same condition of our extraction ( t = 40c and fixed parameters ) , the sc - co2 extraction yields depended on the variability of plant material ( seeds ) of two opuntia forms . at t
= 40c , the extraction yield was gradually increased with increasing time , for the two forms of o. ficus indica , the majority of the extract oil was recovered at an interval time of 15 to 75  min , and the extraction yields were 79.42% and 78.82% , respectively , for the spiny and the thornless form .
the oils soxhlet n - hexane extract yield for the wild spiny form ( 10.32% ) and cultivated form ( 8.91% ) of tunisian o. ficus indica were higher than the sc - co2 extract .
the performance of sc - co2 extraction was relatively low compared to soxhlet n - hexane extract and other extraction methods that exist in the literature , the extraction solvent chloroform / methanol recovers 9.88% . according to snyder et al .
, moisture content between 3% and 12% has very little effect on the extractability of oils from seeds with sc - co2 . in the literature
, it was reported that o. ficus indica contain a good quality of edible oil [ 4 , 5 , 15 , 16 ] such as fatty acids .
the fatty acid profiles of two forms of o. ficus indica seeds were analyzed by gc - fid , and the results are summarized in table 1 .
the results enabled us to identify and compare the constituent fatty acids of total lipids extracted from seeds of the o. ficus indica ( table 1 ) .
the percentage of each compound was calculated from the peak areas given by the gc- fid .
the sc - co2 extraction of fatty acids profile showed eleven peaks ( figure 3(a ) ) ; the soxhlet n - hexane extract of fatty acids profile illustrated only 8 peaks ( figure 3(b ) ) .
the main components for two forms were linoleic acid : sc - co2 ( spiny 57.60% , thornless 59.98% ) and soxhlet
n - hexane ( spiny 57.54% , thornless 60.66 ) , oleic acid : sc - co2 ( spiny 22.31% , thornless 22.40% ) and soxhlet
n - hexane ( spiny 25.28% , thornless 20.58% ) and palmitic acid : sc - co2 ( spiny 14.3% , thornless 12.92% ) and soxhlet n - hexane ( spiny 11.33% , thornless 31.08% ) . according to the ramadan and mrsel work
, solvent extraction using a chloroform / methanol showed that palmitic acid was the second compound after linoleic acid in terms of amount .
reported that the oils extracted with hexane using a soxhlet extractor , linoleic acid contents were higher than our results ( 70.3% ) , oleic acid was very much lower ( 16.8% ) and palmitic acid ( 9.3% ) .
cokuner and tekin   reported that the seed oil extracted from turkish o. ficus indica ( mersin ) with petroleum ether by soxhlet extractor , palmitic acid content ( 12% ) , and linoleic ( 52% ) .
the observed difference is probably due to extraction process and the maturation stage of fruits .
cokuner and tekin   suggested that there was an increase in saturated fatty acid content towards the end of fruit maturation .
the solvent extraction of o. ficus indica seeds has been studied at different conditions previously , but , to the best of our knowledge , there is not any research about the sc - co2 extraction of tunisian o. ficus indica .
the results of sc - co2 extract showed that the saturated fatty acids composition was slightly more important for wild spiny form ( 14.30% for palmitic acid and 3.12% for stearic acid ) than the cultivated thornless form ( 12.92% for palmitic acid and 2.38% for stearic acid ) .
in contrast , the soxhlet n - hexane extract of palmitic acid was most important for the thornless form ( 13.08% for ) than the spiny form ( 11.33% ) .
for the extraction process , the results showed that the oils were found to be highly unsaturated ( us = 4.445.25 ) .
the soxhlet n - hexane extract showed an us more important for the spiny form ( 5.22 ) , the soxhlet n - hexane extract allowed a better extraction of unsaturated fatty acids for the wild form .
the sc - co2 extract showed an us more important for the thornless form ( 5.25 ) , and the sc - co2 extract allowed a better extraction of unsaturated fatty acids for the cultivated form ( table 1 ) . compared to ramadan and mrsel results , the sc - co2 profiles fatty acids of tunisian o. ficus indica seeds showed the richness of this profile with other compounds such as myristic , arachidic , eicosenoic , eicosadienoic , and behenic fatty acid . compared to our soxhlet n - hexane extract results ,
the sc - co2 profiles fatty acids showed the richness of small proportion compounds such as eicosenoic , eicosadienoic , and behenic fatty acid .
( cactaceae ) by supercritical co2 revealed other compounds found in low percentage such as vaccenic and margaric fatty acid .
these results showed that the supercritical co2 extraction ensures a better extraction of small proportion compounds .
the composition of the spiny tunisian o. ficus indica seeds oils revealed the similar fatty acid constituents at different temperature ( table 2 ) .
the temperature of sc - co2 extraction seemed has not effect on the selectivity of fatty acids . the increasing temperature from 40c ( u / s = 4.44 ) to 70c ( u / s = 4.14 ) tended to decrease the sc - co2 extraction yield but not affect the solubility of fatty acids .
the sc - co2 and soxhlet n - hexane extraction was applied to the extraction of oil seeds from wild and cultivated tunisian o. ficus indica .
the oil yields were higher by soxhlet n - hexane extraction , and the wild form gave the best results .
the fatty acids profiles were investigated , and linoleic acid was the major compound followed by oleic acid and palmitic acid for the two forms and for the two extraction process .
the sc - co2 extract allowed a better extraction of unsaturated fatty acids for the cultivated form , and oils revealed a better extraction of small proportion compounds .
in contrast , the soxhlet n - hexane extract allowed a better extraction of unsaturated fatty acids for the wild form , and the fatty acids profile did not identify some compound of lower proportion .
the low extractable compounds of fatty acids and the purity of the sc - co2 extract are giving a better quality of oil .
we can extend our research on the various parameters of supercritical extraction for optimizing better extraction efficiency . with a better extraction of small proportion compounds of fatty acids seed oils
, the sc - co2 process could be more advantageous than the soxhlet n - hexane .
the fatty acids seed oil would seem to have a potential use and be valuable in different areas of bioindustrial implementation , nutraceutical and pharmaceutical .
these results provide a better opportunity to improve and to enhance the natural heritage that characterizes the tunisian o. ficus indica .
 the fatty acids profiles of tunisian opuntia ficus indica seeds ( spiny and thornless form ) were investigated . 
 results of supercritical carbon dioxide ( sc - co2 ) and soxhlet n - hexane extract were compared . 
 quantitatively , the better yield was obtained through soxhlet n - hexane : 10.32% ( spiny ) and 8.91% ( thornless ) against 3.4% ( spiny ) and 1.94% ( thornless ) by sc - co2 extract ( t  =  40c , p = 180 bar , time  =  135  mn , co2 flow rate  =  15  mls1 ) . 
 qualitatively , the main fatty acids components were the same for the two types of extraction . 
 linoleic acid was the major compound , sc - co2 :  57.60% ( spiny ) , 59.98% ( thornless ) , soxhlet n - hexane : 57.54% ( spiny ) , 60.66% ( thornless ) , followed by oleic acid , sc - co2 :  22.31% ( spiny ) , 22.40% ( thornless ) , soxhlet 
 n - hexane : 25.28% ( spiny ) , 20.58% ( thornless ) and palmitic acid , sc - co2 :  14.3% ( spiny ) , 12.92% ( thornless ) , soxhlet n - hexane : 11.33% ( spiny ) , 13.08% ( thornless ) . 
 the sc - co2 profiles fatty acids showed a richness with other minority compounds such as c20:1 , c20:2 , and c22.the seeds oil was highly unsaturated ( us  =  4.445.25 ) , and the rising temperatures donot affect the selectivity of fatty acids extract by sc - co2 : us  =  4.44 ( t  =  40c ) and 4.13 ( t  =  70c ) . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion