Flame Ionization

Cell or tissue lipids were extracted by the procedures similar to the Folch method [4 (link)]. Chloroform/methanol (2:1, v/v) containing 0.005% butylated hydroxytoluene (as antioxidant) was added (usually 5 ml solvent added to 50–100 μl sample) and mixed vigorously for 1 min then left at 4°C overnight. One ml of 0.9% NaCl was added and mixed again. The chloroform phase containing lipids was collected. The remains were extracted with another 2 ml chloroform. The chloroform was pooled and dried under nitrogen and subjected to methylation. To monitor the recovery rate, the fatty acid C23:0 was added to the samples (usually 1 μg added to 2 mg tissue sample) as an internal standard.
Fatty acid methyl esters were prepared by methods similar to those described previously [5 (link),6 (link)] using BF₃/methanol reagent (14% Boron Trifluoride). Lipid sample was mixed with 1 ml hexane in 16 ml glass tubes with Teflon-lined caps. BF₃/MeOH reagent (1 ml) was added and the mixture was heated at 90–110°C in a metal block or a sand bath for 1 hour, cooled to room temperature and methyl esters extracted in the hexane phase after addition of 1 ml H₂O. Samples were allowed to stand for 20–30 min, and then the upper hexane layer was removed and concentrated under nitrogen.
Fatty acid methyl esters were analyzed by gas chromatography using a fully automated HP5890 system equipped with a flame-ionization detector, as described previously [7 (link)] The chromatography utilized an Omegawax 250 capillary column (30 m × 0.25 mm I.D.). Peaks were identified by comparison with fatty acid standards (Nu-chek-Prep, Elysian, MN), and area and its percentage for each resolved peak were analyzed using a Perkin-Elmer M1 integrator.

The erythrocyte fatty acid membrane profile analysis was carried out as previously described, using the erythrocyte membrane pellet obtained by standard methods [39] (link). For this study, selection of the erythrocyte fraction was made by modification of a literature procedure for the selection of aged erythrocytes (red blood cell age >3 months), with cells selected for high density and small diameter compared to the average erythrocyte population [40] .
One mL of whole blood was first centrifuged at 2000 g for 5 min to eliminate the plasma, and a second round of centrifugation was then carried out at 4000 g at 4°C for 5 min in order to yield a stratification by cell density. The bottom layer (2.5 mm from the bottom of tube) consisted of erythrocyte cells, which were evaluated for their diameter using a Scepter™ 2.0 Cell Counter (Merck Millipore, Milan, Italy) to characterize the cell selection from each blood sample. The results were also compared with the cell population obtained from standard density gradient separation [41] (link), [42] .
Briefly, lipids were extracted from erythrocyte membranes according to the method of Bligh and Dyer [43] . The phospholipid fraction was controlled by TLC as previously described [39] (link), then treated with KOH/MeOH solution (0.5 M) for 10 min at room temperature and under stirring [44] (link).
Fatty acid methyl esters (FAME) were extracted using n-hexane; the hexane phase was collected and dried with anhydrous Na₂SO₄. After filtration, the solvent was eliminated by evaporation using a rotating evaporator, and the thin white film of the FAME was subsequently dissolved in a small volume of n-hexane. Approximately 1 µL of this solution was injected into the GC. A Varian CP-3800 gas chromatograph, with a flame ionization detector and an Rtx-2330 column (90% biscyanopropyl-10% phenylcyanopropyl polysiloxane capillary column; 60 m, 0.25 mm i.d., 0.20 µm film thickness) was used for the analysis. Temperature was held at 165°C held for the initial 3 min, followed by an increase of 1°C/min up to 195°C, held for 40 min, followed by a second increase of 10°C/min up to 250°C, held for 5 min. The carrier gas was helium, held at a constant pressure of 29 psi. Methyl esters were identified by comparison with the retention times of commercially available standards or trans fatty acid references, obtained as described elsewhere [45] (link).

Fishes allocated for flesh analysis were filleted (denuded of skin and bone) and stored at −20°C until used for fillet proximate analysis. Fishes allocated for fatty acids analysis were stored at −80°C. Proximate analysis was conducted using standard procedures (AOAC, 1990 ), percentage moisture (dried at 80°C to constant weight), protein (Kjeldahl nitrogen; N × 6.25) in an automated Kjeltech (Model 2300, Tecator, Sweden), total lipid by chloroform/methanol extraction (2:1 v/v) (Folch et al., 1957 (link)) as modified by Ways and Hanahan (1964 (link)) and ash by incineration in a muffle furnace (Model WIT, C & LTetlow, Australia) at 550°C for 18 h. Fatty acid analysis was carried out on each of the added dietary oils, experimental diets and fillet samples from each of the replicates. Fatty acid methyl esters (FAMEs) were prepared from aliquots of total lipids by acid catalyzed transmethylation with sulfuric acid in methanol overnight at 50°C (Christie, 1982 ). FAMEs were purified by TLC using hexane/diethyl ether/acetic acid (85:15:15 v/v/v) as solvent (Tocher and Harvie, 1988 (link)). Separation of FAMEs was carried out in a Gas Chromatograph system (Agilent Technologies, 6890 N, USA) equipped with a flame ionization detector (FID), and a cross-linked silica capillary column HP-88 (100 m, 250 μm ID, 0.2 μm film thickness), on-column injection and using helium as the carrier gas with a flow rate of 1.1 ml min^-1. The column was programmed for an initial temperature of 140°C held for 5 min, rising at a rate of 4°C min^-1 to the final temperature of 240°C and held for 10 min. Injector and detector temperatures were 230°C and 260°C, respectively. The flow rates of compressed air and hydrogen were 300 ml min^-1 and 30 ml min^-1, respectively. Identification and quantification of FAMEs were based on the comparison of the sample retention time with known standards (Sigma Chemicals, St. Louis, USA).

Fresh C. oleifera seeds collected at different treatment groups were dried in an oven at 60°C to a constant weight. All dried seeds were powdered by a mill (FOSS Scino (Suzhou) Co Ltd., Suzhou, China), and 5 g of three independent samples of dry seeds were weighed. Total tea oil was extracted according to the manufacturer’s protocol (Gong et al., 2020 (link)). Approximately 50 mL of petroleum ether was added to the aluminum cup in Soxtec device for extraction, and the machine temperature was set to 75°C. The program was as follows: 30 minutes of digestion, 150 minutes of extraction, and 60 minutes of solvent evaporation and recovery. The extracted oil is stored in the centrifuge tube for further use. Oil content was calculated according to the following formula: Oil content = [(weight of filter paper bag before extraction − weight of filter paper bag after extraction)/weight of powder] × 100%. Each sample had three biological replicates, and all oil contents in this study are performed with three biological replicates.
Fatty acid composition was determined using gas chromatography (Shimadzu GC-2014, Shimadzu, Kyoto, Japan) following the manufacturer’s instructions (Gong et al., 2020 (link)). Each sample had three biological replicates. Fatty acid methyl esters were prepared using NaOH/methanol method. A total of 60 mg of oil was transferred into a ground glass stoppered test tube, dissolved by 4 mL isooctane. Internal standard solution (triglyceride undecanoate, 100 μL) and 200 μL of potassium hydroxide methanol solution were added to the sample. The samples were mixed for 30 s on vortex mixer, and then allowed to clarify. One gram of sodium bisulfate was added, and the test tube was shaken vigorously to neutralize the potassium hydroxide. After the salt settled, the upper layer solution was used for chromatographic analysis. The gas chromatograph program was as follows: flame ionization detector temperature, 250°C; sample inlet temperature, 250°C; chromatographic column, 60 cm × 0.25 mm × 0.2 μm; carrier gas, nitrogen; split ratio, 1:50; sample injection volume, 1 μL; heating process, 50°C (2 min), 170°C (10°C/min, stored for 10 min), 180°C (2°C/min, stored for 10 min), and 220°C (4°C/min, stored for 22 min).

We collected volatiles from the leaves of the two morphotypes under the same growth conditions and ambient temperature, in biological triplicates. Approximately 100 g of dry leaves from the two morphotypes, were extracted with 1000 mL of reverse osmosis water using a Clevenger apparatus⁸⁷ , following four hours of extraction by hydro-distillation. Samples of the essential oils extracted from the leaves were analyzed using gas chromatography with a flame ionization detector (GC-FID) (Shimadzu GC-2010 Plus) and gas chromatography coupled to mass spectrometry (GC–MS) (Shimadzu GCMS-QP2010 SE).
We conducted the analyses according to the following conditions: helium (He) as the carrier gas for both detectors, with the flow and linear speeds of 2.80 mL min⁻¹ and 50.8 cm s⁻¹ (GC-FID), and 1.98 mL min⁻¹ and 50.9 cm s⁻¹ (GC–MS), respectively; injection port temperature of 220 °C with a split ratio of 1:30; fused silica capillary column (30 m × 0.25 mm); stationary phase Rtx^®-5MS (0.25 μm film thickness); oven with an initial temperature of 40 °C, maintained for 3 min, then gradually increased by 3 °C min⁻¹ until 180 °C, where it remained for 10 min (total analysis time: 59.67 min); and FID and MS detector temperature of 240 °C and 200 °C, respectively^{49 (link)}. The used samples were taken from the vials in 1 μL of a solution containing 3% essential oil dissolved in hexane with 0.1 mol L⁻¹ dimethylacetamide (DMA; external standard for reproducibility control).
The GC–MS analyses were performed using electron impact equipment with an impact energy of 70 eV, scanning speed of 1000, scanning interval of 0.50 fragments s⁻¹, and fragments detected from 29 to 400 (m/z). The GC-FID analyses were carried out in a flame formed by H2 and atmospheric air at a temperature of 300 °C. Flow rates of 40 mL min⁻¹ and 400 mL min⁻¹ were used for H2 and air, respectively. Identification of the compounds in the essential oils was accomplished by comparing the obtained mass spectra with those available in the spectral library database (Wiley 7, NIST 05, and NIST 05 s) and retention indices (RI). To calculate the RIs, we used a mixture of saturated alkanes C7–C40 (Supelco-USA) and adjusted retention time of each compound, obtained by GC-FID. The values calculated for each compound were compared with those reported in literature^{88 –90} .
We calculated the relative percentage of each compound in the essential oil using the ratio between the integral area of the peaks and the total area of all sample constituents obtained via GC-FID analyses. The compounds with a relative area above 2% were identified and considered predominant if above 10%.