Lipid stability, antioxidant potential and fatty acid composition of broilers breast meat as influenced by quercetin in combination with α-tocopherol enriched diets

Background Dietary supplementation of antioxidants is a vital route to affect the oxidative stability and fatty acid profile of broiler meat. The supplementation of feed with antioxidants decreases degradation of lipids in muscles thereby enhances meat stability. Methods The present study was carried out to investigate the influence of dietary quercetin in combination with α-tocopherol on growth performance, antioxidant potential, lipid stability and fatty acid composition in breast meat of birds. Accordingly, one day old 300 Hubbard strain male broiler birds were given three levels of quercetin @100, 200 and 300 mg/kg feed in combination with α-tocopherol @150, 225 and 300 mg/kg feed. The resultant meat was subjected to antioxidant assay, lipid stability, quantification of antioxidants followed by fatty acid profile of broiler breast meat. Results The results explicated that feed treatments imparted momentous effect on gain in weight, and feed conversion efficiency however, intake of feed in birds affected non-momentously. The highest weight gain recorded in T9 as 2374.67 & 2388 g/bird followed by T8 & T6 2350 & 2353.33 and 2293.33 & 2307 g/bird, respectively whilst the lowest in T0 as 1992.67 & 1999 g/bird during the experimental year 2013 and 2014. The results regarding antioxidant potential revealed that among treatments, T9 exhibited highest values for total phenolic contents (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) & ferric reducing antioxidant power assay (FRAP) i.e. 158.70 ± 0.84 mg GAE/100 g, 82.40 ± 0.93 % and 682 ± 2.11 μmol/Fe+2/g, respectively as compared to T0 104.27 ± 1.64 mg GAE/100 g, 54.71 ± 0.64 % and 542.67 ± 1.74 μmol/Fe+2 /g of meat, correspondingly. The TBARS assay indicated that malondialdehydes production in meat increased during storage however, antioxidants deposition varied significantly among treatments. Fatty acid compositional analysis revealed that addition of quercetin with α-tocopherol in the bird’s diet decreased the fatty acid generation particularly saturated fatty acids. Conclusion Conclusively, dietary supplementation of quercetin along with α-tocopherol improves growth performance, antioxidant capacity, stability of lipids and fatty acid composition in breast meat of birds.


Introduction
Poultry meat is among the most popular meats in the world owing to its low price, short production time and ease of preparation [1]. The consumption of poultry meat is gaining popularity being an excellent source of high quality protein, possesses balanced amount of fatty acids mainly polyunsaturated fatty acids (PUFA) as well as provides essential vitamins and minerals. This meat contains higher levels of polyunsaturated fatty acids (PUFA) that trigger oxidative deterioration, resultantly decrease the quality of meat products [2]. The lipid oxidation is one of the major route for quality degradation in meat, apart from microbial spoilage. The mechanistic approach involves generation of reactive oxygen species and formation of free radicals, which produce rancid odor, off-flavor and surface discoloration of meat and meat products. The rate of lipid oxidation in fresh and cooked meat products depends on various internal factors such as fat content, fatty acid composition, level of antioxidants, heme pigment and iron contents. The endproducts of this process impair color, aroma, flavor and texture of meat and allied products, hence reduce the nutritive value [3].
Oxidation problems in poultry meat and meat based products can be controlled through antioxidants such as synthetic ones like butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate (PG). However, theses synthetic counterparts are consider harmful for human health as well as controlled by regulatory agencies therefore, meat industry is looking for replacements of these synthetic antioxidants [4]. The quercetin [2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one] is one of flavonol belongs to class of flavonoids, ubiquitously present in fruits and vegetables specially red onion, caper, apple and in some medicinal & aromatic plants [5]. Several in vitro and in vivo studies conducted on human and experimental animals have revealed that quercetin possesses antioxidant and anti-inflammatory prospectives. The antioxidant activity of quercetin is attributed to its ability to scavenge free radicals, donate hydrogen atoms or electrons or chelate metal cations [6]. The pharmacokinetics of quercetin have proven that it ameliorates the absorption and metabolism of nutrients when enter the physiological system. The quercetin and its glycosidic metabolites possess antioxidant properties and modulate biological processes such as cell signaling pathways and reduction of oxidative DNA damage [7]. The oxidative deterioration of low density lipoproteins (LDL) can be prevented by dieatary quercetin as it possesses ability to scavenge free radicals and chelate transition metal ions generated through the oxidation of lipids. The α-tocopherol is the natural antioxidant that protects cells and tissues from oxidative damage induced by free radicals [8]. Several studies have reported that αtocopherol is not deposited to a toxic level unlike other fat soluble antioxidants. Its accumulation at tissue level is strongly regulated through increasing hepatic metabolism that also regulates environmental toxins and speed of drug metabolism [9]. The supplementation of α-tocopherol in poultry meat been found to be one of pragmatic choice to improve oxidative stability of meat lipids and inhibiting oxidation of cholesterol. The inclusion of α-tocopherol in feed protects the broiler birds against stress-induced increase in thiobarbituric acid reactive substances (TBARS) by limiting oxidation as well as preserving animal health. It also enhances the lipid stability and quality of Beijingyou chicken muscles when supplemented through diet [10]. The supplementation of α-tocopherol at a level of 200 IU is considered effective to increase the oxidative stability of meat and its concentration in broiler muscles gradually raise in a dose dependent manner. Alpha tocopherol supplementation through feed improve the oxidative stability and quality of poultry meat and meat based products [11]. The objective of instant exploration was to examine the effect of dietary quercetin and α-tocopherol on growth performance, lipid stability, antioxidative potentialand fatty acid composition of broiler breast meat.

Results and discussion
Growth performance of birds The results (  (Table 2). It is evident from results in Table 3 that feed conversion ratio (FCR) of birds varied significantly among treatments and weeks. The means indicated that birds fed on combinations of α-tocopherol and quercetin enriched diet reported lowest FCR in T9 as 1.686 ± 0.035 followed by T8 and T6 as 1.701 ± 0.031 & 1.722 ± 0.027 whilst, highest in T0 group (1.938 ± 0.026), respectively.
The findings of instant study are supported by the findings of [12], noticed weight gain in broilers fed on αtocopherol @200 mg/kg of feed. One of the researchers groups, [13] indicated that higher concentration of αtocopherol in feed improved growth performance by yielding higher weight gain. Earlier, [14] stated that dietary supplementation of quercetin enhanced the weight gain and growth efficiency in broilers at a level of 200 ppm/kg via diet. Recently, [15] reported that dietary supplementation of quercetin at a concentration of 0.1 and 1 g/kg feed enhanced weight gain of chickens however, reprted nonsignificant effect for feed intake of birds. Similar findings were reported by [16], observed non-momentous effect of hesperidin (3 g/kg of feed) on feed intake of birds. One of scientists groups, [17] delineated that feeding αtocopherol supplemented diet has non momentous impact on birds feed intake. The previous findings of [18] also indicated non substancial effect on feed intake by supplementation of α-tocopherol. Current results regarding FCR of broilers administrated quercetin and αtocopherol supplemented feed are concordant with the work of [19], reported that inclusion of carvacrol and quercetin @ 200 mg/kg feed improved FCR of broilers. Likewise, [17] observed improved FCR of broiler birds linearly with increasing concentration of α-tocopherol. One of the researchers groups, [15] noticed lower feed conversion ratio (p < 0.05) when birds were provided feed containing higher level of quercetin. The results of previous findings of [20] also indicated that efficiency of feed was increased with α-tocopherol fortification in birds. In the current exploration, the average mortality rate for broilers fed on combination of dietary quercetin and alpha tocopherol enriched feed was ranged from 5-6.1 %, however, 7.3 % for control group birds.

Antioxidant potential of breast meat Total phenolic contents
The results indicated that total phenolic contents (TPC) in samples of birds varied significantly among treatments Data are means ± SE. Means sharing similar letters in a column do not differ significantly from one another (p˃0.05) T0 = control without antioxidants, T1 = 100 mg quercetin + 150 mg α-tocopherol/kg feed; T2 = 100 mg quercetin + 225 mg α-tocopherol/kg feed; T3 = 100 mg quercetin + 300 mg α-tocopherol/kg feed; T4 = 200 mg quercetin + 150 mg α-tocopherol/kg feed; T5 = 200 mg quercetin + 225 mg α-tocopherol/kg feed; T6 = 200 mg quercetin + 300 mg α-tocopherol/kg feed, T7 = 300 mg quercetin + 150 mg α-tocopherol/kg feed; T8 = 300 mg quercetin + 225 mg α-tocopherol/kg feed, T9 = 300 mg quercetin + 300 mg α-tocopherol/kg feed but differed non-significantly due to years (  [21] who reported that antioxidants provided through diet are deposited in muscles thereby alleviate oxidative stress of animals by increasing concentration of phenolic compounds. They further stated that deposition of phenolic compounds is higher in breast muscles than that of leg meat of birds. Similarly, [22] indicated that broilers fed on α-tocopherol supplemented feed resulted higher antiradical power due to the accumulation of phenolic compounds. Likewise, [23] also showed that feed containing exogenous antioxidants like vitamin C & E, ubiquinols and polyphenols enhance total phenolic potential of poultry meat when birds are fed on antioxidant enriched feed. One of the researchers groups, [24] observed that total phenolic potential of pig meat was enhanced by increasing concentration of phenolic compounds through feed.
The mechanistic approach elaborates that antioxidants combine with free radicals, inactivate them thus decreases the free radicals intracellular concentration which enhances oxidative stability of meat. Similarly, [27] noticed that vitamin E and sage extract increased the antiradical power of poultry meat than that of control.  1). The current findings are concordant with work of, [28] also found higher ferric reducing power in meat containing natural antioxidants compared to synthetic counterparts.

Ferric reducing antioxidant power
They further elaborated that antioxidants in meat decrease the reductants in meat that are involved in conversion of Fe3+ to Fe2+. Similarly, [29] delineated that total antioxidant capacity of broiler meat as measured by FRAP is significantly varied when birds are provided feed enriched with 300 & 600 mg quercetin/kg body weight.
Oxidative stability by thiobarbituric acid reactive substances assay The results regarding thiobarbituric acid reactive substances (TBARS) of breast meat of broiler showed significant differences due to treatments (Fig. 2). The means in Fig. 2  Previous studies showed that quercetin and α-tocopherol have ability to attenuate the process of lipid peroxidation. It has been observed from findings of [15], noticed the impact of dietary quercetin @ 0.5 and 1 g/kg of feed to the birds. It has been inferred that oxidative stability of broiler meat under refrigerated as measured by TBARS was enhanced (p < 0.05) when birds were fed quercetin One of the researchers groups, [30] observed concomitant increase in lipid peroxidation in pig meat as indicated by higher (p < 0.05) concentrations of TBARS and 8-iso-PGF2 nevertheless, it was ameliorated (p < 0.05) by dietary fortification of quercetin. Likewise, [14] also found that supplementing poultry diet with quercetin @200 ppm/kg feed substantially reduced TBARS. Recently, [31] reported that quercetin alone or in combination with flaxseed reduces the formation of oxysterol in meat (p < 0.05), an oxidation product of cholesterol and lipids after 7 days of refrigerated storage. One of the researchers groups, [32] expounded that α-tocopherol alone or in combination with rosemary & green tea extract retards lipid oxidation in meat.

Quercetin contents
The results in Table 5 [29], reported that quercetin and its metabolites such as quercetin glucuronides and sulfonates are deposited in liver, breast and thigh muscles when birds are provided quercetin supplemented feed. Likewise, [33] also found that quercetin level of turkey roll increased by the addition of onion juice brine (OJB) directly in meat @25 (OJB25) and 50 % (OJB50) solution. They further reported quercetin content of turkey rolls 8.17 and 16.3 mg/kg meat in 25 and 50 % brine solution, respectively. However, cooking process depleted quercetin in resultant meat after cooking (30 min), quercetin content was dropped by 24 % for OJB25 and 38 % for OJB50 group. Present study findings indicated that deposition of quercetin was enhanced when broiler birds were provided diet containing α-tocopherol and quercetin.

Alpha tocopherol contents
The findings (  of instant study are supported by [34] who indicated that α-tocopherol concentration of breast and thigh muscles of birds was enhanced by increasing the level of dietary α-tocopherol. They further stated that feed supplementation @200 or 400 IU of α-tocopherol is effective in retarding the oxidative degradation of lipids and cholesterol. Likewise, [35] also reported fortification feed with α-tocopherol increases the deposition of α-tocopherol in meat. One of the researchers groups, [36] listed that dietary supplementation of α-tocopherol acetate significantly amplified its level in pectoralis muscles indicating its deposition in tissues. They also stated that αtocopherol level of muscles was not decreased with postmortem time. One of the scientists groups, [37] reported that vitamin E supplemented feed to the broiler birds increased vitamin E concentration in breast muscle.

Fatty acid profile of broiler breast meat
The results (  (Table 7). Overall, fatty acid production decreased with increasing level of quercetin and αtocopherol in a dose dependent manner that depicted their lipid lowering potential however, reduction in SFA fatty acids was more obvious than that of PUFA in broiler meat. The findings of instant study are in harmony with [38] they stated that dietary supplementation of quercetin @200 mg/kg feed to broilers affect fatty acid composition of pectoralis muscle of birds. They recorded oleic (18:1n-9, 33-35.2 %), palmitic (16:0, 26.1-27.9 %), linoleic (18:2n-6, 14.0-14.6 %) and stearic acid (18:0, 11.8-13.7 %) in pectoralis muscle, respectively and further elaborated that quercetin addition significantly (p < 0.05) diminishes palmitic, oleic and linoleic acid production. Likewise, [39] noticed that dietary administration of quercetin @1 % in rat affected fatty acid synthesis rate and diminished free fatty acids production. The mechanistic approach for reduction of SFA in broiler meat due to antioxidants supplementation is their potential to inhibit activity of 9-desaturase complex that converts SFA to MUFA One of the scientists groups [40], delineated that quercetin decreases the rate of fatty acid and triacylglycerol (TAG) synthesis in rats.

Conclusion
The findings of instant exploration revealed that antioxidant potential of broilers breast meat can be improved by the supplementing quercetin in combination with alpha tocopherol. The supplementation of birds feed with 300 mg/kg feed dietary level of quercetin in combination with alpha tocopherol improved the growth performance by increasing gain in weight and lowering FCR of birds. Accordingly, the antioxidant potential was also improved through supplementation of quercetin and alpha tocopherol that increases the stability of lipid against oxidation in meat. The results further elaborates that fatty acid production decreased with increasing level of quercetin and α-tocopherol in dose dependent manner however, reduction in SFA fatty acids was more pronounced than that of PUFA content of broiler breast meat.

Materials and methods
The current study was conducted at the National Institute of Food Science and Technology (NIFSAT) and Nutrition Research Center, University of Agriculture, Faisalabad, Pakistan. In present research, the influence of dietary quercetin and α-tocopherol via feed supplementation in broiler was explored to enhance the antioxidant potential and lipid stability of broiler breast meat. The instant study was approved by the National Institute of Food Science and Technology review board. Materials and protocols followed to carry out this study are described herein.

Procurement of raw materials
All reagents and chemicals required for the instant exploration were purchased from Sigma Aldrich (Tokyo, Japan) and Merck (Merck KGaA, Darmstadt, Germany). The quercetin was acquired from Shaanxi Jintai Biological Engineering Co. Ltd. China. Alongside, 300 one day old broiler chicks (50 ± 5 g body weight) were procured from Jadeed Chicks Pvt. Ltd. Faisalabad, Pakistan.
The study was conducted in duplicate to enhance the authenticity of results.

Diet, study plan and management of birds
In the current study, 300 one day old broiler chicks were used as experimental animals for the production of functional broiler meat. The animals were treated by following guidelines of the ethical committee as approved by the university. For the intention, they were weighed individually and divided randomly into 10 groups each consisting of 30 birds reared for a period of six weeks ( Table 8). The composition of the control feed provided to the birds is mentioned in Table 9. Prior to research, the research area and all pens were thoroughly cleaned. For disinfection, bromosept and formalin aqueous solution with 1:12 ratio was used in the experimental premises. Likewise, 2-3 in. thick layer of saw dust was spread in each pen as a litter to keep the bed dry and soft. Moreover, all drinkers and feeders were thoroughly washed and disinfected during the course of research trial. All the pens were tagged with respective treatments and replication numbers. The temperature of the experimental room was maintained at 95 ± 2°F during the first week of trial followed by a decrease of 5°F till reached to 75 ± 2°F. The light and proper ventilation were also maintained in the experimental room. The experimental birds were provided free access to feed and fresh water.

Vaccination schedule of chicks
The glucose solution (50 g/5 L) was given to the chicks after 2 h of distribution in pens for waste removal. On 2nd day, cotrim-50 solution (1 g/5 L of water) was administrated to chicks as an antibacterial agent. The chicks were vaccinated for new castle disease (N.D) and infectious bronchitis (I.B) at 3rd and 4th day for the prevention from respective diseases. Afterwards, on 10th and 18th day, birds were vaccinated against gamboro disease by respective vaccine. Lastly, on 24th day, birds were vaccinated against new castle disease with Lasuta vaccine.

Slaughtering, samples collection and preparation
The experimental birds were reared up to six weeks. For acclimatization, the chicks were fed on control diet during the first two weeks of study. Afterwards, they were fed on diet supplemented with quercetin and α-tocopherol as per treatment plan. At termination, the broiler birds were slaughtered by adopting Halal Islamic Ethical Guidelines. After slaughtering, breast muscles of broilers were separated, deboned, wrapped with aluminum foil and packed in polythene zip lock bags followed by storage at −18°C for analysis. For sample preparation, 5 g meat sample was taken in 50 mL capped polypropylene tube and homogenized by using phosphate buffer and glycerol (20 %) at pH 7.4 through homogenizer. The tubes were placed in ice cold water to prevent the oxidation of muscle samples. Afterwards, filtration of samples was carried out to remove connective tissues.

Growth parameters
The birds were weighed on weekly basis to calculate the weight gain. A measured quantity of feed was provided to chicks during each week for exploring the influence of added antioxidants on feed intake of broilers. Additionally, the mortality rate of the broiler birds during the course of research trial was also calculated. At the termination of research trial, feed conversion ratio of broilers (FCR) was calculated by dividing the feed consumed by  weight gained in the respective week using the following expression; FCR ¼ Feed consumed by the bird inoneweek =Weight gain by the bird in respective week

Antioxidant potential of meat
All the methods used to carry out the instant research were officially approved to carry out analysis. Antioxidant potential of breast meat samples of birds was estimated by using respective analytical methods;

Total phenolic contents
The total phenolic contents (TPC) in breast broiler meat samples were determined by adopting the procedure as described by [43]. The homogenized meat sample (100 μL) was mixed with 500 μL (95 % ethanol), distilled water (2.5 mL) and 250 μL of 50 % Folin-Ciocalteu reagent. After 5 min, 250 μL of 5 % Na2CO3 was added to the resultant mixture, vortex and placed in the dark room for 1 h. Afterwards, absorbance of samples was recorded at 725 nm through UV/Visible Spectrophotometer (CECIL-CE7200) against control. The total phenolic contents of meat samples were estimated as gallic acid equivalent (mg gallic acid/g).

Free radical scavenging activity
The free radical scavenging activity i.e. DPPH (1,1diphenyl-2-picrylhydrazyl) of breast meat samples was measured using the protocol of [44]. Purposely, 1 mL of DPPH solution was added to 4 mL of sample followed by incubation for 30 min at room temperature. The absorbance was measured at 520 nm using UV/Visible Spectrophotometer. The DPPH free radical scavenging activity was calculated by the below mentioned equation; A blank = absorbance of blank sample (t = 0 min) A sample = absorbance of tested solution (t = 15 min)

Ferric reducing antioxidant power
The ferric reducing antioxidant power of breast broiler meat samples was estimated by following the procedure of [45]. The homogenized sample (200 μL) was mixed with 500 μL sodium phosphate buffer (0.2 M, pH 6.6) and 500 μL potassium ferric cyanide (1 %) followed by incubation at 50°C in a water bath for 20 min. After cooling, sample was mixed with 2.5 mL (10 % TCA), distilled water (1.25 mL) and 0.25 mL (0.1 % ferric chloride) for 10 min. The absorbance was measured at 700 nm. During the analysis, an increase in the absorbance of the reaction mixture indicated the higher reducing power of the samples.

Lipid stability by thiobarbituric acid reactive substances assay
The oxidative stability of broiler breast meat samples was measured by using thiobarbituric acid reactive substances (TBARS) according to the guidelines of [46]. In this context, 5 g of ground broiler meat samples were weighed in a 50 mL test tube and homogenized with 50 μL of butylated hydroxytoluene (7.2 %) and 15 mL of deionized distilled water using a homogenizer for 15 s.

Quantification of antioxidants Quercetin contents
The quercetin content of breast meat samples was estimated through HPLC by following the protocol of [47]. Purposely, 2 g meat was mixed with 50 mL of 70 % v/v methanol/water. The mixture was homogenized for 5 min with a blender. The solution was filtered through a Whatman No. 4 filter paper under reduced pressure. The residue was extracted again with 50 mL of 70 % v/v methanol/water for 5 min followed by filtration through a Whatman No. 4 filter paper and in the resulting filtrate, methanol was added to make a volume of 100 mL. For HPLC analysis, the solution was filtered through a 0.45 mL of nylon filter disc prior to analysis. Afterwards, 1 mL of 1000 mg/mL sorbic acid solution (internal standard) was added and the total volume (25 mL) was made with methanol. The solution was filtered through a 0.45 mm nylon filter before analysis. The mobile phase comprised of 0.1 % formic acid in water and methanol with gradient of 20:80 %. The stock and working standards of quercetin were prepared in methanol. The analyses were performed with an Agilent series 1100 quaternary solvent delivery system with cooled autosampler (4°C) and photodiode array detector (Agilent, Waldron Germany) connected to a thermo electron ion trap mass spectrometer operating in negative ion electrospray mode (Thermo Electron, San Jose, USA). The column was maintained at 30°C and mobile phase consisted of (A) 0.1 % formic acid in water and (B) 0.1 % formic acid in methanol with the following gradient;

Alpha tocopherol content
The α-tocopherol content of meat samples was measured by the protocol of [48]. The homogenized meat sample (500 μL) was taken in a test tube followed by the addition of 1.5 mL of urea (6 M) to dissolve the meat tissue. Later, 0.5 mL of ascorbic acid (5 %) was added in the reaction mixture to prevent the oxidation of α-tocopherol in the meat samples along with 1 mL of 6 M urea. The tubes were flushed with N 2 and resultant mixture was vortex for 12 min to extract the tocopherol components of samples. Next, 1 mL of 0.1 M sodium dodecyl sulfate (SDS) solution was added and vortex for 1 min to disintegrate the meat tissue. For deproteination and release of αtocopherol, 4 mL of ethyl alcohol containing 1 % pyrogallol was added in the resulting mixture. Thereafter, petroleum ether (10 mL) was added and the resultant mixture was centrifuged at 5000 × g for 5 min to facilitate the separation of phases. The solvent layer containing α-tocopherol was separated in the vial and the pooled solvent was evaporated under nitrogen. Alpha tocopherol content was dissolved in the mobile phase (100 % methanol) and then filtered through 0.45 μm microfilter, centrifuged at 5000 × g for 5 min to collect the filtrate and stored for HPLC analysis. The mobile phase comprised of methyl alcohol (HPLC grade); 100 % methanol was prepared by filtering through typhlon filter assembly and then adjusted according to requirements of HPLC. The standard of α-tocopherol was prepared by using Sigma Aldrich packed standard 1 mg/mL of αtocopherol as stock solution from which further dilutions (10, 20, 50 and 100 μg/mL of solutions) were prepared. The α-tocopherol was extracted and quantified by using HPLC (PerkinElmer, Series 200, USA) chromatographic system at 290 nm with UV-Visible detector. The HPLC chromatograms were obtained through C 18 column (250 mm × 4.6 mm, 5.0 μm), system controller SCL-10 A, water pump (LC-10 AT) and flow controller valve (FCV-10 AL) with a mobile phase of 100 % methanol at a flow rate of 1 mL/min.

Fatty acids profile
The fatty acid composition of breast meat samples was estimated by adopting the protocol of [49]. Accordingly, 2 g meat samples were weighed into a test tube with 20 mL of Folch solution (10 volumes, chloroform: methanol = 2:1, wt/vol) and homogenized using a polytron for 10 s. Moreover, 24 μL of butylated hydroxyanisole (BHA, 10 % dissolved in 98 % ethanol) was added to each sample prior to homogenization. The homogenate was filtered through whatman no.1 filter paper into a 100 mL graduated cylinder and ¼ volume (on the basis of Folch solution volume) of 0.88 % NaCl solution was added. Afterwards, the cylinder was capped with a glass stopper and the filtrate was mixed well. The cylinder was washed twice with 10 mL of Folch solution (3:47:48/CHCl 3 :CH 3 OH:H 2 O) and the contents were stored up to 6 h until aqueous and organic layers were clearly separated. After separation, upper layer containing methanol was siphoned and 0.5 mL of lower layer (chloroform layer) was moved to a glass scintillation vial and dried at 70°C under nitrogen for 2-3 min. Moreover, 1 mL of BF 3 in methanol was added as methylating agent to cut ester bond to form fatty acids methyl esters and then heated for 50 min followed by cooling at room temperature. Later, 3 mL of hexane and 5 mL of distilled water were added, mixed thoroughly and left overnight for phase separation. The top (hexane) layer, containing methylated fatty acids was used for gas chromatographic analysis. The fatty acid compositional profiling was performed by using Gas Chromatograph (HP 6890) equipped with an auto sampler and flame ionization detector. A capillary column (HP-5; 0.25 mm i.d., 30 m, 0.25-μm film thickness) was used to inject samples (1 μL) into the capillary column. The oven temperature conditions (180°C for 2.5 min, increased to 230°C at 2.5 C/min, then held at 230°C for 7.5 min) were maintained. The temperatures of the inlet and detector were fixed at 280°C. The helium was used as a carrier gas and a constant column flow of 1.1 mL/min was used. The flame ionization detector air, hydrogen (H 2 ) and helium flows were 350, 35, and 43 mL/ min, respectively. The identification of fatty acids was accomplished by comparing mass spectral of fatty acids against their standards. The results of the fatty acid were reported as percentage composition of total lipids and peak area was used to calculate fatty acid composition of samples.

Statistical analysis
The resultant data were analyzed through completely randomized design (CRD) by using package of statistical (Statistic 8.1). Moreover, Analysis of variance (ANOVA) was performed to measure the level of significance [50].