The Ichihara and Fukubayashi (2010) method with some modifications was used to determine the FA compositions of the oils and blends. To summarize, 50 mg oil/blend were used for the preparation of fatty acid methyl esters (FAME) by adding methanolic HCl containing BHT (antioxidant, 50 mg/mL) and subjecting the mixture to 80 ºC (in a water bath) for 2 h. n-hexane was used to extract the formed FAME. After drying the FAME (using nitrogen gas), they were reconstituted in n-hexane and subjected to FA analysis by gas chromatography (GC) (7820A, Agilent, Santa Clara, CA, USA), which was coupled with a Flame Ionization Detector (FID). The stationary phase was HP88 capillary column; 30 m long with i.d. 0.25 mm and the thickness was 0.2 µm. The injector and FID temperature was set at 250 ºC. Oven temperature was set at 140 ^°C for an initial 5 min and with 4 ºC/min rise, until reaching 230 ºC and held at 230 ºC for 12.5 min. 1:25 was the split ratio and nitrogen was the carrier gas at a flow rate of 1.1 mL/min. The FA composition is expressed as % FA.

The acid value (AV, expressed as mg KOH/ g oil), % free fatty acid (% FFA, expressed as % FFA (as lauric acid)) and peroxide value (PV, expressed as milliequivalent O₂/ kg oil) were determined using AOAC official Method 940.28, Ca 5a-40 and Cd 8b-90, respectively (Jagtap et al., 2021, Joshi et al., 2022 and De Boer et al., 2018). Smoke point (SP) was determined according to a method reported by Das et al. (2013).

The CO blends were subjected to heat at 180 ºC for 240 minute (four hours). Samples were collected at 0, 60, 120 and 240 minutes of heating. Using UV/VIS spectrophotometer (UV 3000⁺LABINDIA ANALYTICAL, India), the absorbance of the oils/blends (dissolved in iso-octane) was measured at 232 nm and 268 nm. Values for K₂₃₂ and K₂₆₈ were estimated using the formula cited by Malvis et al. (2019).

The oxidation status of CO blends after four hours of heating was analyzed by estimating the PV, the para-anisidine Value (p-AV) and total oxidation value (TOTOX Value) using AOCS official methods.

THP-1 cells were seeded at the density of 1×10⁵ cells/well on the transparent 96W plate. The concentrations of oils or CO blends used to treat the cells were 125, 62.5 and 31.25 µg/mL. A MTT assay was performed to assess cell viability at the end of 24, 48 and 72 h treatment. In brief, 1 mg/mL MTT stock was added to the cells (200 µL/well) after careful removal of the culture medium. The plates were incubated in the CO₂ incubator for three hours followed by careful removal of the MTT solution from all the wells. DMSO was added to all the cells (100 µL/well) and the plates were kept at room temperature for 20 minute to allow dissolution of formazan crystals. Absorbance was measured at 570 nm. Viability percentage was calculated, assuming the viability of control cells (cells treated with DMSO) to be 100%.

At the end of 48 h treatment with the CO blends (125 µg/mL), THP-1 cells were harvested followed by two, 1X PBS washes. As per Folch et al. (1957), the total lipid extraction was done and the Ichihara and Fukubayashi (2010) method was used for the esterification of the lipids extracted from the cells. The THP-1 FA composition was determined by performing GC on the esterified lipids. The GC conditions as mentioned in section ‘2.3.1’ were used. The data is presented as % FA of the total lipids extracted from the cells.

THP-1 cells (1×10⁵/well in 24 W plate) were treated with individual oils or CO blends (125 µg/mL) for 48 h. For TNFα and IL-6 estimation, oil or blend treated cells were LPS (25 ng/mL) stimulated. The cell supernatant was collected (after LPS stimulation) at 6 h for TNFα and at 24 h for IL-6. ELISAs were performed as per manufacturer’s instructions to determine cytokine levels. The data is graphically represented as fold change compared to +LPS (only LPS stimulation).

GC-FID analysis was used to confirm altered essential FA levels in the blends. The FA composition of the individual oils and CO blends is presented in Table 1. The FA compositions of CO and blends were statistically different than FO which was also reflected in the total SFA, MUFA and PUFA contents (#; p < 0.001 versus FO, $; p < 0.05 versus FO). FO had the highest PUFA content while CO had the highest saturated FA content with lauric acid as the major FA. The reported percentages in lauric acid are in the range of 45.9-52.6 (Kumar et al., 2015; Bhatnagar et al., 2009). CO had meager amounts of both MUFA and PUFA, especially essential FA and LA. CO had no ALA. A similar FA composition for CO has been reported in the literature (Kumar et al., 2015; Bhatnagar et al., 2009; Guillaume et al., 2018, Maurikaa et al., 2020). FO showed ALA as the dominant FA and OA and LA were the next abundant FA. When compared to CO, the blends had significantly modified FA composition except for PA content. As the FO percentage in the CO blends increased, ALA content also increased, resulting in significantly lower LA:ALA. Therefore, from Table 1, it is clear that by blending FO and CO, the FA profile, especially ALA and LA contents and LA:ALA ratio were significantly altered. In addition, total SFA was significantly lower with a significant increase in total MUFA and PUFA contents just by adding 5% FO into the blend.

The physico-chemical parameters of the prepared blends were estimated immediately after blend preparation. These parameters (AV, % FFA, PV and SP) are represented in Table 2. The quality of any oil is indicated by its AV and % FFA (Mahesar et al., 2014). The AV for FO was significantly higher than CO and the blends. Maurikaa et al. (2020) reported the AV of CO at 0.9 mg KOH/g oil. For FO, the reported AV range from 0.21 (Symoniuk et al., 2016) to 0.53 (Bhardwaj et al., 2015). There were no significant differences between and among the AV of the CO and the blends. % FFA values were similar for FO, CO and the blends except for C20, which had a significantly higher value than FO. In the case of CO, reported % FFA values were in the range of 0.53 - 0.65 (Kumar et al., 2015 and Perera et al., 2020) while 0.32 was the % FFA reported for FO by Bhardwaj et al. (2015). PV represents oxidative deterioration of the oil (Perera et al., 2020). No peroxides were detected in CO but blending with FO resulted in significant increase in PV for the blends compared to CO. Among the blends, there were statistically significant differences. The PV for CO was reported in the range of 0.00 -3.9 (Kumar et al., 2009; Moigradean et al., 2012; Kumar et al., 2015; Perera et al., 2020), while the reported values for the PV of FO were 0.98 (Bhardwaj et al., 2015) and 2.34 (Symoniuk et al., 2016). A temperature at which various components of oil undergo breakdown and are noticed in the form of fumes is known as the SP (Guillaume et al., 2018). The SP of FO was significantly lower than CO and its blends. Compared to CO, the blends had significantly lower SP. But compared to the SP of FO, the SP for the blends were significantly higher (p < 0.01 versus FO). The literature reports the SP of CO at 191±3.6 ºC (Guillaume et al., 2018) and 193±3.0 ˚C by Perera et al., (2020). The observed differences in the values of the studied parameters may be due to differences in the raw material, handling, processing and storage conditions and duration ( Mahesar et al., 2014).

For the blends, AV, % FFA and PV increased as the contribution (percentage) of FO in the blend increased, although the SP of the blends decreased. The observed increase in the studied parameters may be because of the presence of pro-oxidants in the oil (Nering 2016). It is important to note that, though the SP for the blends were lower than CO, they were still higher than the routinely used cooking temperatures. Thus, the data from Table 2, indicates that the CO blends containing FO at up to 20% can be used as cooking oils without disturbing their physicochemical characteristics adversely.

Figure 1 represents the changes in the K₂₃₂ and K₂₆₈ values for the oils and blends heated up to 240 min (four hour) at 180 ºC. As shown in Figure 1a, before heating (0 min) as well as after heating, FO had a significantly higher K₂₃₂ value when compared to CO and all the blends (*; p < 0.001) at respective time points. No statistically significant differences were observed between or among CO and all the blends at 0 min. FO, CO and all the blends started to show a significant rise in K₂₃₂ right from 60 min (#; p < 0.001 versus 0 min). Though the fold rises (0 and 240 min) for CO and the blends were higher than FO, FO had higher values than the blends. It is significant to note that when compared to CO, C10 and C20 showed significant deterioration after 120 minutes; whereas in the case of C5, it was at 180 minutes (µ; p < 0.001 and β; p < 0.05 versus CO at respective time points).

Figure 1b represents the effect of heating on K₂₆₈. Before heating (0 min), no significant differences for K₂₆₈ were detected among the individual oils or the CO blends. FO started showing a significant rise in K₂₆₈ right from 60 min (&; p < 0.001 versus 0 min and previous time point). CO did not show any rise in K₂₆₈ at any of the time points of heating. Although the blends did show resistance for significant rise (at 60 min), the significance level rise at later time points (#; p < 0.001 versus 0 min). FO K₂₆₈ values were significantly high at all the time points of heating (except 0 min) when compared to CO and all the blends for respective time points (%; p < 0.001). It is also important to note that, when compared to CO, all the blends showed significant deterioration from 120 min (µ; p < 0.001 versus CO at respective time points). It is clear that though there was no statistical difference at 0 min for K₂₆₈ values, the rise at 240 min was in the order FO > C20 > C10 > C5 > CO, indicating that as the FO levels increased in the blend, K₂₆₈ also increased upon heating. Thus, from Figure1, it is clear that though CO and the blends displayed significant rises in both K₂₃₂ and K₂₆₈, their values were always lower than FO. In addition, the blends resisted these rises up to a certain time period compared to CO.

Table 3 represents the PV, p-AV and TOTOX values for the individual oils and CO blends initially and at the end of four hours heating at 180 ºC. It is clear that all three parameters after four hours heating were very significantly high for all the oils and blends except the p-AV for CO when compared to the before-heating values. Although the fold rise in TOTOX value was the highest for CO, it had the lowest actual value. It is important to note that fold rise for all these three parameters in the blends were higher than FO. This is probably due to the fact that thermo-oxidative deterioration products in the blends are further attacking oxidatively and thermally susceptible components present in the blends. In another study, when CO was heated to 180 ºC, the oxidative stability significantly dropped within the first hour of heating, indicating thermo-oxidative deterioration (Guillaume et al., 2018). The same authors evaluated the performance of coconut oil under two temperature conditions. In the first one, they heated the oil to 180 ºC for six hours and in the second, oil was heated up to 240 ºC. Samples were collected at specific time points or temperatures. In both the cases, CO showed a rise in K₂₃₂ at later time points or temperatures after initial resistance to rise but there was no rise in K₂₆₈ in both cases. Thus, our data is in line with Guillaume et al. (2018), where we also see a rise in K₂₃₂ but no change in K₂₆₈. A significant rise in the primary oxidation but no significant deterioration measured as secondary oxidation products was also well supported by the before and after heating PV and the p-AV values in the case of CO.

From Table 3 and Figure 1 it is clear that though CO alone resisted secondary oxidation, it showed significant deterioration upon heating (PV and K₂₃₂). The thermo-oxidative deterioration reflected by the TOTOX value was highest for the CO blends. In one study, Subramanian (2019) found that CO was the least thermally and thermo-oxidatively stable oil when compared to sunflower oil, groundnut oil and gingelly oil. The thermal and oxidative susceptibility were attributed to factors other than the unsaturation level of the oil. Bhatnagar et al. (2009) experimentally showed that CO had low levels of natural anti-oxidants, especially total tocopherols. Researchers could successfully lower the oxidative deterioration of CO during heating by adding essential oils which were rich in antioxidants. (Perera et al., 2020).

Thus, various chemical parameters were used to determine and confirm the thermal stability of the CO blends. As the FO contribution (percentage) in the CO blends increased, thermal degradation also increased. K₂₃₂ and K₂₆₈ indicated that the blends tolerated different heating durations before demonstrating significant deterioration. The extent of deterioration was indicated either as the level of significance and or the time required to reach that level of significance. Eyres et al. (2016) suggested the use of CO as a cooking oil where intense heat for long duration (like continuous deep frying) was not used but mild or medium heat for short duration (like single shallow fry) was applied.

Varma et al. (2019) used VCO to demonstrate anti-inflammatory and skin protective effects. They found that 200 µg/mL and lower concentrations of VCO were non-toxic in THP-1 cells (24 h treatment). Here, we used commercially available CO. We used three doses of oils and the blends (i.e. 125, 62.5 and 32.25 µg/mL) for three different treatment periods (24 h, 48 h and 72 h). Here, Figure 2 represents data for 125 µg/mL. For the other two concentrations, there were no statistically significant differences wrt Control (data not shown). From Figure 2, it is clear that at 24 h, CO and blends showed higher % viability, which was significant for C10 and C20. But for CO, blends and FO, at later time points, % viability was not significantly different than their respective Controls. Thus, at early time points we observed increased viability, which came down to the level of control cells at later time points. Similar rapid response (after 24 h treatment with CO) for elevated mitochondrial functioning was observed by Gil-Villarino et al. (1999) in hepatic tissue. The authors showed that in chicks fed with CO oil, up-regulation in the mitochondrial function was seen after 24 h but the up-regulated mitochondrial functioning returned back to the level of controls at longer treatment time points of CO feeding. Indeed, lauric acid from CO has been shown to improve mitochondrial functioning and biogenesis in insulin resistant THP-1 derived macrophages (Tham et al., 2020). From Figure 2, it is clear that oils and their blends do not adversely affect cell viability.

The enrichment of FA within the cells is possible by the external supply of that particular FA to the cells. Here, the FA analysis of THP-1 cells was done after treating the cells with individual oils or CO blends. Total lipids were extracted from the treated cells, esterified and subjected to GC-FID. Table 5 presents FA levels represented as a % FA of the total extracted lipids. Here, the major FA available in the individual oils and ALA and LA derived major FA were identified and presented. The FA analysis of the Control and CO-treated cells indicated that ALA was absent in these cells. Compared to these cells, FO-treated cells had significant incorporation of ALA within the cells. It was also reflected in the total ω-3 FA content. CO was the major contributor in the blends with lauric acid as major FA. In CO or blend-treated cells, no alteration in the FA related to CO was observed. In addition, as the FO percentage in the blend increased, there was a rise in the ALA (0.11, 0.13 and 0.47% for C5, C10 and C20, respectively) and total ω-3 FA content (0.64, 0.67 and 1.66% for C5, C10 and C20, respectively). An increase in ALA and total ω-3 FA content was also reflected in the LA: ALA ratio (from 4.3 for C5 to 1.2 for C20), although these changes were not statistically significant. This might be because of the preferential uptake and utilization of MC-SFA present in the CO for energy generation as seen in the liver. It is important to note that percentages of ω-6 FA remained relatively constant in all treatments.

Therefore, it is clear that the external addition of ALA in the form of CO and FO blend can result in a dose-dependent incorporation and rise in ω-3 FA content with a simultaneous lowering of ω-6:ω-3 ratio in THP-1 cells.

ALA (present in FO) is a precursor for Eicosapentaenoic acid and Docosahexaenoic acid. These ω-3 FA are known for their anti-inflammatory potential (Zhao et al., 2007). From the data presented in Table 5, it is clear that externally added ALA as (alone FO or as a CO blend) could alter LA: ALA as well as ω-6:ω-3 ratio in THP-1 cells. Therefore, it was interesting to investigate the effect of CO blends on the release of TNFα and IL-6 inflammatory markers.

As shown in Figures 3a and 3b, when THP-1 cells were stimulated by LPS, there were significant rises in the release of TNFα and IL-6 in the supernatant (denoted as +LPS). For FO pre-treated (before LPS addition) THP-1 cells, there was a significant decrease in the TNFα level in the supernatant but there was no significant alteration in IL-6 level. The blends did not affect either inflammatory marker. This might be because the ω-3 FA levels achieved in the cells are not sufficient to down-regulate LPS-induced inflammation. Indeed, Hintze et al. (2016) have shown that not only ω-6:ω-3 ratio but concentrations of individual FA also regulate cytokine production. In addition, when VCO-treated THP-1 cells were LPS-stimulated, VCO displayed anti-inflammatory potential at the transcriptional and translational levels (Varma et al., 2019). It is known that CO has lower levels of biologically active compounds compared to VCO (Lima and Block, 2019). Thus, the observed differences in the results might be due to the availability of anti-inflammatory bioactive compounds in the commercially-available CO compared to VCO. Here, we observed that although the ω-6:ω-3 ratios were favorably altered in the case of the blends, the concentrations of individual FA were probably not high enough to show anti-inflammatory effects.