Chia seeds and cold-pressed chia seed oil (CPCO) used as control oil were purchased from Yayla Gourmet Cereals (Mersin, Türkiye) and Botalife chia oil (Manolya Natural and Aromatic Products Ltd, Isparta, Türkiye), respectively. H₂SO₄, NaOH, H₃BO₃, Na₂SO₄, MetOH, Folin-Ciocalteu reagent, Na₂CO₃, Gallic acid, NHAC₄, CuCl₂ and other reagents were of analytical purity. All analytical grade chemicals and reagents used in this study were purchased from Merck (Darmstadt, Germany) and Sigma Chem. Co. (St. Louis, MO, USA).

Prior to analysis, chia seeds (see Figure 1) were ground and homogenized using a high-speed blender (7011S model, Waring Laboratory, USA) and ground samples were kept at 4 °C until further use.

100 chia seeds were randomly collected, weighed on an analytical balance (Sartorius ED224S, Sartorius, Germany) and their weight was multiplied by ten. To determine the dimensional properties (length, width) of chia seeds, ten seeds were randomly chosen and measured with a calipper (CD-15CP, Mitutoyo Ltd., Andover, UK). All procedures were repeated at least four times.

Instrumental color values (L, a*, b*) of all chia seed samples were recorded using a Minolta colorimeter (CR-400, Osaka, Japan) calibrated with a white ceramic plate.

The moisture content in ground chia seeds (%) was determined with an infrared moisture analyzer (Ohaus MB45, Switzerland) at 110 °C for 30 min. The contents of ash (%) and protein (%) in the chia seeds were analyzed using the official methods of the Association of Official Agricultural Chemists (AOAC, 2005) and American Association of Cereal Chemists (AACC, 2012). The total oil content in chia seeds (mainly composed of 6.4% palmitic, 2.9% stearic, 7.1% oleic, 18.1% linoleic and 62.8% α-linolenic acid) was measured by 6-h extraction with n-hexane in an automated Soxhlet apparatus (Gerhardt Soxtherm Manager SX, Germany).

The essential oil in chia seeds was extracted using a Clevenger-type apparatus (a glass hydro-distiller) (Uzkuç et al., 2021). Prior to essential oil extraction from chia seeds via hydrodistillation, microwave pre-treatment was conducted. For this purpose, chia seeds were pre-roasted in a household microwave oven (Samsung, ME711K, Malaysia), based on the parameters of our previous work (Aydeniz et al., 2014). In the microwave oven, 360 W power applied to the seeds for 6 min (3 minutes apply/3 minutes wait) for homogeneous heat distribution.

Subsequently, approximately 200 g of ground chia seeds were used for each extraction, and the seed-to-water ratio was set at 1:3 w/v (weight/volume). During the oil extraction process, heating was provided by an electric mantle (Termal Laboratory Equipments, Istanbul, Türkiye), and the condenser temperature was maintained at 8 °C using continuous cold water circulation. The distillation process continued until no further increase was observed in the amount of essential oil collected in the measuring cylinder (see Figure 2). The collected essential oils (HDE) were passed through anhydrous sodium sulfate, stored in amber-colored bottles at -18 °C until analysis, and the extraction procedure was repeated at least twice.

A solvent-free microwave-assisted hydrodistillation method was applied with an advanced microwave extraction system (Milestone ETHOS X, Milestone, Italy) following minor modifications as described in Lucchesi et al. (2004). Before the extraction process, ground chia seeds (4% moisture content) were left to thaw (1:3 w/w, weight/weight) for 60 min in distilled water at room temperature. Excess water was then removed from the seeds, and they were transferred to a glass reactor. Thawing is a crucial step to ensure the initial moisture level required for the seed absorption of microwave irradiation. Hence, a preliminary study was conducted to determine the extraction conditions (Uzkuç et al., 2021). The extraction process was carried out at 750 W until no further essential oil could be extracted. (See Figure 3).

During microwave-assisted hydrodistillation, the software monitored the extraction parameters such as temperature, time, pressure, and power. The extracted essential oils (MAHDE) were dried with anhydrous sodium sulfate to separate them from the water. They were then collected in an amber vial with a PTFE-silicon septum and stored at -18 °C.

Specific gravity values of HDE and MAHDE were determined by the ratio of the weight of 10 μL essential oil to the weight of 10 μL distilled water according to Karakaya et al. (2014). The extraction yields for the essential oils were calculated (Karakaya et al., 2014) and refractive index values were also recorded using a refractometer (Soif Optical instruments, DA 0158, China). Both measurements were made in triplicate at 23 °C.

All essential oil extraction procedures were repeated twice. All physico-chemical analyses in chia seeds and seed essential oils were carried out twice for each replicate sample. All collected data were subjected to analysis of variance (ANOVA) using Minitab ver. 16.1.1 statistical package programs at 5% significance level (Minitab, 2010).

The nutritional benefits of chia seeds, such as gluten-free, high fiber and protein contents, and isoflavone source, are promising characteristics for the development of innovative food and beverage formulations. As a result, the use of whole chia seeds, chia flour, hydrated chia or chia musilage in the food industry is increasing tremendously on a daily basis. Dairy and meat products, gluten-free bakery formulations, chia fresca and hydrocolloid agents containing chia are the best known and most popular products available in local markets (Zettel and Hitzmann, 2016).

Some physical and chemical properties and essential oil yields of chia seeds are listed in Table 1. The seed sizes and 1000-mass weight of chia seeds used for oil production are consistent with those cited in the literature (Porras-Loaiza et al., 2014).

According to the findings stated by different researchers (Goyat et al., 2018; Timilsena et al., 2017), the basic nutritional composition of the seeds varies within a wide range of 30% fat, 18-30% dietary fiber, 15-21% protein, 25-42% carbohydrates and 5% ash. Crude oil content was also found to be lower than reported in the literature. This may be due to species and geographical differences.

Process parameters such as short extraction time, minimum solvent volume and environmental impact, low cost, oil rich in bioactive components and volatile compounds and maximization of oil yield were preferred in the selection of the extraction process. Considering all these process parameters, microwave-assisted hydrodistillation for essential oil extraction has many advantages (Aydeniz et al., 2014).

The efficiency of essential oil extraction is influenced by several parameters such as the extraction method, pre-extraction, pre-treteatment, etc. For example, microwave-assisted distillation had the highest oil yield content, which was significantly different (P < 0.05) from that of hydrodistillation (Table 1).

Direct microwave irradiation during the extraction process can cause structural changes in the seed wall. This can have a significant impact on oil yield. According to Lucchesi et al. (2004), microwave irradiation has a distinct affect on essential oil extraction because microwave beams allow the essential oil to be removed from the seed quickly without leading to major changes or loss in the volatile aromatic compounds. Thus, the main advantages of microwave-assisted extraction are higher oil yield (4.20%), shorter extraction time, minimum energy consumption (kW h/kg oil) and environmental impact (kg CO₂ rejected/kg oil).

The color characteristics of chia seeds are shown in Figure 1 and Table 2. L* and b* values were statistically significantly affected (P > 0.05) by the pre-treatments. Size reduction or grinding of seeds can significantly increase the measured redness and yellowness of seeds due to an increase in surface area.

Relative density and refractive index are important quality parameters for detecting adulteration and also for purity control in essential oils. These criteria are also specified in the European Pharmacopoeia (Conseil de L’Europe, 1996). Table 3 represents the relative density and refractive index values for chia seed oils obtained by different techniques.

Although it is noteworthy that cold-pressed oils have higher relative density and refractive index values, the effect of the distillation technique on the physical properties of essential oils is not statistically significant. The fact that the cold pressing technique is based on a different extraction principle and the pressing temperature is limited to a maximum of 40 °C is thought to be responsible for the higher values.

Rokosik et al. (2020) reported that the relative density and refractive index values of cold-pressed chia seed oil were 0.929 and 1.4825, respectively. The relative density value mentioned by researchers was lower than that of essential oils and cold-pressed chia seed oil.

It is believed that the physical structures of all three oils (essential, cold press, solvent extracted) are completely different (Fathollahi et al., 2021). Similarly, although this result is lower than the refractive index values found in our essential oils, it is similar to the index values of cold-pressed chia oil.

The total phenolic content and antioxidant capacity data obtained in the current study are presented in Table IV. From the chia seed oils produced by different techniques, total phenolic content ranged from minimum to maximum concentrations of 11.37 mg GAE/kg oil (MAHDE) and 102.90 mg GAE/kg oil (CPCO), respectively (Table 4). In general, it was determined that the hydrodistillation technique was found to have three and five times more total phenolic and antioxidant capacity values than the microwave technique, respectively.

Notable statistical differences (P <0.05) in the levels of total phenolic content and antioxidant capacity indicate that hydrodistillation is a superior method for obtaining higher levels of total phenolic content and antioxidant capacity. During the hydrodistillation process, the plant materials are immersed into the boiling water, which leads to heat-induced cell wall degradation and breakdown of phenolic-protein bonds in chia seeds, ensuring a faster and more effective transport of phenolic compounds (Bhalla et al., 2023).

These results are consistent with previously reported studies on the effect of extraction methods on the total phenolic and antioxidant capacity of chia seed oils. Bodoira et al. (2017) determined the total phenolic content of solvent-extracted chia seed oils to be 42 mg GAE/kg oil. Although this value is very close to the total phenolic content (30.56 mg GAE/kg oil) of the essential oil obtained by the hydrodistillation technique, it is 2.5 times lower than the total phenolic content of the cold-pressed chia oil we obtained from the market.

Although essential oils have a complex structure with more than a hundred components, the majority (95%) consists of monoterpenic hydrocarbons and volatile components (Viuda-Martos et al., 2009).

A total of 90 volatile compounds were identified in cold-pressed chia seed oil and chia essential oil samples. The volatile compounds consisted of aldehydes, alcohols, esters, ketones, pyrazine and terpenes (Figure 4).

It was found that the percentage of many of the volatile compounds identified in the chia seed oil samples ranged between 0.5-5% except for some volatiles such as linalool, α-pinene, cumene, α-thujene, o-cymene, mesitylene (Table 5). Considering the volatile composition of the oil samples together, it can be seen that all the oil samples have different characteristics. These different characteristic can be attributed to preferred extraction method and working principles which can affect the volatile composition of the essential oil.

α-pinene is the major component with the highest percentage of 34.56% in the volatile profiles of CPCO. Other major volatiles include hexanal, β-pinene, α-thujene and o-cymene with percentages of 7.15, 8.73, 10.60 and 19.21%, respectively. These volatiles accounted for 80.25% of the total volatile composition while other volatiles accounted of 19.75% with low percentages. In particular, α-pinene, o-cymene, β-pinene and α-thujene associated with woody, terpene, herbal, green pine aroma descriptions were detected in only CPCO.

The results obtained were found to be similar to those reported by Yin et al. (2020), who reported that hexanal, 1-hexanol and ocimene were responsible for the “fresh grass or green vegetable odor” of cold-pressed oils, and by Guneser and Yilmaz (2017), who reported that α-thujene and β-cymene were the main volatiles with the highest concentrations in cold-pressed oils.

In the case of the chia essential oil sample obtained by hydrodistillation (HDE), mesitylene (1,3,5-trimethyl benzene), anethole, cumene, eugenol and benzaldehyde were identified as the major volatiles with the percentages of 14.10, 9.03,7.48, 5.95 and 4.68%, respectively. Mesitylene, anethole and cumene are aromatic compounds that may be responsible for the sweet, anise oil-like and pungent odors of HDE (Pubchem, 2024). The remaining components were identified to range between 0.45-2.91%.

Depending on the extraction methods, some seed oils contain different volatile compounds which can be directly affected by extraction conditions and are accepted as indicators of chemical reactions occurring during oil extraction. Especially furans, pyrazines and also pyrroles are formed by the thermal degradation of sugars and lipid oxidation (Kraljić et al., 2018).

During hydrodistillation, thermal degradation caused by gradually increasing temperature explains the fact that some volatile compounds such as 2-furanaldehyde (3.82%), 2-acetylfuran (2.91%), 5-methylfurfural (2.59%), ethylpyrazine (2.02%) and 2-pentyl furan (1.73%) were identified only in HDE, unlike the other samples.

According to Jakab et al. (2018) the degradation of eucalyptol caused by cleavage of the ether linkage followed by elimination of water leads to the formation of D-limonene. During hydrodistillation, the thermal process leads to the degradation of eucalyptus by dehydration and the formation of d-limonene, which explains the fact that d-limonene and eucalyptol were only detected in HDE.

The major compound identified in the chia essential oil sample obtained by microwave-assisted hydrodistillation (MAHD) was linalool with percentages of 27.99% and followed by hexanol (8.53%), hexanal (7.25%), benzene acetaldehyde (6.40%), hexyl acetate (4.09%) and 3,5-octadiene-2-one (3.98). The percentages of the remaining volatiles in these oil samples ranged from 0.04 to 2.96%.

Linalool is widely used in the perfumery and cosmetic industry for its floral and lavender-like fragrance potential. In addition, the calmimg and anxiolytic effects of linalool inhalation have been clinically demonstrated in animal models (Souto-Maior et al., 2011). Based on these observations in animals, it is thought that MAHD, which contains linalool in about a third of its volatile compositions, may be used for similar purposes.

To the best of our knowledge, there are limited studies in the literature on the volatile composition of chia seed oil and chia seed essential oil. The results of the present study are consistent with previous studies on different Salvia species (Taârit et al., 2014; Elshafie et al., 2018). Wen et al. (2019) revealed that diisobutyl phthalate and phenylacetaldehyde were the major volatiles of chia seed aromatic water while chia seed oil obtained from supercritical CO₂ extraction had mainly aromatic hydrocarbons and alkanes including toluene, nonanal and mesitylene. In another study (Taârit et al., 2014) on the volatile profile of the essential oil from Salvia species in addition to S. hispanica, 𝛼-thujone (14.77%), camphor (13.08%), and 1,8-cineole (6.66%) were determined as major compounds in the essential oils of S. officinalis, while the essential oils of S. verbenaca were characterized by camphor (38.94%), caryophyllene oxide (7.28%), and 13-epi-manool (5.61%) as major volatiles. It was also determined that the essential oil of S. sclarea was composed of linalool (24.25%), 𝛼-thujene (7.48%), linalyl acetate (6.90%), germacrene-D (5.88%), bicyclogermacrene (4.29%), and 𝛼-copaene (4.08%). Similarly, Tulukcu et al. (2019) determined that hexanal, sabinene, α-pinene, α-thujone, borneol, linalyl acetate, β-pinene, camphene, α-thujene, 2,4(10)-thujadien, β-myrcene, limonene, 1,8-cineole and camphor are major volatile compounds, which were similar volatiles determined in the chia seed oil and essential oil in our study.

Similar to our findings, Jung et al. (2021) reported that cold-pressed chia seed oil contained volatiles of acids, alcohols, aldehydes, ketones and terpenes. Hexanoic acid, hexanal, (3E, 5E)-3,5-octadien-2-one, 2-ethylfuran, α-pinene and p-cymene were found in high concentrations in cold-pressed chia seed oil. The researchers reported that the levels of these compounds were 1.23 , 4.74 , 42.6 , 1.06 , 4.42 and 1.55 μ/kg, respectively.