PaPer Ital. J. Food Sci., vol. 28 - 2016 83 - Keywords: flaxseed, folch method, tocopherols, phenolic antioxidants, fatty acids, GC-MS - Variation in physico-chemical/analytical characteristics of oil among different flaxseed (Linum usittatissimum l.) cultiVars nazia yaqoob1,2, ijaz ahmed bhatti1, farooq anwar3,4*, muhammad mushtaq5 and william e. artz6 1Department of Chemistry, University of Agriculture Faisalabad, Pakistan 2Department of Chemistry, Govt College for Women University, Faisalabad, Pakistan 3Department of Chemistry, University of Sargodha, Sargodha, Pakistan 4Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam bin Abdulaziz University, Al-Kharj 11942, Saudi Arabia 5Department of Chemistry, Govt College University Lahore, Pakistan 6Department of Food Science and Human Nutrition, University of Illinois Urbana, Champaign, Illinois, Usa *Corresponding author: fqanwar@yahoo.com AbstrAct the present study evaluates and compares the proximate parameters of flaxseed, as well as the physicochemical characteristics of the extracted flaxseed oils of locally grown eight cultivars. the oil, protein, fiber and ash content of the seeds ranged from32.56-39.98%, 16.02-18.50%, 23.30- 26.88 and 3.20-3.60%, respectively showing considerable variation among cultivars. the quality attributes such as unsaponifiable matter, peroxide value, acid value, para-anisidine value, con- jugated dienes and trienes as well as tocopherols content of the tested flaxseed oils varied signif- icantly (p<0.05) among cultivars. the major tocopherol was γ-tocopherol (173.7 to 257.9 mg/L) followed by relatively low quantities of α-tocopherol (8-12 mg/L), while δ-tocopherol was not de- tected. α-Linolenic acid was found to be the principal fatty acid in the range of 44.51 to 54.87%, while the second major fatty acid present in the oils was oleic acid (21.05 to 30.96%). the varia- tion in the characteristics of oils among different cultivars observed during present investigation might be attributed to difference in genetic makeup and harvesting regimes of the flax plants. mailto:fqanwar%40yahoo.com?subject= 84 Ital. J. Food Sci., vol. 28 - 2016 INtrODUctION Flax (Linum usitatissimum L.) is a multi-pur- pose and economically important oilseed crop. Flaxseeds, which contain approximately 36 to 40% oil, have long been used in human diet and animal feed (tOUrE and XUEMING, 2010). by virtue of the presence of physiologically active components, which provide health benefits be- yond basic nutrition, flaxseed is often grouped as ‘‘functional food’’ (HAsLEr et al., 2000). His- torically, the oil extracted from flaxseeds, has been used as a basic component in the prepa- ration of paints or polymers, linoleum, varnish- es, inks and cosmetics (El-bELtAGI et al., 2007; ZHANG et al., 2008; JHALA and HALL, 2010). How- ever, during the past decade, there has been an increasing interest in the use of flaxseed oil to improve human health status due to its high nutraceutical potential (OOMAH, 2001; cHOO et al., 2007). the potential health benefits of flax- seed oil including reduction in serum choles- terol levels and decreased incidence of diabetes, breast and colon cancer can be ascribed to the presence of high-value antioxidants, tocophe- rols, lignans and essential fatty acids (Muir and Westcott, 2003; HOssEINIAN et al., 2006; cHOO et al., 2007; tOUrE and XUEMING, 2010). Flax- seed oil is one of the richest sources of unsatu- rated fatty acid, especially, linolenic acid (c18:3) with amount in the range of 50-60% of the total fatty acids present (FLAcHOWsKY et al., 1997). the agronomic conditions, such as the soil characteristics, agro-climatic conditions and the cultivar influence the unsaturated fatty ac- ids composition in flaxseed (DUAN et al., 2003). Moreover, studies indicate that the oil content, fatty acids profile and other physicochemical properties vary in the flaxseed crops grown in dif- ferent parts of the world (tAYLOr and MOrrIcE, 1991; WAKJIrA et al., 2004; HALL et al., 2006). traditionally, flaxseed has been grown in the Asian subcontinent for its oil; however in Pa- kistan its applications have been limited to in- dustrial uses. the present study mainly focused on the evaluation and comparison of proximate parameters of flaxseed, as well as the physico- chemical characteristics (such as the refractive index, density, iodine value, acid value, peroxide value, para-anisidine value etc.) and the com- position of tocopherols and fatty acids of flax- seed oil from different flax cultivars (Linum usi- tatissimum L.) indigenous to Pakistan to assess their nutritional value. EXPErIMENtAL Seeds and chemicals/reagents commercially available hybrid varieties (chandni, Ls-108, Ls-105, Ls-99 and Ls-29) of flaxseed used in this study were obtained from the Ayub Agricultural research Institute (AArI), Faisalabad, Pakistan and Ls-33, Ls-31 and Ls-13 were obtained from National Agricul- ture research center (NArc) in Islamabad, Pa- kistan. three different seed samples for each of the flaxseed variety were collected (8×3=24). All the reagents (analytical and HPLc grade) used were from Merck (Darmstadt, Germany) or sig- ma–Aldrich (buchs, switzerland). Pure stand- ards of tocopherols (α-tocopherol, γ-tocopherol, δ-tocopherol), and fatty acid methyl esters were obtained from sigma chemical co. (st. Louis, MO, UsA). Extraction of oil seeds of different flaxseed cultivars were crushed with a domestic electric grinder. the oil from the seeds was extracted using the Folch method (FOLcH et al., 1957). After oil extraction, the solvent was removed under vacuum in a ro- tary evaporator (Eyela, rotary Vacuum Evapora- tor N.N. series equipped with an aspirator and a digital water bath sb-651, 33 Japan) at 45°c. the extracted oil was then stored in a refrigera- tor at 4°c until used for analyses. Analysis of oil seed residues Proximate analyses of oilseed residues (meals), left after oil extraction, were completed accord- ing to standard methods. Protein contents (N- 6.25) were determined according to AOAc meth- od 954.01 (AOAc, 1990), using a Kjeldahl appa- ratus. the fiber contents were determined em- ploying IsO method 5983 (IsO, 1981). briefly, 2 g of finely ground defatted sample was tak- en and boiled with 250 mL of 0.255 M H 2 sO 4 , followed by the filtration and washing of insol- uble residues. the residues were then boiled with 250 mL of 0.313 M NaOH, filtered, washed, and dried. the dried residues were weighed and burnt at 600oc using a muffle furnace (Eyela, tMF-2100, tokyo, Japan) and the loss of mass was determined gravimetrically. Ash contents were determined by following IsO method 749 (IsO, 1977). two grams of meal was carbonized by heating on a gas flame and then ashed in an electric muffle furnace at 600oc, until a constant mass was achieved. Physicochemical properties of oil the extracted oils were analyzed for density, refractive index, peroxide value (PV), acid val- ue, iodine value (IV), saponification value, and unsaponifiable matter following AOcs meth- ods cc10a-25, cc7-25, cd1-25, cd8-53, F-9a- 44, cd3-25, and ca61-40, respectively (AOcs, 1997). the determinations of conjugated dienes (cD) and conjugated trienes (ct) were made us- ing a Hitachi U-2001 spectrophotometer. the oil samples were diluted with isooctane and ab- Ital. J. Food Sci., vol. 28 - 2016 85 sorbance values recorded at 232 and 268 nm for conjugated dienes (cD) and conjugated trienes (ct), respectively. specific extinctions were de- termined following the IUPAc method II D.23 (IUPAc, 1987). the para-anisidine value of the flaxseed oil samples was monitored according to IUPAc method II D.26 (IUPAc, 1987). the oil samples diluted with isooctane were react- ed with p-anisidine solution (0.25% w/v) in ace- tic acid for 10 min and the absorbance of the resulting colored solution recorded at 350 nm using a spectrophotometer. Tocopherols For tocopherol analysis, an HPLc method was adopted from YAQOOb et al. (2010), with some modifications. A Waters Alliance 2695 HPLc sys- tem equipped with YMc-Pack ODs AM-303, c 18 column (250mm x 4.6mm x 5μm) and Agi- lent series 1050 diode array detector, (UV 295 nm) was used. the temperature of the column was maintained at 30oc. the chromatographic separation was performed by isocratic elution with a mixture of acetonitirile and isopropanol (40:60 v/v) at a flow rate of 1 mL/min (Pressure 120 bar). briefly, flaxseed oil (1 g) was accurate- ly weighed into a 5 mL sample vial wrapped in aluminum foil to prevent photo-oxidation. the oil was dissolved in 5 mL acetonitrile before in- jection. samples were injected into the column through an injection loop (20 μL). tocopherols were identified by comparing the retention times of the unknowns with those of pure standards of α-, γ-, and δ-tocopherols. Acquisition of data was made using Agilent chem-station software. the samples were prepared and analyzed sepa- rately in triplicate. Fatty acids profile Fatty acid methyl esters (FAMEs) were pre- pared by IUPAc standard method 2.301 (IUPAc, 1987) which involved the trans-esterification of fatty acids with methanol under base –catalyzed conditions. briefly, 0.2 g of oil was placed in 10 mL capped vials; 5mL of redistilled methanol was added followed by the addition of a pellet of KOH. the content of the vials were heated at 60oc in a heating mantle until the droplets of fats disap- peared. Upon cooling, the reaction mixture was gently transferred to a separating funnel. small amount of n-hexane was added. separating fun- nel was shaken gently. the upper hexane layer was recovered and washed with distilled water. this hexane solution was dried over anhydrous sodium sulfate, filtered and used for gas chro- matographic analysis. FAMEs were separated on an Agilent 5890 series II Gc fitted with a 7673b auto sampler and a capillary column (30 m × 0.25 mm × 0.25 μm) with a Db-WAX (film thickness 0.20 μm) stationary phase and a flame ionizing detector (FID). Helium was used as carrier gas at a flow rate of 1.5 mL/min. Other conditions were as follow: injection volume 1μL, split mode (split ratio1:100), injector temperature, 280ºc, initial oven temperature, 170°c; hold up 2 min, 170- 240ºc (ramp rate 2ºc/min) hold up 10 min, de- tector temperature 260 ºc. FAMEs were identi- fied by comparing their relative and absolute re- tention times with those of authentic standards. the FA composition was reported as a relative percentage of the total peak area. the internal standard used was nonadecanoic acid. All of the quantifications were done by Agilent chem-sta- tion software. Statistical analysis three different seed samples for each of the variety were taken and analyzed individually in triplicate and data reported as mean ± sD (n= 3× 3 =9). An analysis of variance (ANOVA) was performed using Minitab 2000 Version 16.1 sta- tistical software (Minitab Inc. state college, PA, UsA). significant differences (P<0.05) of means were calculated using Duncan’s multiple range tests. rEsULts AND DIscUssION Proximate analysis of seeds the data obtained for the proximate analysis of flaxseeds of eight different cultivars grown in Pakistan is presented in table 1. Oil con- tents varied from 33.25 to 38.38% indicating a significant difference among cultivars selected (p<0.05). the variety chandani had the high- est oil yield whereas Ls-13 contained the low- est. In another study from Pakistan, Anwar et al., (2013) investigated the oil yield for soxhlet- extracted flaxseed to be 42.80%; such variation in oil yield may be linked to the different extrac- tion method used. the oil content of Pakistani flaxseeds are comparable to those grown in canada, North America and Egypt, i.e. 36%, 31.9 to 37.8% and 36-39%, respectively (HEttIArAcHcHY et al., 1990; OOMAH and MAZZA 1998; El-bELt- AGI et al., 2007). However, the present oil con- tents from Pakistani flaxseed cultivars were low- er than those reported for Polish flaxseed culti- vars, i.e., 41.4% (KOZLOWsKA, 1989) but high- er than Ethiopian flaxseed cultivars, 29.1-35.9% (WAKJIrA et al., 2004). such variations in flax- seed oil content with in the countries might be linked to varietal and agro climatic conditions of the regions. the moisture, crude protein, fiber and ash contents of different cultivars of Pakistani flaxseed ranged from 5.98 to 6.22%, 16.02 to 18.50%, 23.30 to 26.80% and 3.21 to 3.60%, respectively. there was no significant difference 86 Ital. J. Food Sci., vol. 28 - 2016 in moisture content for flaxseeds between dif- ferent cultivars (p>0.05). However, crude pro- tein, fiber and ash contents were notably differ- ent among the cultivars of flaxseeds (p<0.05). Depending on the cultivar and growing con- ditions, flaxseed has been reported to contain an average of 23% to 34% protein, 4% ash and 5% fiber (MUIr and WEstcOtt, 2003). Our re- sults are comparable to the previous reports on flaxseed cultivars grown in different regions of the world. Protein contents of Polish and North American cultivars were reported to be greater than 20%, while canadian cultivars had pro- tein generally less than 20% (OOMAH and MAZ- ZA, 1998; cHOO et al., 2007). crude fiber con- tent in different flaxseed residues has been re- ported to be in the range of 7-10% (GUtIErrEZ et al., 2010). the difference in oil, crude protein, and fiber and ash contents of flax seed of differ- ent cultivars might be attributed to differenc- es in growing conditions and genetic makeup of flax plants (OOMAH and MAZZA 1993; DUAN et al., 2003). Physico-chemical properties of oil the physico-chemical parameters determined for oils extracted from eight different cultivars of flaxseeds are presented in table 2. the results indicated that the refractive index (40°c) and density (25°c) ranged from 1.4706 to 1.4737 and 0.9278 to 0.929 mg/mL, respectively, with non- significant (p>0.05) difference among cultivars Our findings are consistent with the previous reports in which the refractive index of flaxseed oil at 20˚c was reported to be 1.475, while the density of flaxseed oil at 25˚c was 0.925 to 0.935 (PrZYbYLsKI, 2005). the density of flaxseed oil is greater than most other vegetable oils, and this might be attributed to the greater content of linolenic acid (GrEEN and MArsHALL, 1984). the iodine value, unsaponifiable matter, sa- ponification number and acid value are charac- teristic for flaxseed oils that contain a large per- centage of polyunsaturated fatty acids. the io- dine values for the tested oils ranged from 195 to 199 g of I 2 /100 g of oil with non-significant table 1 - Proximate analysis of Pakistani flaxseed (Linum usittatissimum. L) cultivars. Variety Parameters Oil content (%) Moisture content (%) Protein content (%) Fiber content (%) Ash content (%) Chandni 38.38±1.80ab 6.02±0.11a 18.50±0.56ab 26.80±1.22a 3.60±0.12ab LS-108 36.38±1.52de 6.12±0.13ab 18.00±0.45d 26.80±0.98a 3.55±0.11d LS-105 37.01±1.46e 6.22±0.15ab 17.98±0.63cd 26.00±1.06ab 3.50±0.11cd LS-99 38.02±1.60c 6.10±0.13a 17.86±0.48bc 25.5±1.14ab 3.50±0.13bc LS-33 35.30±1.35f 5.99±0.12ab 16.02±0.62a 23.50±0.88b 3.21±0.12a LS-31 34.98±1.47a 5.98±0.10ab 16.62±0.54a 23.3±1.16b 3.26±0.14a LS-29 38.00±1.59d 6.02±0.14a 17.99±0.39e 25.75±1.06ab 3.58±0.14e LS-13 33.25±1.31bc 6.00±0.11b 17.00±0.52ab 23.68±1.14b 3.40±0.16ab Values (mean ± SD) are average of triplicate samples of each cultivar, analyzed individually in triplicate (n = 1 x 3 x 3), (P<0.05). Different letters in superscript indicate significant differences. table 2 - Physico-chemical characteristics of oil extracted from Pakistani flaxseed (Linum usittatissimum. L.) cultivars. Parameters Varieties Chandni LS-108 LS-105 LS-99 LS-33 LS-31 LS-29 LS-13 Refractive index (40°C) 1.4729±0.009a 1.4728±0.006a 1.4707±0.005a 1.4737±0.007a 1.4732±0.005a 1.4734±0.006a 1.4706±0.009a 1.4728±0.007a Density g/mL (25°C) 0.928±0.18a 0.929±0.12a 0.9278±0.14a 0.928±0.14a 0.928±0.16a 0.929±0.15a 0.928±0.19a 0.9279±0.21a Iodine Value g 198±3.96a 199±4.26a 195.9±3.24a 201±4.39a 195±3.85a 196±4.96a 197±3.68a 199±3.88a of I/100 g of oil Unsap matter (%) 2.20±0.04c 2.60±0.04a 2.20±0.02c 2.00±0.02d 2.4±0.03b 1.96±0.05d 2.0±0.02d 1.80±0.04e Saponification value mg 189±2.78a 187±3.25a 185.9±3.70a 186±3.46a 185±4.58a 185.86±3.72a 187±3.94a 184±4.71a of KOH/100 g of oil FFA (% as oleic acid) 1.399±0.03d 1.579±0.04c 1.624±0.02bc 1.399±0.03d 1.725±0.05a 1.721±0.04ab 1.447±0.02d 1.732±0.05a Peroxide value 1.00±0.02c 1.20±0.03ab 1.18±0.02ab 1.00±0.04c 1.14±0.03b 1.20±0.02a 1.22±0.03ab 1.14±0.05b (meq/kg of oil) 1cm 1% (λ 232) 5.07±0.20ab 4.70±0.15b 4.81±0.28b 4.79±0.19b 4.80±0.22b 4.75±0.14b 5.55±0.22a 4.76±0.18b 1cm 1% (λ 268) 1.80±0.08ab 2.00±0.09a 1.90±0.08a 1.60±0.04cd 1.50±0.05d 1.80±0.07ab 1.70±0.05bc 1.40±0.06bcd Para-anisidine value 1.13±0.05c 1.41±0.05ab 1.40±0.06ab 1.29±0.05bc 1.36±0.05ab 1.42±0.06ab 1.48±0.05a 1.32±0.04ab Values (mean ± SD) are average of triplicate samples of each cultivar, analyzed individually in triplicate (n = 1 x 3 x 3), (p<0.05). Different letters in superscript indicate significant differences. Ital. J. Food Sci., vol. 28 - 2016 87 (p>0.05) among cultivars. Iodine value for flax- seed oil has been reported to vary between 180 to 203 g of I 2 /100g of oil (PrZYbYLsKI, 2005). LONG et al. (2011) reported iodine value of flax- seed oil to be 162 I 2 /100g. the saponification value and unsaponifiable matter of the test- ed flaxseed oils ranged from 184 to 189 mg of KOH/100g of oil and 1.8 to 2.6%, respectively. saponification values did not differ significant- ly (p>0.05) whereas the unsaponifiable matter varied significantly within the oils of different cultivars (p<0.05). In previous reports, the per- centage of unsaponifiable matter in flaxseed oil was in the range of 0.1 to 1.7% for raw oil, and up to 0.6% for refined flaxseed oil (EsKINEtAL, 1996; cHOO et al., 2007). tEH and bIrcH (2013) reported the unsaponifiable value to be 0.4% for cold pressed flaxseed oil. Free fatty acids (FFA) are produced by the hy- drolysis of triglycerides (LAFONtAN and LANGIN, 2009). the FFA content of the tested flaxseed oils ranged from 1.40 to 1.73%, as oleic acid. the FFA content varied significantly within different flaxseed cultivars (p<0.05). In a previous report, FFA value for flaxseed oil was reported to be 0.1 to 2.0% (PrZYbYLsKI, 2005). LONG et al. (2011) reported the FFA in the flaxseed oil extracted by enzymatic extraction and solvent extraction to be 1.5 and 1.1%, respectively. FFA value for the cold pressed flaxseed oil was reported to be 0.75% (tEH and bIrcH, 2013). FFA in most of the freshly extracted crude vegetable oils is nor- mally below 1.0%. these hydrolytic products are mainly formed as result of chemical hydrolysis (due to presence of moisture in seeds) or enzy- matic hydrolysis. A low value of oil FFA is an in- dication that the seeds have been preserved un- der proper storage conditions with good state. the peroxide value for the flaxseed oils of dif- ferent cultivars ranged from 1.0 to 1.22 meq/ kg of oil that is well below the limit for peroxide value. cHOO et al., (2007) reported the peroxide value ranging from 0.5 to 2.9meq/Kg of cold- pressed flaxseed oil sold in New Zealand. Per- oxide value for the enzymatic, solvent extract- ed flaxseed oils was reported to be 1.2 and 1.0 meq/kg of oil, respectively (LONG et al. 2011) while that for cold pressed flaxseed oil was re- ported to be 2.04 meq/kg of oil (tEH and bIrcH, 2013). Peroxide value is an indicator of prima- ry oxidation products; the extent of these prod- ucts may range up to 10-15 meq/kg of oil (cHOO et al., 2007). the p-anisidine value of the oils extracted from different cultivars of flaxseed ranged from 1.13 to 1.48(p<0.05). the values are higher than those investigated by cHOO et al. (2007) who reported the p-anisidine value to be in the range of 0.36 to 0.4. However, the present values were comparable to the values of en- zymatically and solvent extracted flaxseed oil reported previously, 1.2 and 1.0, respective- ly (LONG et al., 2011). conjugated dienes and trienes produced as a result of secondary ox- idation of polyunsaturated fatty acids can be determined by measuring the absorbance at 232 and 272 nm, respectively (Frankel, 2005). the specific extinction at 232 and 272 nm of different flaxseed oils ranged from 4.70 to 5.55 and 1.40 to 2.00, respectively. cHOO et al., (2007) reported absorbencies at 232 and 270 nm of 1.7 to 2.75 and 0.2 to 0.4, respectively for cold pressed flaxseed oil. tEH and bIrcH (2013) reported absorbencies at 232 and 272 nm for the cold pressed flaxseed oil to be 2.02 and 0.02, respectively which is very low as compared to our present results. Our results are comparable to those reported previously (rEED et al., 2001). Tocopherol content the tocopherol contents of the different flax- seed oils are shown in table 3. Gamma (γ)- tocopherol was the main tocopherol in flaxseed oils, with contribution of approximately 90% of the total tocopherols. the γ-tocopherol content ranged from 173.7 to 257.9 mg/kg of oil and sig- nificantly differed in different cultivars (p<0.05). Alpha (α) tocopherol was the other tocopherol found in the oils (~10%) while delta tocopher- ol was not detected. the contents of α tocoph- erol varied from 39 to 18.7 (mg/kg of oil) show- ing a significant difference among different cul- tivars (p<0.05). the difference in the contents of tocopherols might be due to the varying ge- table 3 - tocopherol contents (mg/kg) of oil extracted from Pakistani flaxseed (Linum usittatissimum. L.) cultivars. Tocopherol Cultivars Chandni LS-108 LS-105 LS-99 LS-33 LS-31 LS-29 LS-13 γ-tocopherol 204.0±5.0bc 217.8±4.8b 179.6±5.8de 173.7±5.5e 201.3±6.2c 192.2±4.9cd 257.9±5.6a 190.8±4.3a α-tocopherol 24.9±0.2e 20.4±0.2f 30.2±0.2c 18.4±0.3g 27.2±0.3d 20.9±0.2f 38.5±0.3a 32.8±0.3b δ-tocopherol ND ND ND ND ND ND ND ND Total tocopherols 228.9 238.2 209.8 192.1 228.5 213.1 296.4 223.6 Values (mean ± SD) are average of triplicate samples of each cultivar, analyzed individually in triplicate (n = 1 x 3 x 3), (P<0.05). Different let- ters in superscript indicate significant differences. ND = not detected. 88 Ital. J. Food Sci., vol. 28 - 2016 netic makeup and growing conditions of differ- ent cultivars (OOMAH and MAZZA, 1997). Our results are consistent with previous reports in which gamma tocopherol was reported as the predominant tocopherol in flaxseed oils (GrEEN and MArsHALL, 1984; HErcHI et al. 2011). the γ- and α-tocopherol content of the Pakistani cul- tivars is considerably greater than that in the flaxseed oil varieties of American, canadian, New Zealand and turkish origin which contain an average127, 93, 140 and 146 (mg/kg of oil) of tocopherols, respectively (bUDINEtAL.,1995; OOMAH and MAZZA, 1997; cHOO et al., 2007; bOZAN and tEMELLI, 2008). the tocopherol contents of Pakistani flaxseed cultivars were comparable to those reported for Egyptian cultivars (210 mg/kg of oil) (EL-bELt- AGI et al., 2007). tEH and bIrcH (2013)reported the contents of γ-tocopherol to be 370 (mg/kg of oil) in cold pressed flaxseed oil, which is consid- erably higher the present finding. Fatty acid profile the fatty acid profile showed a significant var- iation in the contents of fatty acids within the oils of different flaxseed cultivars (p<0.05) as shown in table 4. the amount of total unsat- urated fatty acids in flaxseed oils of the select- ed cultivars was observed to be in the range of 88.79 to 89.78% while the amount of total sat- urated fatty acids ranged from 10.21 to 11.20 % with non-significant (p>0.05) varation among cultivars. One distinct feature of flaxseed oil is the presence of high amount of linolenic acid. In the current study the quantities of linolenic acid were observed to be 44.51 to 54.87%, for different cultivars (p<0.05). the results are comparable to the previous re- ports for American and Egyptian flaxseed varie- ties with 45 to 52% and 46 to 50% alpha linolenic (ALA) acid, respectively (DEcLErcQ et al., 1992; El-bELtAGI et al., 2007). For Ethiopian flaxseed cultivars the ALA contents were 52% (WAKJIrA et al., 2004). However, the ALA contents of the Pakistani cultivars were less than those reported for the flaxseed cultivars grown in New Zealand and canada, i.e. 59.65 and 59%, respectively (HEttIArAcHcHY et al., 1990; cHOO et al., 2007). Moreover, bOZAN and tAMELLI (2008) reported ALA levels to be 56.5 to 61% for flaxseed from turkish origin, i.e. greater than our findings. the trends for FA results in the present study are also in agreement with the reports that with an increase in ALA in flaxseed oil, there is a cor- responding decrease in oleic acid (cHOO et al. 2007). the flaxseed cultivar Ls-33 had the high- est contents of ALA (54.87%) and lowest amount of oleic acid (21.05%), while Ls-99 contained the lowest amount of alpha linolenic acid (44.51%) and the highest amount of oleic acid (30.96%). Overall, the amount of linolenic acid ranged from 44.51 to 54.87%, while that of oleic acid ranged from 21.06 to 30.96% for different cultivars of flaxseed grown in Pakistan. cONcLUsIONs the oil yield considerably varied among the selected flaxseed cultivars. similarly, the signif- icant differences for most of the physico-chemi- cal/analytical characteristics among the tested oils were recorded. such variations in oil qual- ity characteristics might be linked to different genetic makeup of the cultivars as well as to their variable harvesting conditions. Overall, the flaxseed cultivar chandi, Ls-99 and Ls-29 had relatively higher oil yield; the cultivar Ns- 29, Ls-108, chandi and Ls-33 exhibited great- er amount of tocopherols whereas those of Ls- 33, Ls-29 and Ls-105 were rich in alpha lino- lenic acid (ALA) among others. the findings of this comparative study can be useful for selec- tion of economically and nutritionally important flaxseed cultivars, especially, as ingredient for functional foods and nutraceuticals. AcKNOWLEDGEMENts Authors are thankful to the Higher Education commission of Pakistan for the funding of the project under the Indige- nous Ph.D. 5000 scholarship scheme and international re- search support initiative program. table 4 - Fatty acid composition (g/100g of FA) of oil extracted from Pakistani flaxseed (Linum usittatissimum. L) cultivars. Fatty acid (FA) Varieties Chandni LS-108 LS-105 LS-99 LS-33 LS-31 LS-29 LS-13 C 16:0 5.95±0.07cd 5.94±0.07b 5.8±0.06b 6.14±0.07a 6.11±0.19bc 6.52±0.01bc 6.27±0.01d 6.21±0.03cd C 18:0 4.63±0.04c 4.70±0.12d 4.41±0.03cd 4.97±0.08bc 4.83±0.06ab 4.68±0.06a 4.58±0.01d 4.4±0.04bc C 18:1 (n-9) 30.46±0.63ab 28.86±0.53de 26.25±0.35e 30.96±0.47c 21.05±0.45f 28.33±0.56a 24.09±0.58d 26.75±0.59bc C 18:1 (n-7) 1.69±0.58a 1.64±0.51a 1.61±0.54a 1.53±0.41a 1.46±0.44a 1.83±0.64a 1.69±0.49a 1.8±0.63a C 18:2 (n-6) 11.26±0.04ab 11.18±0.01d 7.63±0.74cd 11.88±0.21bc 11.68±0.8a 10.5±0.01a 10.08±0.1e 9.42±0.13ab C 18:3 (n-3) 46.02±0.10f 47.67±0.11d 54.29±0.44c 44.51±0.12e 54.87±0.01a 48.13±0.14g 53.29±0.02b 51.41±0.08e Values (mean ± SD) are average of triplicate samples of each cultivar, analyzed individually in triplicate (n = 1 x 3 x 3), (P<0.05). Different letters in superscript indicate significant differences. Ital. J. 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